Effect of fraxetin on antioxidant defense and stress proteins in human neuroblastoma cell model of rotenone neurotoxicity. Comparative study with myricetin and N-acetylcysteine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Molina-Jimenez, Maria Francisca; Sanchez-Reus, Maria Isabel; Cascales, Maria

2005-12-15

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. Recently, it has been shown that fraxetin (coumarin) and myricetin (flavonoid) have significant neuroprotective effects against apoptosis induced by rotenone, increase the total glutathione levels in vitro, and inhibit lipid peroxidation. Thus, these considerations prompted us to investigate the way in which fraxetin and myricetin affect the endogenous antioxidant defense system, such as Mn and CuZn superoxide dismutase (MnSOD, CuZnSOD), catalase, glutathione reductase (GR), and glutathione peroxidase (GPx) on rotenone neurotoxicity in neuroblastoma cells. N-acetylcysteine (NAC), a potent antioxidant, was employed as a comparative agent. Also,more » the expression and protein levels of HSP70 by Northern and Western blot analysis were assayed in SH-SY5Y cells. After incubation for 16 h, rotenone significantly increased the expression and activity of MnSOD, GPx, and catalase. When cells were preincubated with fraxetin, there was a decrease in the protein levels and activity of both MnSOD and catalase, in comparison with the rotenone treatment. The myricetin effect was less pronounced. Activity and expression of GPx were increased by rotenone and pre-treatment with fraxetin did not modify significantly these levels. The significant enhancement in HSP70 expression at mRNA and protein levels induced by fraxetin was observed by pre-treatment of cells 0.5 h before rotenone insult. These data suggest that major features of rotenone-induced neurotoxicity are partially mediated by free radical formation and oxidative stress, and that fraxetin partially protects against rotenone toxicity affecting the main protection system of the cells against oxidative injury.« less

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility

PubMed Central

Plaza Davila, María; Martin Muñoz, Patricia; Tapia, Jose A.; Ortega Ferrusola, Cristina; Balao da Silva C, Carolina; Peña, Fernando J.

2015-01-01

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37°C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence. PMID:26407142

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility.

PubMed

Plaza Davila, María; Martin Muñoz, Patricia; Tapia, Jose A; Ortega Ferrusola, Cristina; Balao da Silva C, Carolina; Peña, Fernando J

2015-01-01

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37 °C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence.

Neuroprotective effect of asiatic acid on rotenone-induced mitochondrial dysfunction and oxidative stress-mediated apoptosis in differentiated SH-SYS5Y cells.

PubMed

Nataraj, Jagatheesan; Manivasagam, Thamilarasan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed

2017-07-01

Parkinson's disease (PD) is a chronic neurodegenerative disease, manifested due to the loss of dopaminergic neurons, which ultimately leads to impaired movement in elderly populations. The pathogenesis of PD is associated with numerous factors including oxidative stress, mitochondrial dysfunction and apoptosis. There is no effective therapy available to cure or halt the progression of this disease still now. Asiatic acid (AA) is a triterpene extracted from Centella asiatica has been reported as an antioxidant and anti-inflammatory agent, that offers neuroprotection against glutamate toxicity. Therefore, in this study, we have investigated the effect of AA in a rotenone (an inhibitor of mitochondrial complex I) induced in vitro model of PD. Following the exposure of SH-SY5Y cells to rotenone, there was a marked overproduction of ROS, mitochondrial dysfunction (as indexed by the decrease in mitochondrial membrane potential) and apoptosis (Hoechst and dual staining, comet assay; expressions of pro-apoptotic and anti-apoptotic indices). Pre-treatment with AA reversed these changes might be due to its antioxidant, mitoprotective and anti-apoptotic properties. However further extensive studies on in vivo models of PD are warranted to prove AA neuroprotective effect before entering into the clinical trial.

Early ionic and membrane potential changes caused by the pesticide rotenone in striatal cholinergic interneurons.

PubMed

Bonsi, P; Calabresi, P; De Persis, C; Papa, M; Centonze, D; Bernardi, G; Pisani, A

2004-01-01

Mitochondrial metabolism impairment has been implicated in the pathogenesis of several neurodegenerative disorders. In the present work, we combined electrophysiological recordings and microfluorometric measurements from cholinergic interneurons obtained from a rat neostriatal slice preparation. Acute application of the mitochondrial complex I inhibitor rotenone produced an early membrane hyperpolarization coupled to a fall in input resistance, followed by a late depolarizing response. Current-voltage relationship showed a reversal potential of -80 +/- 3 mV, suggesting the involvement of a potassium (K+) current. Simultaneous measurement of intracellular sodium [Na+]i or calcium [Ca2+]i concentrations revealed a striking correlation between [Na+]i elevation and the early membrane hyperpolarization, whereas a significant [Ca2+]i rise matched the depolarizing phase. Interestingly, ion and membrane potential changes were mimicked by ouabain, inhibitor of the Na+-K+ATPase, and were insensitive to tetrodotoxin (TTX) or to a combination of glutamate receptor antagonists. The rotenone effects were partially reduced by blockers of ATP-sensitive K+ channels, glibenclamide and tolbutamide, and largely attenuated by a low Na+-containing solution. Morphological analysis of the rotenone effects on striatal slices showed a significant decrease in the number of choline acetyltransferase (ChAT) immunoreactive cells. These results suggest that rotenone rapidly disrupts the ATP content, leading to a decreased Na+-K+ATPase function and, therefore, to [Na+]i overload. In turn, the hyperpolarizing response might be generated both by the opening of ATP-sensitive K+ channels and by Na+-activated K+ conductances. The increase in [Ca2+]i occurs lately and does not seem to influence the early events.

Analysis of regional brain mitochondrial bioenergetics and susceptibility to mitochondrial inhibition utilizing a microplate based system

PubMed Central

Sauerbeck, Andrew; Pandya, Jignesh; Singh, Indrapal; Bittman, Kevin; Readnower, Ryan; Bing, Guoying; Sullivan, Patrick

2012-01-01

The analysis of mitochondrial bioenergetic function typically has required 50–100 μg of protein per sample and at least 15 min per run when utilizing a Clark-type oxygen electrode. In the present work we describe a method utilizing the Seahorse Biosciences XF24 Flux Analyzer for measuring mitochondrial oxygen consumption simultaneously from multiple samples and utilizing only 5 μg of protein per sample. Utilizing this method we have investigated whether regionally based differences exist in mitochondria isolated from the cortex, striatum, hippocampus, and cerebellum. Analysis of basal mitochondrial bioenergetics revealed that minimal differences exist between the cortex, striatum, and hippocampus. However, the cerebellum exhibited significantly slower basal rates of Complex I and Complex II dependent oxygen consumption (p < 0.05). Mitochondrial inhibitors affected enzyme activity proportionally across all samples tested and only small differences existed in the effect of inhibitors on oxygen consumption. Investigation of the effect of rotenone administration on Complex I dependent oxygen consumption revealed that exposure to 10 pM rotenone led to a clear time dependent decrease in oxygen consumption beginning 12 min after administration (p < 0.05). These studies show that the utilization of this microplate based method for analysis of mitochondrial bioenergetics is effective at quantifying oxygen consumption simultaneously from multiple samples. Additionally, these studies indicate that minimal regional differences exist in mitochondria isolated from the cortex, striatum, or hippocampus. Furthermore, utilization of the mitochondrial inhibitors suggests that previous work indicating regionally specific deficits following systemic mitochondrial toxin exposure may not be the result of differences in the individual mitochondria from the affected regions. PMID:21402103

Mitochondrial-derived hydrogen peroxide inhibits relaxation of bovine coronary arterial smooth muscle to hypoxia through stimulation of ERK MAP kinase.

PubMed

Gao, Qun; Zhao, Xiangmin; Ahmad, Mansoor; Wolin, Michael S

2009-12-01

Mitochondrial reactive oxygen species (ROS) are potentially important in vascular oxygen-sensing mechanisms because hypoxia appears to be a stimulus for mitochondrial ROS generation; however, scavenging of endogenous ROS does not alter relaxation of endothelium-denuded bovine coronary arteries (BCA) to hypoxia. The purpose of this study was to investigate the influence of increasing mitochondrial ROS on the relaxation of BCA to hypoxia. Increasing mitochondrial superoxide with inhibitors of electron transport (10 microM rotenone and antimycin) and by opening mitochondrial ATP-dependent K+ channels with 100 microM diazoxide were observed in this study to attenuate relaxation of BCA precontracted with 30 mM KCl to hypoxia by 68-76% and 38%, respectively. This effect of rotenone is not prevented by inhibiting NADPH oxidase (Nox) activation or scavenging superoxide with Peg-SOD; however, it is reversed 85% and 26% by increasing the consumption of intracellular peroxide by 0.1 mM ebselen and 32.5 U/ml Peg-catalase. Because inhibition of extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase (10 microM PD-98059), but not src kinase or rho kinase, also reverses the effects of rotenone by 69%, the peroxide-elicited force-enhancing effects of ERK appear to be attenuating the response to hypoxia. Rotenone increased the phosphorylation of ERK (by 163%). Activation of ERK in BCA with 0.1 mM peroxide or endogenous peroxide generated by stimulating Nox2 with a stretch treatment or contraction with 100 nM U-46619 also attenuated relaxation to hypoxia. Thus coronary arterial relaxation to hypoxia may be attenuated by pathophysiological conditions associated with increased peroxide generation by mitochondria or other sources that stimulate ERK.

The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria, within the context of a tumour stem cell-like phenotype.

PubMed

Ruggeri, Pierdomenico; Farina, Antonietta R; Di Ianni, Natalia; Cappabianca, Lucia; Ragone, Marzia; Ianni, Giulia; Gulino, Alberto; Mackay, Andrew R

2014-01-01

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.

The TrkAIII Oncoprotein Inhibits Mitochondrial Free Radical ROS-Induced Death of SH-SY5Y Neuroblastoma Cells by Augmenting SOD2 Expression and Activity at the Mitochondria, within the Context of a Tumour Stem Cell-like Phenotype

PubMed Central

Di Ianni, Natalia; Cappabianca, Lucia; Ragone, Marzia; Ianni, Giulia; Gulino, Alberto; Mackay, Andrew R.

2014-01-01

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB. PMID:24736663

Bioenergetic adaptation in response to autophagy regulators during rotenone exposure

PubMed Central

Giordano, Samantha; Dodson, Matthew; Ravi, Saranya; Redmann, Matthew; Ouyang, Xiaosen; Usmar, Victor M Darley; Zhang, Jianhua

2015-01-01

Parkinson’s disease (PD) is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, exposure to the environmental neurotoxin rotenone increases the probability of developing PD. We previously reported that in differentiated SH-SY5Y cells, rotenone-induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10–100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 hr, caused cell death at 24 hr with an LD50 of 10 nM. Overall autophagic flux was decreased by 10 nM rotenone at both 2 and 24 hr, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 hr, suggesting that a mitochondrial-specific lysosomal degradation pathway may be activated. Upregulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3-methyladenine (3-MA) exacerbated cell death. Interestingly, while 3-MA exacerbated the rotenone-dependent effects on bioenergetics, rapamycin did not prevent rotenone-induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor. PMID:25081478

Pro-oxidant mitochondrial matrix-targeted ubiquinone MitoQ10 acts as anti-oxidant at retarded electron transport or proton pumping within Complex I.

PubMed

Plecitá-Hlavatá, Lydie; Jezek, Jan; Jezek, Petr

2009-01-01

Oxidative stress of mitochondrial origin, i.e. elevated mitochondrial superoxide production, belongs to major factors determining aging and oxidative-stress-related diseases. Antioxidants, such as the mitochondria-targeted coenzyme Q, MitoQ(10), may prevent or cure these pathological conditions. To elucidate pro- and anti-oxidant action of MitoQ(10), we studied its effects on HepG2 cell respiration, mitochondrial network morphology, and rates of superoxide release (above that neutralized by superoxide dismutase) to the mitochondrial matrix (J(m)). MitoSOX Red fluorescence confocal microscopy monitoring of J(m) rates showed pro-oxidant effects of 3.5-fold increased J(m) with MitoQ(10). MitoQ(10) induced fission of the mitochondrial network which was recovered after 24h. In rotenone-inhibited HepG2 cells (i.e., already under oxidative stress) MitoQ(10) sharply decreased rotenone-induced J(m), but not together with the Complex II inhibitor thenoyltrifluoroacetone. Respiration of HepG2 cells and isolated rat liver mitochondria with MitoQ(10) increased independently of rotenone. The increase was prevented by thenoyltrifluoroacetone. These results suggest that MitoQ(10) accepts electrons prior to the rotenone-bound Q-site, and the Complex II reverse mode oxidizes MitoQ(10)H(2) to regenerate MitoQ(10). Consequently, MitoQ(10) has a pro-oxidant role in intact cells, whereas it serves as an antioxidant when Complex I-derived superoxide generation is already elevated due to electron flow retardation. Moreover, unlike mitochondrial uncoupling, MitoQ(10) exerted its antioxidant role when Complex I proton pumping was retarded by a hydrophobic amiloride, 5-(N-ethyl-N-isopropyl) amiloride. Consequently, MitoQ(10) may be useful in the treatment of diseases originating from impairment of respiratory chain Complex I due to oxidatively damaged mitochondrial DNA, when its targeted delivery to pathogenic tissues is ensured.

Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.

PubMed

Smolina, Natalia; Bruton, Joseph; Kostareva, Anna; Sejersen, Thomas

2017-01-01

Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The Seahorse equipment allows measuring oxygen consumption rate (OCR) in living cells and estimates key parameters of mitochondrial respiration in real-time mode. Through use of mitochondrial inhibitors, four key mitochondrial respiration parameters can be measured: basal, ATP production-linked, maximal, and proton leak-linked OCR. This approach requires application of mitochondrial inhibitors-oligomycin to block ATP synthase, FCCP-to make the inner mitochondrial membrane permeable for protons and allow maximum electron flux through the electron transport chain, and rotenone and antimycin A-to inhibit complexes I and III, respectively. This chapter describes the protocol of OCR assessment in the culture of primary myotubes obtained upon satellite cell fusion.

DJ-1 deficiency in astrocytes selectively enhances mitochondrial Complex I inhibitor-induced neurotoxicity

PubMed Central

Mullett, Steven J.; Hinkle, David A.

2011-01-01

Parkinson’s disease (PD) brains show evidence of mitochondrial respiratory Complex I deficiency, oxidative stress, and neuronal death. Complex I-inhibiting neurotoxins, such as the pesticide rotenone, cause neuronal death and parkinsonism in animal models. We have previously shown that DJ-1 over-expression in astrocytes augments their capacity to protect neurons against rotenone, that DJ-1 knock-down impairs astrocyte-mediated neuroprotection against rotenone, and that each process involves astrocyte-released factors. To further investigate the mechanism behind these findings, we developed a high-throughput, plate-based bioassay that can be used to assess how genetic manipulations in astrocytes affect their ability to protect co-cultured neurons. We used this bioassay to show that DJ-1 deficiency-induced impairments in astrocyte-mediated neuroprotection occur solely in the presence of pesticides that inhibit Complex I (rotenone, pyridaben, fenazaquin, and fenpyroximate); not with agents that inhibit Complexes II-V, that primarily induce oxidative stress, or that inhibit the proteasome. This is a potentially PD-relevant finding because pesticide exposure is epidemiologically-linked with an increased risk for PD. Further investigations into our model suggested that astrocytic glutathione and heme oxygenase-1 anti-oxidant systems are not central to the neuroprotective mechanism. PMID:21219333

Mechanism of Siglec-8-induced human eosinophil apoptosis: role of caspases and mitochondrial injury.

PubMed

Nutku, Esra; Hudson, Sherry A; Bochner, Bruce S

2005-10-28

Sialic acid binding immunoglobulin like lectin (Siglec)-8 crosslinking with specific antibodies causes human eosinophil apoptosis. Mechanisms by which Siglec-8 crosslinking induces apoptosis are not known. Peripheral blood eosinophils were examined for caspase, mitochondria and reactive oxygen species (ROS) involvement after incubating the cells with anti-Siglec-8 crosslinking Abs or control Abs, in the presence or absence of selective inhibitors. Siglec-8 crosslinking induced rapid cleavage of caspase-3, caspase-8, and caspase-9 in eosinophils. Selective caspase-8 and/or caspase-9 inhibitors inhibited this apoptosis. Siglec-8 crosslinking on eosinophils increased dissipation of mitochondrial membrane potential upstream of caspase activation. Rotenone and antimycin, inhibitors of mitochondrial respiratory chain components, completely inhibited apoptosis. Additional experiments with an inhibitor of ROS, diphenyleneiodonium, demonstrated that ROS was also essential for Siglec-8-mediated apoptosis and preceded Siglec-8-mediated mitochondrial dissipation. These experiments show that Siglec-8-induced apoptosis occurs through the sequential production of ROS, followed by induction of mitochondrial injury and caspase cleavage.

Rotenone Upregulates Alpha-Synuclein and Myocyte Enhancer Factor 2D Independently from Lysosomal Degradation Inhibition

PubMed Central

Stefanoni, Giovanni; Melchionda, Laura; Riva, Chiara; Brighina, Laura

2013-01-01

Dysfunctions of chaperone-mediated autophagy (CMA), the main catabolic pathway for alpha-synuclein, have been linked to the pathogenesis of Parkinson's disease (PD). Since till now there is limited information on how PD-related toxins may affect CMA, in this study we explored the effect of mitochondrial complex I inhibitor rotenone on CMA substrates, alpha-synuclein and MEF2D, and effectors, lamp2A and hsc70, in a human dopaminergic neuroblastoma SH-SY5Y cell line. Rotenone induced an upregulation of alpha-synuclein and MEF2D protein levels through the stimulation of their de novo synthesis rather than through a reduction of their CMA-mediated degradation. Moreover, increased MEF2D transcription resulted in higher nuclear protein levels that exert a protective role against mitochondrial dysfunction and oxidative stress. These results were compared with those obtained after lysosome inhibition with ammonium chloride. As expected, this toxin induced the cytosolic accumulation of both alpha-synuclein and MEF2D proteins, as the result of the inhibition of their lysosome-mediated degradation, while, differently from rotenone, ammonium chloride decreased MEF2D nuclear levels through the downregulation of its transcription, thus reducing its protective function. These results highlight that rotenone affects alpha-synuclein and MEF2D protein levels through a mechanism independent from lysosomal degradation inhibition. PMID:23984410

Mitochondrial electron transport chain is involved in microcystin-RR induced tobacco BY-2 cells apoptosis.

PubMed

Huang, Wenmin; Li, Dunhai; Liu, Yongding

2014-09-01

Microcystin-RR (MC-RR) has been suggested to induce apoptosis in tobacco BY-2 cells through mitochondrial dysfunction including the loss of mitochondrial membrane potential (ΔΨm). To further elucidate the mechanisms involved in MC-RR induced apoptosis in tobacco BY-2 cells, we have investigated the role of mitochondrial electron transport chain (ETC) as a potential source for reactive oxygen species (ROS). Tobacco BY-2 cells after exposure to MC-RR (60mg/L) displayed apoptotic changes in association with an increased production of ROS and loss of ΔΨm. All of these adverse effects were significantly attenuated by ETC inhibitors including Rotenone (2μmol/L, complex I inhibitor) and antimycin A (0.01μmol/L, complex III inhibitor), but not by thenoyltrifluoroacetone (5μmol/L, complex II inhibitor). These results suggest that mitochondrial ETC plays a key role in mediating MC-RR induced apoptosis in tobacco BY-2 cells through an increased mitochondrial production of ROS. Copyright © 2014. Published by Elsevier B.V.

Rotenone-sensitive mitochondrial potential in Phytomonas serpens: electrophoretic Ca(2+) accumulation.

PubMed

Moysés, Danuza Nogueira; Barrabin, Hector

2004-06-07

Phytomonas sp. are flagellated trypanosomatid plant parasites that cause diseases of economic importance in plantations of coffee, oil palm, cassava and coconuts. Here we investigated Ca(2+) uptake by the vanadate-insensitive compartments using permeabilized Phytomonas serpens promastigotes. This uptake occurs at a rate of 1.13+/-0.23 nmol Ca(2+) mg x protein(-1) min(-1). It is completely abolished by the H(+) ionophore FCCP and by valinomycin and nigericin. It is also inhibited by 2 microM ruthenium red, which, at this low concentration, is known to inhibit the mitochondrial calcium uniport. Furthermore, salicylhydroxamic acid (SHAM) and propylgallate, specific inhibitors of the alternative oxidase in plant and parasite mitochondria, are also effective as inhibitors of the Ca(2+) transport. These compounds abolish the membrane potential that is monitored with safranine O. Rotenone, an inhibitor of NADH-CoQ oxidoreductase, can also dissipate 100% of the membrane potential. It is suggested that the mitochondria of P. serpens can be energized via oxidation of NADH in a pathway involving the NADH-CoQ oxidoreductase and the alternative oxidase to regenerate the ubiquinone. The electrochemical H(+) gradient can be used to promote Ca(2+) uptake by the mitochondria.

Biguanides sensitize leukemia cells to ABT-737-induced apoptosis by inhibiting mitochondrial electron transport

PubMed Central

Velez, Juliana; Pan, Rongqing; Lee, Jason T.C.; Enciso, Leonardo; Suarez, Marta; Duque, Jorge Eduardo; Jaramillo, Daniel; Lopez, Catalina; Morales, Ludis; Bornmann, William; Konopleva, Marina; Krystal, Gerald; Andreeff, Michael; Samudio, Ismael

2016-01-01

Metformin displays antileukemic effects partly due to activation of AMPK and subsequent inhibition of mTOR signaling. Nevertheless, Metformin also inhibits mitochondrial electron transport at complex I in an AMPK-independent manner, Here we report that Metformin and rotenone inhibit mitochondrial electron transport and increase triglyceride levels in leukemia cell lines, suggesting impairment of fatty acid oxidation (FAO). We also report that, like other FAO inhibitors, both agents and the related biguanide, Phenformin, increase sensitivity to apoptosis induction by the bcl-2 inhibitor ABT-737 supporting the notion that electron transport antagonizes activation of the intrinsic apoptosis pathway in leukemia cells. Both biguanides and rotenone induce superoxide generation in leukemia cells, indicating that oxidative damage may sensitize toABT-737 induced apoptosis. In addition, we demonstrate that Metformin sensitizes leukemia cells to the oligomerization of Bak, suggesting that the observed synergy with ABT-737 is mediated, at least in part, by enhanced outer mitochondrial membrane permeabilization. Notably, Phenformin was at least 10-fold more potent than Metformin in abrogating electron transport and increasing sensitivity to ABT-737, suggesting that this agent may be better suited for targeting hematological malignancies. Taken together, our results suggest that inhibition of mitochondrial metabolism by Metformin or Phenformin is associated with increased leukemia cell susceptibility to induction of intrinsic apoptosis, and provide a rationale for clinical studies exploring the efficacy of combining biguanides with the orally bioavailable derivative of ABT-737, Venetoclax. PMID:27283492

Biguanides sensitize leukemia cells to ABT-737-induced apoptosis by inhibiting mitochondrial electron transport.

PubMed

Velez, Juliana; Pan, Rongqing; Lee, Jason T C; Enciso, Leonardo; Suarez, Marta; Duque, Jorge Eduardo; Jaramillo, Daniel; Lopez, Catalina; Morales, Ludis; Bornmann, William; Konopleva, Marina; Krystal, Gerald; Andreeff, Michael; Samudio, Ismael

2016-08-09

Metformin displays antileukemic effects partly due to activation of AMPK and subsequent inhibition of mTOR signaling. Nevertheless, Metformin also inhibits mitochondrial electron transport at complex I in an AMPK-independent manner, Here we report that Metformin and rotenone inhibit mitochondrial electron transport and increase triglyceride levels in leukemia cell lines, suggesting impairment of fatty acid oxidation (FAO). We also report that, like other FAO inhibitors, both agents and the related biguanide, Phenformin, increase sensitivity to apoptosis induction by the bcl-2 inhibitor ABT-737 supporting the notion that electron transport antagonizes activation of the intrinsic apoptosis pathway in leukemia cells. Both biguanides and rotenone induce superoxide generation in leukemia cells, indicating that oxidative damage may sensitize toABT-737 induced apoptosis. In addition, we demonstrate that Metformin sensitizes leukemia cells to the oligomerization of Bak, suggesting that the observed synergy with ABT-737 is mediated, at least in part, by enhanced outer mitochondrial membrane permeabilization. Notably, Phenformin was at least 10-fold more potent than Metformin in abrogating electron transport and increasing sensitivity to ABT-737, suggesting that this agent may be better suited for targeting hematological malignancies. Taken together, our results suggest that inhibition of mitochondrial metabolism by Metformin or Phenformin is associated with increased leukemia cell susceptibility to induction of intrinsic apoptosis, and provide a rationale for clinical studies exploring the efficacy of combining biguanides with the orally bioavailable derivative of ABT-737, Venetoclax.

The role of autophagy in Parkinson's disease: rotenone-based modeling

PubMed Central

2013-01-01

Background Autophagy-mediated self-digestion of cytoplasmic inclusions may be protective against neurodegenerative diseases such as Parkinson’s disease (PD). However, excessive autophagic activation evokes autophagic programmed cell death. Methods In this study, we aimed at exploring the role of autophagy in the pathogenesis of rotenone-induced cellular and animal models for PD. Results Reactive oxygen species over-generation, mitochondrial membrane potential reduction or apoptosis rate elevation occurred in a dose-dependent fashion in rotenone-treated human neuroblastoma cell line SH-SY5Y. The time- and dose-dependent increases in autophagic marker microtubule-associated protein1 light chain 3 (LC3) expression and decreases in autophagic adaptor protein P62 were observed in this cellular model. LC3-positive autophagic vacuoles were colocalized with alpha-synuclein-overexpressed aggregations. Moreover, the number of autophagic vacuoles was increased in rotenone-based PD models in vitro and in vivo. Conclusions These data, along with our previous finding showing rotenone-induced toxicity was prevented by the autophagy enhancers and was aggravated by the autophagy inhibitors in SH-SY5Y, suggest that autophagy contributes to the pathogenesis of PD, attenuates the rotenone toxicity and possibly represents a new subcellular target for treating PD. PMID:23497442

Neuroprotective effects of metallothionein against rotenone-induced myenteric neurodegeneration in parkinsonian mice.

PubMed

Murakami, Shinki; Miyazaki, Ikuko; Sogawa, Norio; Miyoshi, Ko; Asanuma, Masato

2014-10-01

Parkinson's disease (PD) is a neurodegenerative disease with motor symptoms as well as non-motor symptoms that precede the onset of motor symptoms. Mitochondrial complex I inhibitor, rotenone, has been widely used to reproduce PD pathology in the central nervous system (CNS) and enteric nervous system (ENS). We reported previously that metallothioneins (MTs) released from astrocytes can protect dopaminergic neurons against oxidative stress. The present study examined the changes in MT expression by chronic systemic rotenone administration in the striatum and colonic myenteric plexus of C57BL mice. In addition, we investigated the effects of MT depletion on rotenone-induced neurodegeneration in CNS and ENS using MT-1 and MT-2 knockout (MT KO) mice, or using primary cultured neurons from MT KO mice. In normal C57BL mice, subcutaneous administration of rotenone for 6 weeks caused neurodegeneration, increased MT expression with astrocytes activation in the striatum and myenteric plexus. MT KO mice showed more severe myenteric neuronal damage by rotenone administration after 4 weeks than wild-type mice, accompanied by reduced astroglial activation. In primary cultured mesencephalic neurons from MT KO mice, rotenone exposure induced neurotoxicity in dopaminergic neurons, which was complemented by addition of recombinant protein. The present results suggest that MT seems to provide protection against neurodegeneration in ENS of rotenone-induced PD model mice.

Muscarinic Receptor Activation Protects Cells from Apoptotic Effects of DNA Damage, Oxidative Stress, and Mitochondrial Inhibition*

PubMed Central

De Sarno, Patrizia; Shestopal, Svetlana A.; King, Taj D.; Zmijewska, Anna; Song, Ling; Jope, Richard S.

2006-01-01

The impact of muscarinic receptor stimulation was examined on apoptotic signaling induced by DNA damage, oxidative stress, and mitochondrial impairment. Exposure of human neuroblastoma SH-SY5Y cells to the DNA-damaging agent camptothecin increased p53 levels, activated caspase-3, and caused cell death. Pretreatment with oxotremorine-M, a selective agonist of muscarinic receptors that are expressed endogenously in these cells, did not affect the accumulation of p53 but greatly attenuated caspase-3 activation and protected from cell death to nearly the same extent as treatment with a general caspase inhibitor. Treatment with 50–200 μm H2O2 caused the activation of caspase-3 beginning after 2–3 h, followed by eventual cell death. Oxotremorine-M pretreatment protected cells from H2O2-inducedcaspase-3 activation and death, and this was equivalent to protection afforded by a caspase inhibitor. Muscarinic receptor stimulation also protected cells from caspase-3 activation induced by exposure to rotenone, a mitochondrial complex 1 inhibitor, but no protection was evident from staurosporine-induced caspase-3 activation. The mechanism of protection afforded by muscarinic receptor activation from camptothecin-induced apoptotic signaling involved blockade of mitochondrial cytochrome c release associated with a bolstering of mitochondrial bcl-2 levels and blockade of the translocation of Bax to mitochondria. Likely the most proximal of these events to muscarinic receptor activation, mitochondrial Bax accumulation, also was attenuated by oxotremorine-M treatment after treatment with H2O2 or rotenone. These results demonstrate that stimulation of muscarinic receptors provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. These findings suggest that neurotransmitter-induced signaling bolsters survival mechanisms, and inadequate neurotransmission may exacerbate neuronal loss. PMID:12538580

Mitochondrial origin of extracelullar transferred electrons in yeast-based biofuel cells.

PubMed

Hubenova, Yolina; Mitov, Mario

2015-12-01

The influence of mitochondrial electron transport chain inhibitors on the electricity outputs of Candida melibiosica yeast-based biofuel cell was investigated. The addition of 30 μM rotenone or antimycin A to the yeast suspension results in a decrease in the current generation, corresponding to 25.7±1.3%, respectively 38.8±1.9% reduction in the electric charge passed through the bioelectrochemical system. The latter percentage coincides with the share of aerobic respiration in the yeast catabolic processes, determined by the decrease of the ethanol production during cultivation in the presence of oxygen compared with that obtained under strict anaerobic conditions. It was established that the presence of both inhibitors leads to almost complete mitochondrial dysfunction, expressed by inactivation of cytochrome c oxidase and NADH:ubiquinone oxidoreductase as well as reduced electrochemical activity of isolated yeast mitochondria. It was also found that methylene blue partially neutralized the rotenone poisoning, probably serving as alternative intracellular electron shuttle for by-passing the complex I blockage. Based on the obtained results, we suppose that electrons generated through the aerobic respiration processes in the mitochondria participate in the extracellular electron transfer from the yeast cells to the biofuel cell anode, which contributes to higher current outputs at aerobic conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Agaricus blazei extract attenuates rotenone-induced apoptosis through its mitochondrial protective and antioxidant properties in SH-SY5Y neuroblastoma cells.

PubMed

Venkatesh Gobi, Veerappan; Rajasankar, Srinivasagam; Ramkumar, Muthu; Dhanalakshmi, Chinnasamy; Manivasagam, Thamilarasan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed; Chidambaram, Ranganathan

2018-02-01

The present study was aimed to find out the effect of Agaricus blazei mushroom extract against rotenone-induced cellular model. SH-SY5Y neuroblastoma cells are divided into four experimental groups (control, rotenone (100 nM), A. blazei (5 μg/ml) + rotenone (100 nM), and A. blazei alone treated) based on MTT assay, cells were allowed to measure the ROS, TBARS levels, and antioxidants activities. Finally, mitochondrial transmembrane potential (MMP) and expressions of apoptotic proteins were also analyzed. Pre-treatment with A. blazei significantly enhanced cell viability, attenuated rotenone-induced ROS, MMP, and apoptosis. Our results indicated that anti-apoptotic properties of this natural compound due to its antioxidant and mitochondrial protective function protect rotenone-induced cytotoxicity. Therefore, it may be concluded that A. blazei can be further developed as a promising drug for the treatment of Parkinson's disease (PD).

Lipophilic Cationic Cyanines Are Potent Complex I Inhibitors and Specific in Vitro Dopaminergic Toxins with Mechanistic Similarities to Both Rotenone and MPP(.).

PubMed

Kadigamuwa, Chamila C; Mapa, Mapa S T; Wimalasena, Kandatege

2016-09-19

We have recently reported that simple lipophilic cationic cyanines are specific and potent dopaminergic toxins with a mechanism of toxicity similar to that of the Parkinsonian toxin MPP(+). In the present study, a group of fluorescent lipophilic cyanines have been used to further exploit the structure-activity relationship of the specific dopaminergic toxicity of cyanines. Here, we report that all cyanines tested were highly toxic to dopaminergic MN9D cells with IC50s in the range of 60-100 nM and not toxic to non-neuronal HepG2 cells parallel to that previously reported for 2,2'- and 4,4'-cyanines. All cyanines nonspecifically accumulate in the mitochondria of both MN9D and HepG2 cells at high concentrations, inhibit the mitochondrial complex I with the inhibition potencies similar to the potent complex I inhibitor, rotenone. They increase the reactive oxygen species (ROS) production specifically in dopaminergic cells causing apoptotic cell death. These and other findings suggest that the complex I inhibition, the expression of low levels of antioxidant enzymes, and presence of high levels of oxidatively labile radical propagator, dopamine, could be responsible for the specific increase in ROS production in dopaminergic cells. Thus, the predisposition of dopaminergic cells to produce high levels of ROS in response to mitochondrial toxins together with their inherent greater demand for energy may contribute to their specific vulnerability toward these toxins. The novel findings that cyanines are an unusual class of potent mitochondrial toxins with specific dopaminergic toxicity suggest that their presence in the environment could contribute to the etiology of PD similar to that of MPP(+) and rotenone.

Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase)

PubMed Central

Miyadera, Hiroko; Shiomi, Kazuro; Ui, Hideaki; Yamaguchi, Yuichi; Masuma, Rokuro; Tomoda, Hiroshi; Miyoshi, Hideto; Osanai, Arihiro; Kita, Kiyoshi; Ōmura, Satoshi

2003-01-01

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues. PMID:12515859

Mitochondrial Respiratory Chain Inhibitors Involved in ROS Production Induced by Acute High Concentrations of Iodide and the Effects of SOD as a Protective Factor

PubMed Central

Wang, Lingyan; Duan, Qi; Wang, Tingting; Ahmed, Mohamed; Zhang, Na; Li, Yongmei; Li, Lanying; Yao, Xiaomei

2015-01-01

A major source of reactive oxygen species (ROS) generation is the mitochondria. By using flow cytometry of the mitochondrial fluorescent probe, MitoSOX Red, western blot of mitochondrial ROS scavenger Peroxiredoxin (Prx) 3 and fluorescence immunostaining, ELISA of cleaved caspases 3 and 9, and TUNEL staining, we demonstrated that exposure to 100 μM KI for 2 hours significantly increased mitochondrial superoxide production and Prx 3 protein expression with increased expressions of cleaved caspases 3 and 9. Besides, we indicated that superoxide dismutase (SOD) at 1000 unit/mL attenuated the increase in mitochondrial superoxide production, Prx 3 protein expression, and lactate dehydrogenase (LDH) release and improved the relative cell viability at 100 μM KI exposure. However, SOD inhibitor diethyldithiocarbamic acid (DETC) (2 mM), Rotenone (0.5 μM), a mitochondrial complex I inhibitor, and Antimycin A (10 μM), a complex III inhibitor, caused an increase in mitochondrial superoxide production, Prx 3 protein expression, and LDH release and decreased the relative cell viability. We conclude that the inhibitors of mitochondrial respiratory chain complex I or III may be involved in oxidative stress caused by elevated concentrations of iodide, and SOD demonstrates its protective effect on the Fischer rat thyroid cell line (FRTL) cells. PMID:26294939

Stereotaxical Infusion of Rotenone: A Reliable Rodent Model for Parkinson's Disease

PubMed Central

Xiong, Nian; Huang, Jinsha; Zhang, Zhentao; Zhang, Zhaowen; Xiong, Jing; Liu, Xingyuan; Jia, Min; Wang, Fang; Chen, Chunnuan; Cao, Xuebing; Liang, Zhihou; Sun, Shenggang; Lin, Zhicheng; Wang, Tao

2009-01-01

A clinically-related animal model of Parkinson's disease (PD) may enable the elucidation of the etiology of the disease and assist the development of medications. However, none of the current neurotoxin-based models recapitulates the main clinical features of the disease or the pathological hallmarks, such as dopamine (DA) neuron specificity of degeneration and Lewy body formation, which limits the use of these models in PD research. To overcome these limitations, we developed a rat model by stereotaxically (ST) infusing small doses of the mitochondrial complex-I inhibitor, rotenone, into two brain sites: the right ventral tegmental area and the substantia nigra. Four weeks after ST rotenone administration, tyrosine hydroxylase (TH) immunoreactivity in the infusion side decreased by 43.7%, in contrast to a 75.8% decrease observed in rats treated systemically with rotenone (SYS). The rotenone infusion also reduced the DA content, the glutathione and superoxide dismutase activities, and induced alpha-synuclein expression, when compared to the contralateral side. This ST model displays neither peripheral toxicity or mortality and has a high success rate. This rotenone-based ST model thus recapitulates the slow and specific loss of DA neurons and better mimics the clinical features of idiopathic PD, representing a reliable and more clinically-related model for PD research. PMID:19924288

Detoxified extract of Rhus verniciflua stokes inhibits rotenone-induced apoptosis in human dopaminergic cells, SH-SY5Y.

PubMed

Sapkota, Kumar; Kim, Seung; Park, Se-Eun; Kim, Sung-Jun

2011-03-01

Rhus verniciflua Stokes (RVS), traditionally used as a food supplement and in traditional herbal medicine for centuries in Korea, is known to possess various pharmacological properties. Environmental neurotoxins such as rotenone, a specific inhibitor of complex I provide models of Parkinson's disease (PD) both in vivo and in vitro. In this study, we investigated the neuroprotective effect of RVS against rotenone-induced toxicity in human dopaminergic cells, SH-SY5Y. Cells exposed to rotenone for 24 h-induced cellular injury and apoptotic cell death. Pretreatment of cells with RVS provided significant protection to SH-SY5Y cells. Further, RVS offered remarkable protection against rotenone-induced oxidative stress and markedly inhibited mitochondrial membrane potential (MMP) disruption. RVS also attenuated the up-regulation of Bax, Caspase-9 and Caspase-3 and down-regulation of Bcl-2. Moreover, pretreatment with RVS prevented the decrease in tyrosine hydroxylase (TH) levels in SH-SY5Y cells. Interestingly, RVS conferred profound protection to human dopaminergic cells by preventing the downregulation of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). These results suggest that RVS may protect dopaminergic neurons against rotenone-induced apoptosis by multiple functions and contribute to neuroprotection in neurodegenerative diseases, such as PD.

Lowered iPLA2γ activity causes increased mitochondrial lipid peroxidation and mitochondrial dysfunction in a rotenone-induced model of Parkinson's disease.

PubMed

Chao, Honglu; Liu, Yinlong; Fu, Xian; Xu, Xiupeng; Bao, Zhongyuan; Lin, Chao; Li, Zheng; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

2018-02-01

iPLA 2 γ, calcium-independent phospholipase A 2 γ, discerningly hydrolyses glycerophospholipids to liberate free fatty acids. iPLA 2 γ-deficiency has been associated with abnormal mitochondrial function. More importantly, the iPLA 2 family is causative proteins in mitochondrial neurodegenerative disorders such as parkinsonian disorders. However, the mechanisms by which iPLA 2 γ affects Parkinson's disease (PD) remain unknown. Mitochondrion stress has a key part in rotenone-induced dopaminergic neuronal degeneration. The present evaluation revealed that lowered iPLA 2 γ function provokes the parkinsonian phenotype and leads to the reduction of dopamine and its metabolites, lowered survival, locomotor deficiencies, and organismal hypersensitivity to rotenone-induced oxidative stress. In addition, lowered iPLA 2 γ function escalated the amount of mitochondrial irregularities, including mitochondrial reactive oxygen species (ROS) regeneration, reduced ATP synthesis, reduced glutathione levels, and abnormal mitochondrial morphology. Further, lowered iPLA 2 γ function was tightly linked with strengthened lipid peroxidation and mitochondrial membrane flaws following rotenone treatment, which can cause cytochrome c release and eventually apoptosis. These results confirmed the important role of iPLA 2 γ, whereby decreasing iPLA 2 γ activity aggravates mitochondrial degeneration to induce neurodegenerative disorders in a rotenone rat model of Parkinson's disease. These findings may be useful in the design of rational approaches for the prevention and treatment of PD-associated symptoms. Copyright © 2017 Elsevier Inc. All rights reserved.

Mic60/Mitofilin Overexpression Alters Mitochondrial Dynamics and Attenuates Vulnerability of Dopaminergic Cells to Dopamine and Rotenone

PubMed Central

Van Laar, Victor S.; Berman, Sarah B.; Hastings, Teresa G.

2017-01-01

Mitochondrial dysfunction has been implicated in Parkinson’s disease (PD) neuropathology. Mic60, also known as mitofilin, is a protein of the inner mitochondrial membrane and a key component of the mitochondrial contact site and cristae junction organizing system (MICOS). Mic60 is critical for maintaining mitochondrial membrane structure and function. We previously demonstrated that mitochondrial Mic60 protein is susceptible to both covalent modification and loss in abundance following exposure to dopamine quinone. In this study, we utilized neuronally-differentiated SH-SY5Y and PC12 dopaminergic cell lines to examine the effects of altered Mic60 levels on mitochondrial function and cellular vulnerability in response to PD-relevant stressors. Short hairpin RNA (shRNA)-mediated knockdown of endogenous Mic60 protein in neuronal SH-SY5Y cells significantly potentiated dopamine-induced cell death, which was rescued by co-expressing shRNA-insensitive Mic60. Conversely, in PC12 and SH-SY5Y cells, Mic60 overexpression significantly attenuated both dopamine- and rotenone-induced cell death as compared to controls. Mic60 overexpression in SH-SY5Y cells was also associated with increased mitochondrial respiration, and, following rotenone exposure, increased spare respiratory capacity. Mic60 knockdown cells exhibited suppressed respiration and, following rotenone treatment, decreased spare respiratory capacity. Mic60 overexpression also affected mitochondrial fission/fusion dynamics. PC12 cells overexpressing Mic60 exhibited increased mitochondrial interconnectivity. Further, both PC12 cells and primary rat cortical neurons overexpressing Mic60 displayed suppressed mitochondrial fission and increased mitochondrial length in neurites. These results suggest that altering levels of Mic60 in dopaminergic neuronal cells significantly affects both mitochondrial homeostasis and cellular vulnerability to the PD-relevant stressors dopamine and rotenone, carrying implications for PD pathogenesis. PMID:27001148

Air pollution induces enhanced mitochondrial oxidative stress in cystic fibrosis airway epithelium.

PubMed

Kamdar, O; Le, Wei; Zhang, J; Ghio, A J; Rosen, G D; Upadhyay, D

2008-10-29

We studied the effects of airborne particulate matters (PM) on cystic fibrosis (CF) epithelium. We noted that PM enhanced human CF bronchial epithelial apoptosis, activated caspase-9 and PARP-1; and reduced mitochondrial membrane potential. Mitochondrial inhibitors (4,4-diisothiocyanatostilbene-2,2'disulfonic acid, rotenone and thenoyltrifluoroacetone) blocked PM-induced generation of reactive oxygen species and apoptosis. PM upregulated pro-apoptotic Bad, Bax, p53 and p21; and enhanced mitochondrial localization of Bax. The anti-apoptotic Bcl-2, Bcl-xl, Mcl-1 and Xiap remained unchanged; however, overexpression of Bcl-xl blocked PM-induced apoptosis. Accordingly, we provide the evidence that PM enhances oxidative stress and mitochondrial signaling mediated apoptosis via the modulation of Bcl family proteins in CF.

Mitochondrial uncoupling agents antagonize rotenone actions in rat substantia nigra dopamine neurons.

PubMed

Wu, Yan-Na; Munhall, Adam C; Johnson, Steven W

2011-06-13

Mild uncoupling of oxidative phosphorylation has been reported to reduce generation of reactive oxygen species (ROS) and therefore may be neuroprotective. We reported previously that the mitochondrial poison rotenone enhanced currents evoked by N-methyl-D-aspartate (NMDA) by a ROS-dependent mechanism in rat midbrain dopamine neurons. Thus, rotenone, which produces a model of Parkinson's disease in rodents, may increase the risk of dopamine neuron excitotoxicity. The purpose of this study was to test the hypothesis that oxidative phosphorylation uncoupling agents would antagonize the effect of rotenone on NMDA current. We used patch pipettes to record whole-cell currents under voltage-clamp (-60 mV) in substantia nigra dopamine neurons in slices of rat brain. Rotenone, NMDA and uncoupling agents were added to the brain slice superfusate. Inward currents evoked by NMDA (30 μM) more than doubled in amplitude after slices were superfused for 30 min with 100 nM rotenone. Continuous superfusion with the uncoupling agent carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (1-3 nM) or 2,4-dinitrophenol (100 nM) significantly antagonized and delayed the ability of rotenone to potentiate NMDA currents. Coenzyme Q₁₀ (1-10 nM), which has been reported to facilitate uncoupling protein activity, also antagonized this action of rotenone. These results suggest that mild uncoupling of oxidative phosphorylation may protect dopamine neurons against injury from mitochondrial poisons such as rotenone. Published by Elsevier B.V.

Acute action of rotenone on excitability of catecholaminergic neurons in rostral ventrolateral medulla.

PubMed

Zhang, Zhaoqiang; Shi, Limin; Du, Xixun; Jiao, Qian; Jiang, Hong

2017-09-01

The degeneration of the rostral ventrolateral medulla (RVLM) catecholaminergic neurons was responsible for some cardiovascular symptoms in Parkinson's disease (PD). Our previous study had observed the impairment of these neurons in the early stage of PD in the rotenone-induced PD rat model, but the related mechanisms remain unclear. Rotenone is a mitochondrial inhibitor, influencing the neuronal electrophysiological activity through activation of K-ATP channels that potentially participate in cell death processes. In the present study, effects of rotenone on electrophysiological properties of RVLM catecholaminergic neurons and its underlying mechanisms were investigated. In coronal slices of brain containing the RVLM through patch clamp technique, rotenone (0.5μM) induced gradual postsynaptic inhibition on the spontaneous firing and cell membrane hyperpolarization with outward currents of catecholaminergic neurons. The electrophysiological changes were blocked by glibenclamide (30μM), a blocker of K-ATP channels, and were nearly unchanged by diazoxide (100μM), an opener of K-ATP channels. Our results also showed that effects of rotenone on catecholaminergic neurons including reactive oxygen species (ROS) generation were prevented by pretreatment of coenzyme Q10 (CoQ10, 100μM), a scavenger of ROS. These suggest that rotenone-induced electrophysiological changes of RVLM catecholaminergic neurons are caused by the opening of K-ATP channels, which are partly related to ROS generation. The changes of K-ATP channels might account for the vulnerability of RVLM catecholaminergic neurons. Copyright © 2017 Elsevier Inc. All rights reserved.

Integrating multiple aspects of mitochondrial dynamics in neurons: Age-related differences and dynamic changes in a chronic rotenone model

PubMed Central

Arnold, Beth; Cassady, Steven J.; Van Laar, Victor S.; Berman, Sarah B.

2010-01-01

Changes in dynamic properties of mitochondria are increasingly implicated in neurodegenerative diseases, particularly Parkinson’s disease (PD). Static changes in mitochondrial morphology, often under acutely toxic conditions, are commonly utilized as indicators of changes in mitochondrial fission and fusion. However, in neurons, mitochondrial fission and fusion occur in a dynamic system of axonal/dendritic transport, biogenesis and degradation, and thus, likely interact and change over time. We sought to explore this using a chronic neuronal model (nonlethal low-concentration rotenone over several weeks), examining distal neurites, which may give insight into the earliest changes occurring in PD. Using this model, in live primary neurons, we directly quantified mitochondrial fission, fusion, and transport over time and integrated multiple aspects of mitochondrial dynamics, including morphology and growth/mitophagy. We found that rates of mitochondrial fission and fusion change as neurons age. In addition, we found that chronic rotenone exposure initially increased the ratio of fusion to fission, but later, this was reversed. Surprisingly, despite changes in rates of fission and fusion, mitochondrial morphology was minimally affected, demonstrating that morphology can be an inaccurate indicator of fission/fusion changes. In addition, we found evidence of subcellular compartmentalization of compensatory changes, as mitochondrial density increased in distal neurites first, which may be important in PD, where pathology may begin distally. We propose that rotenone-induced early changes such as in mitochondrial fusion are compensatory, accompanied later by detrimental fission. As evidence, in a dopaminergic neuronal model, in which chronic rotenone caused loss of neurites before cell death (like PD pathology), inhibiting fission protected against the neurite loss. This suggests that aberrant mitochondrial dynamics may contribute to the earliest neuropathologic mechanisms in PD. These data also emphasize that mitochondrial fission and fusion do not occur in isolation, and highlight the importance of analysis and integration of multiple mitochondrial dynamic functions in neurons. PMID:20850532

Aging Neural Progenitor Cells Have Decreased Mitochondrial Content and Lower Oxidative Metabolism*

PubMed Central

Stoll, Elizabeth A.; Cheung, Willy; Mikheev, Andrei M.; Sweet, Ian R.; Bielas, Jason H.; Zhang, Jing; Rostomily, Robert C.; Horner, Philip J.

2011-01-01

Although neurogenesis occurs in discrete areas of the adult mammalian brain, neural progenitor cells (NPCs) produce fewer new neurons with age. To characterize the molecular changes that occur during aging, we performed a proteomic comparison between primary-cultured NPCs from the young adult and aged mouse forebrain. This analysis yielded changes in proteins necessary for cellular metabolism. Mitochondrial quantity and oxygen consumption rates decrease with aging, although mitochondrial DNA in aged NPCs does not have increased mutation rates. In addition, aged cells are resistant to the mitochondrial inhibitor rotenone and proliferate in response to lowered oxygen conditions. These results demonstrate that aging NPCs display an altered metabolic phenotype, characterized by a coordinated shift in protein expression, subcellular structure, and metabolic physiology. PMID:21900249

Neurosupportive Role of Vanillin, a Natural Phenolic Compound, on Rotenone Induced Neurotoxicity in SH-SY5Y Neuroblastoma Cells

PubMed Central

Dhanalakshmi, Chinnasamy; Manivasagam, Thamilarasan; Nataraj, Jagatheesan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed

2015-01-01

Vanillin, a phenolic compound, has been reported to offer neuroprotection against experimental Huntington's disease and global ischemia by virtue of its antioxidant, anti-inflammatory, and antiapoptotic properties. The present study aims to elucidate the underlying neuroprotective mechanism of vanillin in rotenone induced neurotoxicity. Cell viability was assessed by exposing SH-SY5Y cells to various concentrations of rotenone (5–200 nM) for 24 h. The therapeutic effectiveness of vanillin against rotenone was measured by pretreatment of vanillin at various concentrations (5–200 nM) and then incubation with rotenone (100 nM). Using effective dose of vanillin (100 nM), mitochondrial membrane potential, levels of reactive oxygen species (ROS), and expression patterns of apoptotic markers were assessed. Toxicity of rotenone was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, release of cyt-c, and enhanced expressions of proapoptotic and downregulation of antiapoptotic indices via the upregulation of p38 and JNK-MAPK pathway proteins. Our results indicated that the pretreatment of vanillin attenuated rotenone induced mitochondrial dysfunction, oxidative stress, and apoptosis. Thus, vanillin may serve as a potent therapeutic agent in the future by virtue of its multiple pharmacological properties in the treatment of neurodegenerative diseases including PD. PMID:26664453

Impairment of Atg5-Dependent Autophagic Flux Promotes Paraquat- and MPP+-Induced Apoptosis But Not Rotenone or 6-Hydroxydopamine Toxicity

PubMed Central

Franco, Rodrigo

2013-01-01

Controversial reports on the role of autophagy as a survival or cell death mechanism in dopaminergic cell death induced by parkinsonian toxins exist. We investigated the alterations in autophagic flux and the role of autophagy protein 5 (Atg5)-dependent autophagy in dopaminergic cell death induced by parkinsonian toxins. Dopaminergic cell death induced by the mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium (MPP+) and rotenone, the pesticide paraquat, and the dopamine analog 6-hydroxydopamine (6-OHDA) was paralleled by increased autophagosome accumulation. However, when compared with basal autophagy levels using chloroquine, autophagosome accumulation was a result of impaired autophagic flux. Only 6-OHDA induced an increase in autophagosome formation. Overexpression of a dominant negative form of Atg5 increased paraquat- and MPP+-induced cell death. Stimulation of mammalian target of rapamycin (mTOR)-dependent signaling protected against cell death induced by paraquat, whereas MPP+-induced toxicity was enhanced by wortmannin, a phosphoinositide 3-kinase class III inhibitor, rapamycin, and trehalose, an mTOR-independent autophagy activator. Modulation of autophagy by either pharmacological or genetic approaches had no effect on rotenone or 6-OHDA toxicity. Cell death induced by parkinsonian neurotoxins was inhibited by the pan caspase inhibitor (Z-VAD), but only caspase-3 inhibition was able to decrease MPP+-induced cell death. Finally, inhibition of the lysosomal hydrolases, cathepsins, increased the toxicity by paraquat and MPP+, supporting a protective role of Atg5-dependent autophagy and lysosomes degradation pathways on dopaminegic cell death. These results demonstrate that in dopaminergic cells, Atg5-dependent autophagy acts as a protective mechanism during apoptotic cell death induced by paraquat and MPP+ but not during rotenone or 6-OHDA toxicity. PMID:23997112

Oxidative damage to macromolecules in human Parkinson’s disease and the rotenone model

PubMed Central

Sanders, Laurie H.; Greenamyre, J. Timothy

2013-01-01

Parkinson’s disease (PD), the most common neurodegenerative movement disorder, is associated with selective degeneration of nigrostriatal dopamine neurons. While the underlying mechanisms contributing to neurodegeneration in PD appear to be multifactorial, mitochondrial impairment and oxidative stress are widely considered to be central to many forms of the disease. Whether oxidative stress is a cause or consequence of dopaminergic death, there is substantial evidence for oxidative stress in both human PD patients and in animal models of PD, especially using rotenone, a complex I inhibitor. There are many indices of oxidative stress, but this review covers the recent evidence for oxidative damage to nucleic acids, lipids and proteins in both the brain and peripheral tissues in human PD and in the rotenone model. Limitations of the existing literature and future perspectives are discussed. Understanding how each particular macromolecule is damaged by oxidative stress and the interplay of secondary damage to other biomolecules may help design better targets for treatment of PD. PMID:23328732

Bioenergetic and proteolytic defects in fibroblasts from patients with sporadic Parkinson's disease.

PubMed

Ambrosi, Giulia; Ghezzi, Cristina; Sepe, Sara; Milanese, Chiara; Payan-Gomez, Cesar; Bombardieri, Cintia R; Armentero, Marie-Therese; Zangaglia, Roberta; Pacchetti, Claudio; Mastroberardino, Pier Giorgio; Blandini, Fabio

2014-09-01

Parkinson's disease (PD) is a complex disease and the current interest and focus of scientific research is both investigating the variety of causes that underlie PD pathogenesis, and identifying reliable biomarkers to diagnose and monitor the progression of pathology. Investigation on pathogenic mechanisms in peripheral cells, such as fibroblasts derived from patients with sporadic PD and age/gender matched controls, might generate deeper understanding of the deficits affecting dopaminergic neurons and, possibly, new tools applicable to clinical practice. Primary fibroblast cultures were established from skin biopsies. Increased susceptibility to the PD-related toxin rotenone was determined with apoptosis- and necrosis-specific cell death assays. Protein quality control was evaluated assessing the efficiency of the Ubiquitin Proteasome System (UPS) and protein levels of autophagic markers. Changes in cellular bioenergetics were monitored by measuring oxygen consumption and glycolysis-dependent medium acidification. The oxido-reductive status was determined by detecting mitochondrial superoxide production and oxidation levels in proteins and lipids. PD fibroblasts showed higher vulnerability to necrotic cell death induced by complex I inhibitor rotenone, reduced UPS function and decreased maximal and rotenone-sensitive mitochondrial respiration. No changes in autophagy and redox markers were detected. Our study shows that increased susceptibility to rotenone and the presence of proteolytic and bioenergetic deficits that typically sustain the neurodegenerative process of PD can be detected in fibroblasts from idiopathic PD patients. Fibroblasts might therefore represent a powerful and minimally invasive tool to investigate PD pathogenic mechanisms, which might translate into considerable advances in clinical management of the disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Ketones prevent synaptic dysfunction induced by mitochondrial respiratory complex inhibitors

PubMed Central

Kim, Do Young; Vallejo, Johana; Rho, Jong M

2010-01-01

Abstract Ketones have previously shown beneficial effects in models of neurodegenerative disorders, particularly against associated mitochondrial dysfunction and cognitive impairment. However, evidence of a synaptic protective effect of ketones remains lacking. We tested the effects of ketones on synaptic impairment induced by mitochondrial respiratory complex (MRC) inhibitors using electrophysiological, reactive oxygen species (ROS) imaging and biochemical techniques. MRC inhibitors dose-dependently suppressed both population spike (PS) and field potential amplitudes in the CA1 hippocampus. Pre-treatment with ketones strongly prevented changes in the PS, whereas partial protection was seen in the field potential. Rotenone (Rot; 100 nmol/L), a MRC I inhibitor, suppressed synaptic function without altering ROS levels and PS depression by Rot was unaffected by antioxidants. In contrast, antioxidant-induced PS recovery against the MRC II inhibitor 3-nitropropionic acid (3-NP; 1 mmol/L) was similar to the synaptic protective effects of ketones. Ketones also suppressed ROS generation induced by 3-NP. Finally, ketones reversed the decreases in ATP levels caused by Rot and 3-NP. In summary, our data demonstrate that ketones can preserve synaptic function in CA1 hippocampus induced by MRC dysfunction, likely through an antioxidant action and enhanced ATP generation. PMID:20374433

Differential effects of buffer pH on Ca2+-induced ROS emission with inhibited mitochondrial complexes I and III

PubMed Central

Lindsay, Daniel P.; Camara, Amadou K. S.; Stowe, David F.; Lubbe, Ryan; Aldakkak, Mohammed

2015-01-01

Excessive mitochondrial reactive oxygen species (ROS) emission is a critical component in the etiology of ischemic injury. Complex I and complex III of the electron transport chain are considered the primary sources of ROS emission during cardiac ischemia and reperfusion (IR) injury. Several factors modulate ischemic ROS emission, such as an increase in extra-matrix Ca2+, a decrease in extra-matrix pH, and a change in substrate utilization. Here we examined the combined effects of these factors on ROS emission from respiratory complexes I and III under conditions of simulated IR injury. Guinea pig heart mitochondria were suspended in experimental buffer at a given pH and incubated with or without CaCl2. Mitochondria were then treated with either pyruvate, a complex I substrate, followed by rotenone, a complex I inhibitor, or succinate, a complex II substrate, followed by antimycin A, a complex III inhibitor. H2O2 release rate and matrix volume were compared with and without adding CaCl2 and at pH 7.15, 6.9, or 6.5 with pyruvate + rotenone or succinate + antimycin A to simulate conditions that may occur during in vivo cardiac IR injury. We found a large increase in H2O2 release with high [CaCl2] and pyruvate + rotenone at pH 6.9, but not at pHs 7.15 or 6.5. Large increases in H2O2 release rate also occurred at each pH with high [CaCl2] and succinate + antimycin A, with the highest levels observed at pH 7.15. The increases in H2O2 release were associated with significant mitochondrial swelling, and both H2O2 release and swelling were abolished by cyclosporine A, a desensitizer of the mitochondrial permeability transition pore (mPTP). These results indicate that ROS production by complex I and by complex III is differently affected by buffer pH and Ca2+ loading with mPTP opening. The study suggests that changes in the levels of cytosolic Ca2+ and pH during IR alter the relative amounts of ROS produced at mitochondrial respiratory complex I and complex III. PMID:25805998

Energy metabolism determines the sensitivity of human hepatocellular carcinoma cells to mitochondrial inhibitors and biguanide drugs.

PubMed

Hsu, Chia-Chi; Wu, Ling-Chia; Hsia, Cheng-Yuan; Yin, Pen-Hui; Chi, Chin-Wen; Yeh, Tien-Shun; Lee, Hsin-Chen

2015-09-01

Human hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide particularly in Asia. Deregulation of cellular energetics was recently included as one of the cancer hallmarks. Compounds that target the mitochondria in cancer cells were proposed to have therapeutic potential. Biguanide drugs which inhibit mitochondrial complex I and repress mTOR signaling are clinically used to treat type 2 diabetes mellitus patients (T2DM) and were recently found to reduce the risk of HCC in T2DM patients. However, whether alteration of energy metabolism is involved in regulating the sensitivity of HCC to biguanide drugs is still unclear. In the present study, we treated four HCC cell lines with mitochondrial inhibitors (rotenone and oligomycin) and biguanide drugs (metformin and phenformin), and found that the HCC cells which had a higher mitochondrial respiration rate were more sensitive to these treatments; whereas the HCC cells which exhibited higher glycolysis were more resistant. When glucose was replaced by galactose in the medium, the altered energy metabolism from glycolysis to mitochondrial respiration in the HCC cells enhanced the cellular sensitivity to mitochondrial inhibitors and biguanides. The energy metabolism change enhanced AMP-activated protein kinase (AMPK) activation, mTOR repression and downregulation of cyclin D1 and Mcl-1 in response to the mitochondrial inhibitors and biguanides. In conclusion, our results suggest that increased mitochondrial oxidative metabolism upregulates the sensitivity of HCC to biguanide drugs. Enhancing the mitochondrial oxidative metabolism in combination with biguanide drugs may be a therapeutic strategy for HCC.

Inhibition of neuroinflammation and mitochondrial dysfunctions by carbenoxolone in the rotenone model of Parkinson's disease.

PubMed

Thakur, Poonam; Nehru, Bimla

2015-02-01

α-Synuclein aggregation contributes to the Parkinson's disease (PD) pathology in multiple ways-the two most important being the activation of neuroinflammation and mitochondrial dysfunction. Our recent studies have shown the beneficial effects of a heat shock protein (HSP) inducer, carbenoxolone (Cbx), in reducing the aggregation of α-synuclein in a rotenone-based rat model of PD. The present study was designed to explore its ability to attenuate the α-synuclein-mediated alterations in neuroinflammation and mitochondrial functions. The PD model was generated by the rotenone administration (2 mg/kg b.wt.) to the male SD rats for a period of 5 weeks. Cbx (20 mg/kg b.wt.) co-administration was seen to reduce the activation of astrocytes incited by rotenone. Subsequently, the release of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β was inhibited. Further, the expression level of various inflammatory mediators such as COX-2, iNOS, and NF-κB was also reduced following Cbx co-treatment. Cbx was also shown to reduce the rotenone-induced decline in activity of mitochondrial complexes-I, -II, and -IV. Protection of mitochondrial functions and reduction in neuroinflammation lead to the lesser production of ROS and subsequently reduced oxidative stress. This was reflected by the increase in both the cytosolic and mitochondrial GSH levels as well as SOD activity during Cbx co-treatment. Thus, Cbx reduces the inflammatory response and improves the mitochondrial dysfunctions by reducing α-synuclein aggregation. In addition, it also reduces the associated oxidative stress. Due to its ability to target the multiple pathways implicated in the PD, Cbx can serve as a highly beneficial prophylactic agent.

Ursodeoxycholic Acid Ameliorates Apoptotic Cascade in the Rotenone Model of Parkinson's Disease: Modulation of Mitochondrial Perturbations.

PubMed

Abdelkader, Noha F; Safar, Marwa M; Salem, Hesham A

2016-03-01

The recent emergence of ursodeoxycholic acid (UDCA) as a contender in modifying neurotoxicity in human dopaminergic cells as well as its recognized anti-apoptotic and anti-inflammatory potentials in various hepatic pathologies raised impetus in investigating its anti-parkinsonian effect in rat rotenone model. UDCA prominently improved motor performance in the open field test and halted the decline in the striatal dopamine content. Meanwhile, it improved mitochondrial function as verified by elevation of ATP associated with preservation of mitochondrial integrity as portrayed in the electron microscope examination. In addition, through its anti-inflammatory potential, UDCA reduced the rotenone-induced nuclear factor-κB expression and tumor necrosis factor alpha level. Furthermore, UDCA amended alterations in Bax and Bcl-2 and reduced the activities of caspase-8, caspase-9, and caspase-3, indicating that it suppressed rotenone-induced apoptosis via modulating both intrinsic and extrinsic pathways. In conclusion, UDCA can be introduced as a novel approach for the management of Parkinson's disease via anti-apoptotic and anti-inflammatory mechanisms. These effects are probably linked to dopamine synthesis and mitochondrial regulation.

Agmatine effects on mitochondrial membrane potential and NF-κB activation protect against rotenone-induced cell damage in human neuronal-like SH-SY5Y cells.

PubMed

Condello, Salvatore; Currò, Monica; Ferlazzo, Nadia; Caccamo, Daniela; Satriano, Joseph; Ientile, Riccardo

2011-01-01

Agmatine, an endogenous arginine metabolite, has been proposed as a novel neuromodulator that plays protective roles in the CNS in several models of cellular damage. However, the mechanisms involved in these protective effects in neurodegenerative diseases are poorly understood. The present study was undertaken to investigate the effects of agmatine on cell injury induced by rotenone, commonly used in establishing in vivo and in vitro models of Parkinson's disease, in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). We report that agmatine dose-dependently suppressed rotenone-induced cellular injury through a reduction of oxidative stress. Similar effects were obtained by spermine, suggesting a scavenging effect for these compounds. However, unlike spermine, agmatine also prevented rotenone-induced nuclear factor-κB nuclear translocation and mitochondrial membrane potential dissipation. Furthermore, rotenone-induced increase in apoptotic markers, such as caspase 3 activity, Bax expression and cytochrome c release, was significantly attenuated with agmatine treatment. These findings demonstrate mitochondrial preservation with agmatine in a rotenone model of apoptotic cell death, and that the neuroprotective action of agmatine appears because of suppressing apoptotic signalling mechanisms. Thus, agmatine may have therapeutic potential in the treatment of Parkinson's disease by protecting dopaminergic neurons.

Neuroprotective effects of phloretin and its glycosylated derivative on rotenone-induced toxicity in human SH-SY5Y neuronal-like cells.

PubMed

Barreca, Davide; Currò, Monica; Bellocco, Ersilia; Ficarra, Silvana; Laganà, Giuseppina; Tellone, Ester; Laura Giunta, Maria; Visalli, Giuseppa; Caccamo, Daniela; Galtieri, Antonio; Ientile, Riccardo

2017-07-08

Phloretin and phlorizin are the two strong natural antioxidants whose biological and pharmacological applications are rapidly growing in different human pathological conditions. The neuroprotective activity of the two flavonoids has been analyzed on cell culture of neuroblastoma cells. The neuroprotective activity of the two flavonoids has been analyzed on cell culture of neuroblastoma cells and evaluated by testing cell vitality, mitochondrial transmembrane potential and ROS production, antioxidant enzymes detection, activation of caspase 3, DNA damage, protein carbonylation, lipid peroxidation, and superoxide anion scavenging activity. Incubation of cells with rotenone caused cell death and significant increase in intracellular reactive oxygen species, activation of caspase 3, and variation in mitochondrial transmembrane potential. Although, rotenone exposure caused a significant increase of antioxidant enzymes, high levels of lipid peroxidation were also observed. Phloretin or phlorizin, at micromolar concentration, reduced rotenone-induced cell death by scavenging ability against superoxide anion radical, one of the main effectors of rotenone toxicity at level of mitochondrial respiratory chain complex I. Under our experimental conditions, a reduction of the intracellular ROS levels with consequent normalization of the aforementioned antioxidant enzymes occurred. Concomitantly, we observed the inhibition of caspase 3 activity and DNA damage. This study shows the promising neuroprotective ability of the two dihydrochalcones able to protect human differentiated neuroblastoma cells (commonly used as model of Parkinson's disease) from injury induced by rotenone, actively scavenging ROS, normalizing mitochondrial transmembrane potential and consequently avoiding energy depletion. © 2017 BioFactors, 43(4):549-557, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Redox signaling in the growth and development of colonial hydroids.

PubMed

Blackstone, Neil W

2003-02-01

Redox signaling provides a quick and efficient mechanism for clonal or colonial organisms to adapt their growth and development to aspects of the environment, e.g. the food supply. A 'signature' of mitochondrial redox signaling, particularly as mediated by reactive oxygen species (ROS), can be elucidated by experimental manipulation of the electron transport chain. The major sites of ROS formation are found at NADH dehydrogenase of complex I and at the interface between coenzyme Q and complex III. Inhibitors of complex III should thus upregulate ROS from both sites; inhibitors of complex I should upregulate ROS from the first but not the second site, while uncouplers of oxidative phosphorylation should downregulate ROS from both sites. To investigate the possibility of such redox signaling, perturbations of colony growth and development were carried out using the hydroid Podocoryna carnea. Oxygen uptake of colonies was measured to determine comparable physiological doses of antimycin A(1) (an inhibitor of complex III), rotenone (an inhibitor of complex I) and carbonyl cyanide m-chlorophenylhydrazone (CCCP; an uncoupler of oxidative phosphorylation). Using these doses, clear effects on colony growth and development were obtained. Treatment with antimycin A(1) results in 'runner-like' colony growth, with widely spaced polyps and stolon branches, while treatment with CCCP results in 'sheet-like' growth, with closely spaced polyps and stolon branches. Parallel results have been obtained previously with azide, an inhibitor of complex IV, and dinitrophenol, another uncoupler of oxidative phosphorylation. Perhaps surprisingly, rotenone produced effects on colony development similar to those of CCCP. Assays of peroxides using 2',7'-dichlorofluorescin diacetate and fluorescent microscopy suggest a moderate difference in ROS formation between the antimycin and rotenone treatments. The second site of ROS formation (the interface between coenzyme Q and complex III) may thus predominate in the signaling that regulates colony development. The fat-rich, brine shrimp diet of these hydroids may be relevant in this context. Acyl CoA dehydrogenase, which catalyzes the first step in the mitochondrial beta-oxidation of fatty acids, carries electrons to coenzyme Q, thus bypassing complex I. These results support a role for redox signaling, mediated by ROS, in colony development. Nevertheless, other redox sensors between complexes I and III may yet be found.

Febuxostat ameliorates secondary progressive experimental autoimmune encephalomyelitis by restoring mitochondrial energy production in a GOT2-dependent manner

PubMed Central

Kinoshita, Makoto; Sumi-Akamaru, Hisae; Sasaki, Tsutomu; Takata, Kazushiro; Koda, Toru; Namba, Akiko; Yamashita, Kazuya; Sanda, Eri; Sakaguchi, Manabu; Kumanogoh, Atsushi; Shirakura, Takashi; Tamura, Mizuho; Sakoda, Saburo; Mochizuki, Hideki

2017-01-01

Oxidative stress and mitochondrial dysfunction are important determinants of neurodegeneration in secondary progressive multiple sclerosis (SPMS). We previously showed that febuxostat, a xanthine oxidase inhibitor, ameliorated both relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis (EAE) by preventing neurodegeneration in mice. In this study, we investigated how febuxostat protects neuron in secondary progressive EAE. A DNA microarray analysis revealed that febuxostat treatment increased the CNS expression of several mitochondria-related genes in EAE mice, most notably including GOT2, which encodes glutamate oxaloacetate transaminase 2 (GOT2). GOT2 is a mitochondrial enzyme that oxidizes glutamate to produce α-ketoglutarate for the Krebs cycle, eventually leading to the production of adenosine triphosphate (ATP). Whereas GOT2 expression was decreased in the spinal cord during the chronic progressive phase of EAE, febuxostat-treated EAE mice showed increased GOT2 expression. Moreover, febuxostat treatment of Neuro2a cells in vitro ameliorated ATP exhaustion induced by rotenone application. The ability of febuxostat to preserve ATP production in the presence of rotenone was significantly reduced by GOT2 siRNA. GOT2-mediated ATP synthesis may be a pivotal mechanism underlying the protective effect of febuxostat against neurodegeneration in EAE. Accordingly, febuxostat may also have clinical utility as a disease-modifying drug in SPMS. PMID:29107957

Febuxostat ameliorates secondary progressive experimental autoimmune encephalomyelitis by restoring mitochondrial energy production in a GOT2-dependent manner.

PubMed

Honorat, Josephe A; Nakatsuji, Yuji; Shimizu, Mikito; Kinoshita, Makoto; Sumi-Akamaru, Hisae; Sasaki, Tsutomu; Takata, Kazushiro; Koda, Toru; Namba, Akiko; Yamashita, Kazuya; Sanda, Eri; Sakaguchi, Manabu; Kumanogoh, Atsushi; Shirakura, Takashi; Tamura, Mizuho; Sakoda, Saburo; Mochizuki, Hideki; Okuno, Tatsusada

2017-01-01

Oxidative stress and mitochondrial dysfunction are important determinants of neurodegeneration in secondary progressive multiple sclerosis (SPMS). We previously showed that febuxostat, a xanthine oxidase inhibitor, ameliorated both relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis (EAE) by preventing neurodegeneration in mice. In this study, we investigated how febuxostat protects neuron in secondary progressive EAE. A DNA microarray analysis revealed that febuxostat treatment increased the CNS expression of several mitochondria-related genes in EAE mice, most notably including GOT2, which encodes glutamate oxaloacetate transaminase 2 (GOT2). GOT2 is a mitochondrial enzyme that oxidizes glutamate to produce α-ketoglutarate for the Krebs cycle, eventually leading to the production of adenosine triphosphate (ATP). Whereas GOT2 expression was decreased in the spinal cord during the chronic progressive phase of EAE, febuxostat-treated EAE mice showed increased GOT2 expression. Moreover, febuxostat treatment of Neuro2a cells in vitro ameliorated ATP exhaustion induced by rotenone application. The ability of febuxostat to preserve ATP production in the presence of rotenone was significantly reduced by GOT2 siRNA. GOT2-mediated ATP synthesis may be a pivotal mechanism underlying the protective effect of febuxostat against neurodegeneration in EAE. Accordingly, febuxostat may also have clinical utility as a disease-modifying drug in SPMS.

[Role of ATP-sensitive potassium channel activators in liver mitochondrial function in rats with different resistance to hypoxia].

PubMed

Tkachenko, H M; Kurhaliuk, N M; Vovkanych, L S

2003-01-01

Effects of ATP-sensitive potassium (KATP) channels opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) in rats with different resistance to hypoxia on indices of ADP-stimulation of mitochondrial respiration by Chance, calcium capacity and processes of lipid peroxidation in liver has been investigated. We used next substrates of oxidation: 0.35 mM succinate, 1 mM alpha-ketoglutarate. Additional analyses contain the next inhibitors: mitochondrial fermentative complex I-10 mkM rotenone, succinate dehydrogenase 2 mM malonic acid. It was shown that effects of pinacidil induced the increasing of oxidative phosporylation efficacy and ATP synthesis together with lowering of calcium capacity in rats with low resistance to hypoxia. Effects of pinacidil were leveled by glibenclamide. These changes are connected with the increasing of respiratory rate, calcium overload and intensification of lipid peroxidation processes. A conclusion was made about protective effect of pinacidil on mitochondrial functioning by economization of oxygen-dependent processes, adaptive potentialities of organisms with low resistance to hypoxia being increased.

Mitochondrial Targeted Coenzyme Q, Superoxide, and Fuel Selectivity in Endothelial Cells

PubMed Central

Fink, Brian D.; O'Malley, Yunxia; Dake, Brian L.; Ross, Nicolette C.; Prisinzano, Thomas E.; Sivitz, William I.

2009-01-01

Background Previously, we reported that the “antioxidant” compound “mitoQ” (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates. Methods and Results To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity. Conclusions In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over fatty acid oxidation at the intact cell level. PMID:19158951

Mitochondrial targeted coenzyme Q, superoxide, and fuel selectivity in endothelial cells.

PubMed

Fink, Brian D; O'Malley, Yunxia; Dake, Brian L; Ross, Nicolette C; Prisinzano, Thomas E; Sivitz, William I

2009-01-01

Previously, we reported that the "antioxidant" compound "mitoQ" (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates. To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity. In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over fatty acid oxidation at the intact cell level.

In Vitro Evaluation of Mitochondrial Function and Estrogen Signaling in Cell Lines Exposed to the Antiseptic Cetylpyridinium Chloride

PubMed Central

Datta, Sandipan; He, Guochun; Tomilov, Alexey; Sahdeo, Sunil; Denison, Michael S.

2017-01-01

Background: Quaternary ammonium salts (QUATS), such as cetylpyridinium chloride (CPC) and benzalkonium chloride (BAK), are frequently used in antiseptic formulations, including toothpastes, mouthwashes, lozenges, throat and nasal sprays, and as biocides. Although in a recent ruling, the U.S. Food and Drug Administration (FDA) banned CPC from certain products and requested more data on BAK’s efficacy and safety profile, QUATS, in general, and CPC and BAK, in particular, continue to be used in personal health care, food, and pharmaceutical and cleaning industries. Objectives: We aimed to assess CPC's effects on mitochondrial toxicity and endocrine disruption in vitro. Method: Mitochondrial O2 consumption and adenosine triphosphate (ATP) synthesis rates of osteosarcoma cybrid cells were measured before and after CPC and BAK treatment. Antiestrogenic effects of the compounds were measured by a luciferase-based assay using recombinant human breast carcinoma cells (VM7Luc4E2, ERalpha-positive). Results: CPC inhibited both mitochondrial O2 consumption [half maximal inhibitory concentration (IC50): 3.8μM] and ATP synthesis (IC50: 0.9μM), and additional findings supported inhibition of mitochondrial complex 1 as the underlying mechanism for these effects. In addition, CPC showed concentration-dependent antiestrogenic activity half maximal effective concentration [(EC50): 4.5μM)]. BAK, another antimicrobial QUATS that is structurally similar to CPC, and the pesticide rotenone, a known complex 1 inhibitor, also showed mitochondrial inhibitory and antiestrogenic effects. In all three cases, there was overlap of the antiestrogenic activity with the mitochondrial inhibitory activity. Conclusions: Mitochondrial inhibition in vitro occurred at a CPC concentration that may be relevant to human exposures. The antiestrogenic activity of CPC, BAK, rotenone, and triclosan may be related to their mitochondrial inhibitory activity. Our findings support the need for additional research on the mitochondrial inhibitory and antiestrogenic effects of QUATS, including CPC and BAK. https://doi.org/10.1289/EHP1404 PMID:28885978

Transcriptome Analysis of a Rotenone Model of Parkinsonism Reveals Complex I-Tied and -Untied Toxicity Mechanisms Common to Neurodegenerative Diseases

PubMed Central

Cabeza-Arvelaiz, Yofre; Schiestl, Robert H.

2012-01-01

The pesticide rotenone, a neurotoxin that inhibits the mitochondrial complex I, and destabilizes microtubules (MT) has been linked to Parkinson disease (PD) etiology and is often used to model this neurodegenerative disease (ND). Many of the mechanisms of action of rotenone are posited mechanisms of neurodegeneration; however, they are not fully understood. Therefore, the study of rotenone-affected functional pathways is pertinent to the understanding of NDs pathogenesis. This report describes the transcriptome analysis of a neuroblastoma (NB) cell line chronically exposed to marginally toxic and moderately toxic doses of rotenone. The results revealed a complex pleiotropic response to rotenone that impacts a variety of cellular events, including cell cycle, DNA damage response, proliferation, differentiation, senescence and cell death, which could lead to survival or neurodegeneration depending on the dose and time of exposure and cell phenotype. The response encompasses an array of physiological pathways, modulated by transcriptional and epigenetic regulatory networks, likely activated by homeostatic alterations. Pathways that incorporate the contribution of MT destabilization to rotenone toxicity are suggested to explain complex I-independent rotenone-induced alterations of metabolism and redox homeostasis. The postulated mechanisms involve the blockage of mitochondrial voltage-dependent anions channels (VDACs) by tubulin, which coupled with other rotenone-induced organelle dysfunctions may underlie many presumed neurodegeneration mechanisms associated with pathophysiological aspects of various NDs including PD, AD and their variant forms. Thus, further investigation of such pathways may help identify novel therapeutic paths for these NDs. PMID:22970289

Neuroprotective effects of anthocyanin- and proanthocyanidin-rich extracts in cellular models of Parkinson’s disease

PubMed Central

Strathearn, Katherine E.; Yousef, Gad G.; Grace, Mary H.; Roy, Susan L.; Tambe, Mitali A.; Ferruzzi, Mario G.; Wu, Qing-Li; Simon, James E.; Lila, Mary Ann; Rochet, Jean-Christophe

2014-01-01

Neuropathological evidence indicates that dopaminergic cell death in Parkinson’s disease (PD) involves impairment of mitochondrial complex I, oxidative stress, microglial activation, and the formation of Lewy bodies. Epidemiological findings suggest that the consumption of berries rich in anthocyanins and proanthocyanidins may reduce PD risk. In this study, we investigated whether extracts rich in anthocyanins, proanthocyanidins, or other polyphenols suppress the neurotoxic effects of rotenone in a primary cell culture model of PD. Dopaminergic cell death elicited by rotenone was suppressed by extracts prepared from blueberries, grape seed, hibiscus, blackcurrant, and Chinese mulberry. Extracts rich in anthocyanins and proanthocyanidins exhibited greater neuroprotective activity than extracts rich in other polyphenols, and a number of individual anthocyanins interfered with rotenone neurotoxicity. The blueberry and grape seed extracts rescued rotenone-induced defects in mitochondrial respiration in a dopaminergic cell line, and a purple basal extract attenuated nitrite release from microglial cells stimulated by lipopolysaccharide. These findings suggest that anthocyanin- and proanthocyanidin-rich botanical extracts may alleviate neurodegeneration in PD via enhancement of mitochondrial function. PMID:24502982

Superoxide produced in the matrix of mitochondria enhances methylmercury toxicity in human neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mailloux, Ryan J.; Yumvihoze, Emmanuel; Chan, Hing Man, E-mail: laurie.chan@uottawa.ca

2015-12-15

The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O{sub 2}·{sup −}), the proximal reactive oxygen species (ROS) generated by mitochondria, is responsible for MeHg demethylation. Here, we investigated the impact of different mitochondrial respiratory inhibitors, namely rotenone and antimycin A, on the O{sub 2}·{sup −} mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O{sub 2}·{sup −} in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O{sub 2}·{sup −} productionmore » by mitochondria. Both rotenone and PQ, which increase O{sub 2}·{sup −} in the mitochondrial matrix at a dose-dependent manner, enhanced the conversion of MeHg to inorganic mercury (iHg). Surprisingly, antimycin A, which prompts emission of O{sub 2}·{sup −} into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O{sub 2}·{sup −} emission. Rotenone and PQ also enhanced the toxicity of sub-toxic doses (0.1 μM) MeHg which correlated with the accumulation of iHg in mitochondria and depletion of mitochondrial protein thiols. Taken together, our results demonstrate that MeHg degradation is mediated by mitochondrial O{sub 2}·{sup −}, specifically within the matrix of mitochondria when O{sub 2}·{sup −} is in adequate supply. Our results also show that O{sub 2}·{sup −} amplifies MeHg toxicity specifically through its conversion to iHg and subsequent interaction with protein cysteine thiols (R-SH). The implications of our findings in mercury neurotoxicity are discussed herein. - Highlights: • Superoxide produced in the matrix of mitochondria degrades MeHg. • Superoxide produced in intermembrane space does not degrade MeHg. • Matrix-generated superoxide enhances Hg toxicity by converting MeHg to iHg.« less

Dopamine D2 receptor-mediated neuroprotection in a G2019S Lrrk2 genetic model of Parkinson's disease.

PubMed

Tozzi, Alessandro; Tantucci, Michela; Marchi, Saverio; Mazzocchetti, Petra; Morari, Michele; Pinton, Paolo; Mancini, Andrea; Calabresi, Paolo

2018-02-12

Parkinson's disease (PD) is a neurodegenerative disorder in which genetic and environmental factors synergistically lead to loss of midbrain dopamine (DA) neurons. Mutation of leucine-rich repeated kinase2 (Lrrk2) genes is responsible for the majority of inherited familial cases of PD and can also be found in sporadic cases. The pathophysiological role of this kinase has to be fully understood yet. Hyperactivation of Lrrk2 kinase domain might represent a predisposing factor for both enhanced striatal glutamatergic release and mitochondrial vulnerability to environmental factors that are observed in PD. To investigate possible alterations of striatal susceptibility to mitochondrial dysfunction, we performed electrophysiological recordings from the nucleus striatum of a G2019S Lrrk2 mouse model of PD, as well as molecular and morphological analyses of G2019S Lrrk2-expressing SH-SY5Y neuroblastoma cells. In G2019S mice, we found reduced striatal DA levels, according to the hypothesis of alteration of dopaminergic transmission, and increased loss of field potential induced by the mitochondrial complex I inhibitor rotenone. This detrimental effect is reversed by the D2 DA receptor agonist quinpirole via the inhibition of the cAMP/PKA intracellular pathway. Analysis of mitochondrial functions in G2019S Lrrk2-expressing SH-SY5Y cells revealed strong rotenone-induced oxidative stress characterized by reduced Ca 2+ buffering capability and ATP synthesis, production of reactive oxygen species, and increased mitochondrial fragmentation. Importantly, quinpirole was able to prevent all these changes. We suggest that the G2019S-Lrrk2 mutation is a predisposing factor for enhanced striatal susceptibility to mitochondrial dysfunction induced by exposure to mitochondrial environmental toxins and that the D2 receptor stimulation is neuroprotective on mitochondrial function, via the inhibition of cAMP/PKA intracellular pathway. We suggest new possible neuroprotective strategies for patients carrying this genetic alteration based on drugs specifically targeting Lrrk2 kinase domain and mitochondrial functionality.

Mortalin inhibition in experimental Parkinson's disease.

PubMed

Chiasserini, Davide; Tozzi, Alessandro; de Iure, Antonio; Tantucci, Michela; Susta, Federica; Orvietani, Pier Luigi; Koya, Keizo; Binaglia, Luciano; Calabresi, Paolo

2011-08-01

Among heat shock proteins, mortalin has been linked to the pathogenesis of Parkinson's disease. In the present work a rat model of Parkinson's disease was used to analyze the expression of striatal proteins and, more specifically, mortalin expression. The possible involvement of mortalin in Parkinson's disease pathogenesis was further investigated by utilizing an electrophysiological approach and pharmacological inhibition of mortalin in both the physiological and the parkinsonian states. Proteomic analysis was used to investigate changes in striatal protein expression in the 6-hydroxydopamine rat model of Parkinson's disease. The electrophysiological effects of MKT-077, a rhodamine-123 analogue acting as an inhibitor of mortalin, were measured by field potential recordings from corticostriatal brain slices obtained from control, sham-operated, and 6-hydroxydopamine-denervated animals. Slices in the presence of rotenone, an inhibitor of mitochondrial complex I, were also analyzed. Proteomic analysis revealed downregulation of mortalin in the striata of 6-hydroxydopamine-treated rats in comparison with sham-operated animals. MKT-077 reduced corticostriatal field potential amplitude in physiological conditions, inducing membrane depolarization and inward current in striatal medium spiny neurons. In addition, we observed that concentrations of MKT-077 not inducing any electrophysiological effect in physiological conditions caused significant changes in striatal slices from parkinsonian animals as well as in slices treated with a submaximal concentration of rotenone. These findings suggest a critical link between mortalin function and mitochondrial activity in both physiological and pathological conditions mimicking Parkinson's disease. Copyright © 2011 Movement Disorder Society.

Neuroprotective effect of curcumin as evinced by abrogation of rotenone-induced motor deficits, oxidative and mitochondrial dysfunctions in mouse model of Parkinson's disease.

PubMed

Khatri, Dharmendra K; Juvekar, Archana R

Curcumin, a natural polyphenolic compound extracted from rhizomes of Curcuma longa (turmeric), a plant in the ginger family (Zingiberaceae) has been used worldwide and extensively in Southeast Asia. Curcumin exhibited numerous biological and pharmacological activities including potent antioxidant, cardiovascular disease, anticancer, anti-inflammatory effects and neurodegenerative disorders in cell cultures and animal models. Hence, the present study was designed in order to explore the possible neuroprotective role of curcumin against rotenone induced cognitive impairment, oxidative and mitochondrial dysfunction in mice. Chronic administration of rotenone (1mg/kg i.p.) for a period of three weeks significantly impaired cognitive function (actophotometer, rotarod and open field test), oxidative defense (increased lipid peroxidation, nitrite concentration and decreased activity of superoxide dismutase, catalase and reduced glutathione level) and mitochondrial complex (II and III) enzymes activities as compared to normal control group. Three weeks of curcumin (50, 100 and 200mg/kg, p.o.) treatment significantly improved behavioral alterations, oxidative damage and mitochondrial enzyme complex activities as compared to negative control (rotenone treated) group. Curcumin treated mice also mitigated enhanced acetylcholine esterase enzyme level as compared to negative control group. We found that curcumin restored motor deficits and enhanced the activities of antioxidant enzymes suggesting its antioxidant potential in vivo. The findings of the present study conclude neuroprotective role of curcumin against rotenone induced Parkinson's in mice and offer strong justification for the therapeutic prospective of this compound in the management of PD. Copyright © 2016. Published by Elsevier Inc.

Resveratrol Suppresses Rotenone-induced Neurotoxicity Through Activation of SIRT1/Akt1 Signaling Pathway.

PubMed

Wang, Hui; Dong, Xiaoguang; Liu, Zengxun; Zhu, Shaowei; Liu, Haili; Fan, Wenchuang; Hu, Yanlai; Hu, Tao; Yu, Yonghui; Li, Yizhao; Liu, Tianwei; Xie, Chengjia; Gao, Qing; Li, Guibao; Zhang, Jing; Ding, Zhaoxi; Sun, Jinhao

2018-06-01

Rotenone is a common pesticide and has been reported as one of the risk factors for Parkinson disease. Rotenone can cause neuronal death or apoptosis through inducing oxidative injury and inhibiting mitochondrial function. As a natural polyphenolic compound, resveratrol possesses the antioxidant capacity and neuroprotective effect. However, the mechanism underlying the neuroprotective effect of resveratrol against rotenone-induced neurotoxicity remains elusive. Here, we treated PC12 cells with rotenone to induce neurotoxicity, and the neurotoxic cells were subjected to resveratrol treatment. The CCK8 and LDH activity assays demonstrated that resveratrol could suppress neurotoxicity induced by rotenone (P < 0.01). The DCFH-DA assay indicated that resveratrol reduced the production of reactive oxygen species (ROS). JC-1 and Hoechst 33342/PI staining revealed that resveratrol attenuated mitochondrial dysfunction and cell apoptosis. Moreover, resveratrol reversed rotenone-induced decrease in SIRT1 expression and Akt1 phosphorylation (P < 0.05). Furthermore, when the SIRT1 and Akt1 activity was inhibited by niacinamide and LY294002, respectively, the neuroprotective effect of resveratrol was remarkably attenuated, which implied that SIRT1 and Akt1 could mediate this process and may be potential molecular targets for intervening rotenone-induced neurotoxicity. In summary, our study demonstrated that resveratrol reduced rotenone-induced oxidative damage, which was partly mediated through activation of the SIRT1/Akt1 signaling pathway. Our study launched a promising avenue for the potential application of resveratrol as a neuroprotective therapeutic agent in Parkinson disease. Anat Rec, 301:1115-1125, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Oxidative stress in duckweed (Lemna minor L.) induced by glyphosate: Is the mitochondrial electron transport chain a target of this herbicide?

PubMed

Gomes, Marcelo Pedrosa; Juneau, Philippe

2016-11-01

We investigated the physiological responses of Lemna minor plants exposed to glyphosate. The deleterious effects of this herbicide on photosynthesis, respiration, and pigment concentrations were related to glyphosate-induced oxidative stress through hydrogen peroxide (H 2 O 2 ) accumulation. By using photosynthetic and respiratory electron transport chain (ETC) inhibitors we located the primary site of reactive oxygen species (ROS) production in plants exposed to 500 mg glyphosate l -1 . Inhibition of mitochondrial ETC Complex I by rotenone reduced H 2 O 2 concentrations in glyphosate-treated plants. Complex III activity was very sensitive to glyphosate which appears to act much like antimycin A (an inhibitor of mitochondrial ETC Complex III) by shunting electrons from semiquinone to oxygen, with resulting ROS formation. Confocal evaluations for ROS localization showed that ROS are initially produced outside of the chloroplasts upon initial glyphosate exposure. Our results indicate that in addition to interfering with the shikimate pathway, glyphosate can induce oxidative stress in plants through H 2 O 2 formation by targeting the mitochondrial ETC, which would explain its observed effects on non-target organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Alterations in mitochondrial dynamics induced by tebufenpyrad and pyridaben in a dopaminergic neuronal cell culture model

PubMed Central

Charli, Adhithiya; Jin, Huajun; Anantharam, Vellareddy; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

2015-01-01

Tebufenpyrad and pyridaben are two agro-chemically important acaricides that function like the known mitochondrial toxicant rotenone. Although these two compounds have been commonly used to kill populations of mites and ticks in commercial greenhouses, their neurotoxic profiles remain largely unknown. Therefore, we investigated the effects of these two pesticides on mitochondrial structure and function in an in vitro cell culture model using the Seahorse bioanalyzer and confocal fluorescence imaging. The effects were compared with rotenone. Exposing rat dopaminergic neuronal cells (N27 cells) to tebufenpyrad and pyridaben for 3 h induced dose-dependent cell death with an EC50 of 3.98 μM and 3.77 μM, respectively. Also, tebufenpyrad and pyridaben (3 μM) exposure induced reactive oxygen species (ROS) generation and m-aconitase damage, suggesting that the pesticide toxicity is associated with oxidative damage. Morphometric image analysis with the MitoTracker red fluorescent probe indicated that tebufenpyrad and pyridaben, as well as rotenone, caused abnormalities in mitochondrial morphology, including reduced mitochondrial length and circularity. Functional bioenergetic experiments using the Seahorse XF96 analyzer revealed that tebufenpyrad and pyridaben very rapidly suppressed the basal mitochondrial oxygen consumption rate similar to that of rotenone. Further analysis of bioenergetic curves also revealed dose-dependent decreases in ATP-linked respiration and respiratory capacity. The luminescence-based ATP measurement further confirmed that pesticide-induced mitochondrial inhibition of respiration is accompanied by the loss of cellular ATP. Collectively, our results suggest that exposure to the pesticides tebufenpyrad and pyridaben induces neurotoxicity by rapidly initiating mitochondrial dysfunction and oxidative damage in dopaminergic neuronal cells. Our findings also reveal that monitoring the kinetics of mitochondrial respiration with Seahorse could be used as an early neurotoxicological high-throughput index for assessing the risk that pesticides pose to the dopaminergic neuronal system. PMID:26141520

Effects of hypnotic bromovalerylurea on microglial BV2 cells.

PubMed

Kawasaki, Shun; Abe, Naoki; Ohtake, Fumito; Islam, Afsana; Choudhury, Mohammed Emamussalehin; Utsunomiya, Ryo; Kikuchi, Satoshi; Nishihara, Tasuku; Kuwabara, Jun; Yano, Hajime; Watanabe, Yuji; Aibiki, Mayuki; Yorozuya, Toshihiro; Tanaka, Junya

2017-06-01

An old sedative and hypnotic bromovalerylurea (BU) has anti-inflammatory effects. BU suppressed nitric oxide (NO) release and proinflammatory cytokine expression by lipopolysaccharide (LPS)-treated BV2 cells, a murine microglial cell line. However, BU did not inhibit LPS-induced nuclear translocation of nuclear factor-κB and subsequent transcription. BU suppressed LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). The Janus kinase 1 (JAK1) inhibitor filgotinib suppressed the NO release much more weakly than that of BU, although filgotinib almost completely prevented LPS-induced STAT1 phosphorylation. Knockdown of JAK1, STAT1, or IRF1 did not affect the suppressive effects of BU on LPS-induced NO release by BV2 cells. A combination of BU and filgotinib synergistically suppressed the NO release. The mitochondrial complex I inhibitor rotenone, which did not prevent STAT1 phosphorylation or IRF1 expression, suppressed proinflammatory mediator expression less significantly than BU. BU and rotenone reduced intracellular ATP (iATP) levels to a similar extent. A combination of rotenone and filgotinib suppressed NO release by LPS-treated BV2 cells as strongly as BU. These results suggest that anti-inflammatory actions of BU may be attributable to the synergism of inhibition of JAK1/STAT1-dependent pathways and reduction in iATP level. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Rotenone induces oxidative stress and dopaminergic neuron damage in organotypic substantia nigra cultures.

PubMed

Testa, Claudia M; Sherer, Todd B; Greenamyre, J Timothy

2005-03-24

Rotenone, a pesticide and complex I inhibitor, causes nigrostriatal degeneration similar to Parkinson disease pathology in a chronic, systemic, in vivo rodent model [M. Alam, W.J. Schmidt, Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats, Behav. Brain Res. 136 (2002) 317-324; R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson's disease, Nat. Neurosci. 3 (2000) 1301-1306; S.M. Fleming, C. Zhu, P.O. Fernagut, A. Mehta, C.D. DiCarlo, R.L. Seaman, M.F. Chesselet, Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone, Exp. Neurol. 187 (2004) 418-429; T.B. Sherer, J.H. Kim, R. Betarbet, J.T. Greenamyre, Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation, Exp. Neurol. 179 (2003) 9-16.]. To better investigate the role of mitochondria and complex I inhibition in chronic, progressive neurodegenerative disease, we developed methods for long-term culture of rodent postnatal midbrain organotypic slices. Chronic complex I inhibition over weeks by low dose (10-50 nM) rotenone in this system lead to dose- and time-dependent destruction of substantia nigra pars compacta neuron processes, morphologic changes, some neuronal loss, and decreased tyrosine hydroxylase (TH) protein levels. Chronic complex I inhibition also caused oxidative damage to proteins, measured by protein carbonyl levels. This oxidative damage was blocked by the antioxidant alpha-tocopherol (vitamin E). At the same time, alpha-tocopherol also blocked rotenone-induced reductions in TH protein and TH immunohistochemical changes. Thus, oxidative damage is a primary mechanism of mitochondrial toxicity in intact dopaminergic neurons. The organotypic culture system allows close study of this and other interacting mechanisms over a prolonged time period in mature dopaminergic neurons with intact processes, surrounding glia, and synaptic connections.

Patients with chronic fatigue syndrome performed worse than controls in a controlled repeated exercise study despite a normal oxidative phosphorylation capacity.

PubMed

Vermeulen, Ruud C W; Kurk, Ruud M; Visser, Frans C; Sluiter, Wim; Scholte, Hans R

2010-10-11

The aim of this study was to investigate the possibility that a decreased mitochondrial ATP synthesis causes muscular and mental fatigue and plays a role in the pathophysiology of the chronic fatigue syndrome (CFS/ME). Female patients (n = 15) and controls (n = 15) performed a cardiopulmonary exercise test (CPET) by cycling at a continuously increased work rate till maximal exertion. The CPET was repeated 24 h later. Before the tests, blood was taken for the isolation of peripheral blood mononuclear cells (PBMC), which were processed in a special way to preserve their oxidative phosphorylation, which was tested later in the presence of ADP and phosphate in permeabilized cells with glutamate, malate and malonate plus or minus the complex I inhibitor rotenone, and succinate with rotenone plus or minus the complex II inhibitor malonate in order to measure the ATP production via Complex I and II, respectively. Plasma CK was determined as a surrogate measure of a decreased oxidative phosphorylation in muscle, since the previous finding that in a group of patients with external ophthalmoplegia the oxygen consumption by isolated muscle mitochondria correlated negatively with plasma creatine kinase, 24 h after exercise. At both exercise tests the patients reached the anaerobic threshold and the maximal exercise at a much lower oxygen consumption than the controls and this worsened in the second test. This implies an increase of lactate, the product of anaerobic glycolysis, and a decrease of the mitochondrial ATP production in the patients. In the past this was also found in patients with defects in the mitochondrial oxidative phosphorylation. However the oxidative phosphorylation in PBMC was similar in CFS/ME patients and controls. The plasma creatine kinase levels before and 24 h after exercise were low in patients and controls, suggesting normality of the muscular mitochondrial oxidative phosphorylation. The decrease in mitochondrial ATP synthesis in the CFS/ME patients is not caused by a defect in the enzyme complexes catalyzing oxidative phosphorylation, but in another factor. NL16031.040.07.

Leaf extract of Rhus verniciflua Stokes protects dopaminergic neuronal cells in a rotenone model of Parkinson's disease.

PubMed

Kim, Seung; Park, Se-Eun; Sapkota, Kumar; Kim, Myung-Kon; Kim, Sung-Jun

2011-10-01

The present study investigated the neuroprotective effects of Rhus verniciflua Stokes (RVS) leaf extract on rotenone-induced apoptosis in human dopaminergic cells, SH-SY5Y. Cells were pretreated with RVS extract for 1 h then treated with vehicle or rotenone for 24 h. Cell viability, cell cytotoxicity, cell morphology and nuclear morphology were examined by MTT assay, lactate dehydrogenase release assay, phase contrast microscopy and staining with Hoechast 33342, respectively. Reactive oxygen species were measured by 2'7'-dichlorofluorescein diacetate and fragmented DNA was observed by TUNEL assay. Mitochondrial membrane potential was determined by Rhodamine 123. Pro-apoptotic and anti-apoptotic proteins and tyrosine hydroxylase were analysed by Western blotting. Results showed that RVS suppressed rotenone-induced reactive oxygen species generation, cellular injury and apoptotic cell death. RVS also prevented rotenone-mediated changes in Bax/Bcl-2 levels, mitochondrial membrane potential dissipation and Caspase 3 activation. Moreover, RVS pretreatment increased the tyrosine hydroxylase levels in SH-SY5Y cells. These findings demonstrate that RVS protects SH-SY5Y cells against rotenone-induced injury and suggest that RVS may have potential therapeutic value for neurodegenerative disease associated with oxidative stress. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Caenorhabditis elegans neuron degeneration and mitochondrial suppression caused by selected environmental chemicals

PubMed Central

Zhou, Shaoyu; Wang, Zemin; Klaunig, James E

2013-01-01

Mitochondrial alterations have been documented for many years in the brains of Parkinson’s disease (PD), a disorder that is characterized by the selective loss of dopamine neurons. Recent studies have demonstrated that Parkinson’s disease-associated proteins are either present in mitochondria or translocated into mitochondria in response to stress, further reinforcing the importance of the mitochondrial function in the pathogenesis of Parkinson’s disease. Exposure to environmental chemicals such as pesticides and heavy metals has been suggested as risk factors in the development of Parkinson’s disease. It has been reported that a number of environmental agents including tobacco smoke and perfluorinated compounds, pesticides, as well as metals (Mn2+ and Pb2+) modulate mitochondrial function. However the exact mechanism of mitochondrial alteration has not been defined in the context of the development and progression of Parkinson’s disease. The complexity of the mammalian system has made it difficult to dissect the molecular components involved in the pathogenesis of Parkinson’s disease. In the present study we used the nematode Caenorhabditis elegans (C. elegans) model of neuron degeneration and investigated the effect of environmental chemicals on mitochondrial biogenesis and mitochondrial gene regulation. Chronic exposure to low concentration (2 or 4 μM) of pesticide rotenone, resulted in significant loss of dopamine neuron in C. elegans, a classic feature of Parkinson’s disease. We then determined if the rotenone-induced neuron degeneration is accompanied by a change in mitochondria biogenesis. Analysis of mitochondrial genomic replication by quantitative PCR showed a dramatic decrease in mitochondrial DNA (mtDNA) copies of rotenone-treated C. elegans compared to control. This decreased mitochondrial biogenesis occurred prior to the development of loss of dopamine neurons, and was persistent. The inhibition of mtDNA replication was also found in C. elegans exposed to another neuron toxicant Mn2+ at the concentration 50 or 100 mM. We further examined the mitochondrial gene expression and found significant lower level of mitochondrial complex IV subunits COI and COII in C. elegans exposed to rotenone. These results demonstrate that environmental chemicals cause persistent suppression of mitochondrial biogenesis and mitochondrial gene expression, and suggest a critical role of modifying mitochondrial biogenesis in toxicants-induced neuron degeneration in C. elegans model. PMID:24380023

Selective inhibition of deactivated mitochondrial complex I by biguanides.

PubMed

Matsuzaki, Satoshi; Humphries, Kenneth M

2015-03-24

Biguanides are widely used antihyperglycemic agents for diabetes mellitus and prediabetes treatment. Complex I is the rate-limiting step of the mitochondrial electron transport chain (ETC), a major source of mitochondrial free radical production, and a known target of biguanides. Complex I has two reversible conformational states, active and de-active. The deactivated state is promoted in the absence of substrates but is rapidly and fully reversed to the active state in the presence of NADH. The objective of this study was to determine the relative sensitivity of active/de-active complex I to biguanide-mediated inhibition and resulting superoxide radical (O₂(•⁻)) production. Using isolated rat heart mitochondria, we show that deactivation of complex I sensitizes it to metformin and phenformin (4- and 3-fold, respectively), but not to other known complex I inhibitors, such as rotenone. Mitochondrial O₂(•⁻) production by deactivated complex I was measured fluorescently by NADH-dependent 2-hydroxyethidium formation at alkaline pH to impede reactivation. Superoxide production was 260.4% higher than in active complex I at pH 9.4. However, phenformin treatment of de-active complex I decreased O₂(•⁻) production by 14.9%, while rotenone increased production by 42.9%. Mitochondria isolated from rat hearts subjected to cardiac ischemia, a condition known to induce complex I deactivation, were sensitized to phenformin-mediated complex I inhibition. This supports the idea that the effects of biguanides are likely to be influenced by the complex I state in vivo. These results demonstrate that the complex I active and de-active states are a determinant in biguanide-mediated inhibition.

Vitamin K2 suppresses rotenone-induced microglial activation in vitro

PubMed Central

Yu, Yan-xia; Li, Yi-pei; Gao, Feng; Hu, Qing-song; Zhang, Yan; Chen, Dong; Wang, Guang-hui

2016-01-01

Aim: Increasing evidence has shown that environmental factors such as rotenone and paraquat induce neuroinflammation, which contributes to the pathogenesis of Parkinson's disease (PD). In this study, we investigated the molecular mechanisms underlying the repression by menaquinone-4 (MK-4), a subtype of vitamin K2, of rotenone-induced microglial activation in vitro. Methods: A microglial cell line (BV2) was exposed to rotenone (1 μmol/L) with or without MK-4 treatment. The levels of TNF-α or IL-1β in 100 μL of cultured media of BV2 cells were measured using ELISA kits. BV2 cells treated with rotenone with or without MK4 were subjected to mitochondrial membrane potential, ROS production, immunofluorescence or immunoblot assays. The neuroblastoma SH-SY5Y cells were treated with conditioned media (CM) of BV2 cells that were exposed to rotenone with or without MK-4 treatment, and the cell viability was assessed using MTT assay. Results: In rotenone-treated BV2 cells, MK-4 (0.5–20 μmol/L) dose-dependently suppressed the upregulation in the expression of iNOS and COX-2 in the cells, as well as the production of TNF-α and IL-1β in the cultured media. MK-4 (5–20 μmol/L) significantly inhibited rotenone-induced nuclear translocation of NF-κB in BV2 cells. MK-4 (5–20 μmol/L) significantly inhibited rotenone-induced p38 activation, ROS production, and caspase-1 activation in BV2 cells. MK-4 (5–20 μmol/L) also restored the mitochondrial membrane potential that had been damaged by rotenone. Exposure to CM from rotenone-treated BV2 cells markedly decreased the viability of SH-SY5Y cells. However, this rotenone-activated microglia-mediated death of SH-SY5Y cells was significantly attenuated when the BV2 cells were co-treated with MK-4 (5–20 μmol/L). Conclusion: Vitamin K2 can directly suppress rotenone-induced activation of microglial BV2 cells in vitro by repressing ROS production and p38 activation. PMID:27498777

Neuroprotective effect of CNB-001, a novel pyrazole derivative of curcumin on biochemical and apoptotic markers against rotenone-induced SK-N-SH cellular model of Parkinson's disease.

PubMed

Jayaraj, Richard L; Tamilselvam, Kuppusamy; Manivasagam, Thamilarasan; Elangovan, Namasivayam

2013-11-01

Oxidative stress and mitochondrial dysfunction are underpinned for initiating a cascade of toxic events leading to dopaminergic neuronal death in Parkinson's disease (PD) and identified as vital target for therapeutic intervention. Curcumin, a potent antioxidant has been reported to display diverse neuroprotective properties against various neurodegenerative diseases including PD. In this present study, we investigated the protective effect of CNB-001, a pyrazole derivative of curcumin on rotenone-induced toxicity and its possible mechanisms in neuroblastoma SK-N-SH cells. Rotenone insult significantly reduced cell viability (MTT assay) and resulted in 78 % apoptosis (dual staining) by altering Bcl-2, Bax, caspase-3, and cytochrome C expression. Moreover, rotenone enhanced ROS production and disrupts mitochondrial membrane potential. These resultant phenotypes were distinctly alleviated by CNB-001. Pretreatment with CNB-001(2 μM) 2 h before rotenone exposure (100 nM) increased cell viability, decreased ROS formation, maintained normal physiological mitochondrial membrane potential, and reduced apoptosis. Furthermore, CNB-001 inhibited downstream apoptotic cascade by increasing the expression of vital antiapoptotic protein Bcl-2 and decreased the expression of Bax, caspase-3, and cytochrome C. Collectively, the results suggest that CNB-001 protects neuronal cell against toxicity through antioxidant and antiapoptotic properties through its action on mitochondria. Therefore, it may be concluded that CNB-001 can be further developed as a promising drug for treatment of PD.

SNJ-1945, a calpain inhibitor, protects SH-SY5Y cells against MPP(+) and rotenone.

PubMed

Knaryan, Varduhi H; Samantaray, Supriti; Park, Sookyoung; Azuma, Mitsuyoshi; Inoue, Jun; Banik, Naren L

2014-07-01

Complex pathophysiology of Parkinson's disease involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in Parkinson's disease and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. To unravel these mechanisms SH-SY5Y neuroblastoma cells were differentiated into dopaminergic and cholinergic phenotypes, respectively, and used as cell culture model following exposure to two parkinsonian neurotoxicants MPP(+) and rotenone. SNJ-1945, a cell-permeable calpain inhibitor was tested for its neuroprotective efficacy. MPP(+) and rotenone dose-dependently elevated the levels of intracellular free Ca(2+) and induced a concomitant rise in the levels of active calpain. SNJ-1945 pre-treatment significantly protected cell viability and preserved cellular morphology following MPP(+) and rotenone exposure. The neurotoxicants elevated the levels of reactive oxygen species more profoundly in SH-SY5Y cells differentiated into dopaminergic phenotype, and this effect could be attenuated with SNJ-1945 pre-treatment. In contrast, significant levels of inflammatory mediators cyclooxygenase-2 (Cox-2 and cleaved p10 fragment of caspase-1) were up-regulated in the cholinergic phenotype, which could be dose-dependently attenuated by the calpain inhibitor. Overall, SNJ-1945 was efficacious against MPP(+) or rotenone-induced reactive oxygen species generation, inflammatory mediators, and proteolysis. A post-treatment regimen of SNJ-1945 was also examined in cells and partial protection was attained with calpain inhibitor administration 1-3 h after exposure to MPP(+) or rotenone. Taken together, these results indicate that calpain inhibition is a valid target for protection against parkinsonian neurotoxicants, and SNJ-1945 is an efficacious calpain inhibitor in this context. SH-SY5Y cells, differentiated as dopaminergic (TH positive) and cholinergic (ChAT positive), were used as in vitro models for Parkinson's disease. MPP+ and rotenone induced up-regulation of calpain, expression, and activity as a common mechanism of neurodegeneration. SNJ-1945, a novel calpain inhibitor, protected both the cell phenotypes against MPP+ and rotenone. © 2013 International Society for Neurochemistry.

Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat

PubMed Central

Choi, Won-Seok; Kruse, Shane E.; Palmiter, Richard D.; Xia, Zhengui

2008-01-01

Inhibition of mitochondrial complex I is one of the leading hypotheses for dopaminergic neuron death associated with Parkinson's disease (PD). To test this hypothesis genetically, we used a mouse strain lacking functional Ndufs4, a gene encoding a subunit required for complete assembly and function of complex I. Deletion of the Ndufs4 gene abolished complex I activity in midbrain mesencephalic neurons cultured from embryonic day (E) 14 mice, but did not affect the survival of dopaminergic neurons in culture. Although dopaminergic neurons were more sensitive than other neurons in these cultures to cell death induced by rotenone, MPP+, or paraquat treatments, the absence of complex I activity did not protect the dopaminergic neurons, as would be expected if these compounds act by inhibiting complex 1. In fact, the dopaminergic neurons were more sensitive to rotenone. These data suggest that dopaminergic neuron death induced by treatment with rotenone, MPP+, or paraquat is independent of complex I inhibition. PMID:18812510

Nitrosative stress mediated misfolded protein aggregation mitigated by Na-D-β-hydroxybutyrate intervention.

PubMed

Kabiraj, Parijat; Pal, Rituraj; Varela-Ramirez, Armando; Miranda, Manuel; Narayan, Mahesh

2012-09-28

Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species, is associated with the pathogenesis of neurodegenerative disorders. Rotenone, a mitochondrial stressor induces caspase-9 and caspase-3 activation leading proteolytic cleavage of substrate nuclear poly(ADP-ribose) polymerase (PARP). PARP cleavage is directly related to apoptotic cell death. In this study, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1, as a rotenone-induced Parkinsonia-onset biomarker. We report that the innate ketone body, Na-D-β-hydroxybutyrate (NaβHB) reduces markedly the incidence of synphilin-1 aggregation. Furthermore, our data reveal that the metabolic byproduct also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells. Together, these results suggest that NaβHB is neuroprotective; it attenuates effects originating from mitochondrial insult and can serve as a scaffold for the design and development of sporadic neuropathies. Copyright © 2012 Elsevier Inc. All rights reserved.

Mitochondrial mechanisms of neural cell death and neuroprotective interventions in Parkinson's disease.

PubMed

Fiskum, Gary; Starkov, Anatoly; Polster, Brian M; Chinopoulos, Christos

2003-06-01

Mitochondrial dysfunction, due to either environmental or genetic factors, can result in excessive production of reactive oxygen species, triggering the apoptotic death of dopaminergic cells in Parkinson's disease. Mitochondrial free radical production is promoted by the inhibition of electron transport at any point distal to the sites of superoxide production. Neurotoxins that induce parkinsonian neuropathology, such as MPP(+) and rotenone, stimulate superoxide production at complex I of the electron transport chain and also stimulate free radical production at proximal redox sites including mitochondrial matrix dehydrogenases. The oxidative stress caused by elevated mitochondrial production of reactive oxygen species promotes the expression and (or) intracellular distribution of the proapoptotic protein Bax to the mitochondrial outer membrane. Interactions between Bax and BH3 death domain proteins such as tBid result in Bax membrane integration, oligomerization, and permeabilization of the outer membrane to intermembrane proteins such as cytochrome c. Once released into the cytosol, cytochrome c together with other proteins activates the caspase cascade of protease activities that mediate the biochemical and morphological alterations characteristic of apoptosis. In addition, loss of mitochondrial cytochrome c stimulates mitochondrial free radical production, further promoting cell death pathways. Excessive mitochondrial Ca(2+) accumulation can also release cytochrome c and promote superoxide production through a mechanism distinctly different from that of Bax. Ca(2+) activates a mitochondrial inner membrane permeability transition causing osmotic swelling, rupture of the outer membrane, and complete loss of mitochondrial structural and functional integrity. While amphiphilic cations, such as dibucaine and propranolol, inhibit Bax-mediated cytochrome c release, transient receptor potential channel inhibitors inhibit mitochondrial swelling and cytochrome c release induced by the inner membrane permeability transition. These advances in the knowledge of mitochondrial cell death mechanisms and their inhibitors may lead to neuroprotective interventions applicable to Parkinsons's disease.

Reverse electron transport effects on NADH formation and metmyoglobin reduction.

PubMed

Belskie, K M; Van Buiten, C B; Ramanathan, R; Mancini, R A

2015-07-01

The objective was to determine if NADH generated via reverse electron flow in beef mitochondria can be used for electron transport-mediated reduction and metmyoglobin reductase pathways. Beef mitochondria were isolated from bovine hearts (n=5) and reacted with combinations of succinate, NAD, and mitochondrial inhibitors to measure oxygen consumption and NADH formation. Mitochondria and metmyoglobin were reacted with succinate, NAD, and mitochondrial inhibitors to measure electron transport-mediated metmyoglobin reduction and metmyoglobin reductase activity. Addition of succinate and NAD increased oxygen consumption, NADH formation, electron transport-mediated metmyoglobin reduction, and reductase activity (p<0.05). Addition of antimycin A prevented electron flow beyond complex III, therefore, decreasing oxygen consumption and electron transport-mediated metmyoglobin reduction. Addition of rotenone prevented reverse electron flow, increased oxygen consumption, increased electron transport-mediated metmyoglobin reduction, and decreased NADH formation. Succinate and NAD can generate NADH in bovine tissue postmortem via reverse electron flow and this NADH can be used by both electron transport-mediated and metmyoglobin reductase pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mitochondria and NMDA Receptor-Dependent Toxicity of Berberine Sensitizes Neurons to Glutamate and Rotenone Injury

PubMed Central

Kysenius, Kai; Brunello, Cecilia A.; Huttunen, Henri J.

2014-01-01

The global incidence of metabolic and age-related diseases, including type 2 diabetes and Alzheimer's disease, is on the rise. In addition to traditional pharmacotherapy, drug candidates from complementary and alternative medicine are actively being pursued for further drug development. Berberine, a nutraceutical traditionally used as an antibiotic, has recently been proposed to act as a multi-target protective agent against type 2 diabetes, dyslipidemias, ischemic brain injury and neurodegenerative diseases, such as Parkinson's and Alzheimer's disease. However, the safety profile of berberine remains controversial, as isolated reports suggest risks with acute toxicity, bradycardia and exacerbation of neurodegeneration. We report that low micromolar berberine causes rapid mitochondria-dependent toxicity in primary neurons characterized by mitochondrial swelling, increased oxidative stress, decreased mitochondrial membrane potential and depletion of ATP content. Berberine does not induce caspase-3 activation and the resulting neurotoxicity remains unaffected by pan-caspase inhibitor treatment. Interestingly, inhibition of NMDA receptors by memantine and MK-801 completely blocked berberine-induced neurotoxicity. Additionally, subtoxic nanomolar concentrations of berberine were sufficient to sensitize neurons to glutamate excitotoxicity and rotenone injury. Our study highlights the need for further safety assessment of berberine, especially due to its tendency to accumulate in the CNS and the risk of potential neurotoxicity as a consequence of increasing bioavailability of berberine. PMID:25192195

Nitrosative stress mediated misfolded protein aggregation mitigated by Na-D-{beta}-hydroxybutyrate intervention

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kabiraj, Parijat; Pal, Rituraj; Varela-Ramirez, Armando

2012-09-28

Highlights: Black-Right-Pointing-Pointer Rotenone is a model for inducing apoptosis and synphilin-1 accumulation in Parkinson Prime s studies. Black-Right-Pointing-Pointer The metabolite sodium betahydroxybutryate mitigates these effects in SHSY5Y cell lines. Black-Right-Pointing-Pointer Results reveal a novel and innate mechanism to prevent neurodegeneration/cell death. -- Abstract: Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species, is associated with the pathogenesis of neurodegenerative disorders. Rotenone, a mitochondrial stressor induces caspase-9 and caspase-3 activation leading proteolytic cleavage of substrate nuclear poly(ADP-ribose) polymerase (PARP). PARP cleavage is directly related to apoptotic cell death. In this study, we have monitored the aggregation of green-fluorescent protein (GFP)-taggedmore » synphilin-1, as a rotenone-induced Parkinsonia-onset biomarker. We report that the innate ketone body, Na-D-{beta}-hydroxybutyrate (Na{beta}HB) reduces markedly the incidence of synphilin-1 aggregation. Furthermore, our data reveal that the metabolic byproduct also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells. Together, these results suggest that Na{beta}HB is neuroprotective; it attenuates effects originating from mitochondrial insult and can serve as a scaffold for the design and development of sporadic neuropathies.« less

Baicalein antagonizes rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to Parkinsonism.

PubMed

Song, Ju-Xian; Choi, Mandy Yuen-Man; Wong, Kavin Chun-Kit; Chung, Winkie Wing-Yan; Sze, Stephen Cho-Wing; Ng, Tzi-Bun; Zhang, Kalin Yan-Bo

2012-01-21

Two active compounds, baicalein and its glycoside baicalin were found in the dried root of Scutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluate the protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to parkinsonism. Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis was evaluated with a fluorescent DNA-binding probe Hoechst 33258. The production of reactive oxidative species (ROS) and loss of mitochondrial membrane potential (ΔΨm) were determined by fluorescent staining with DCFH-DA and Rhodanmine 123, respectively. The expression of Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 was determined by the Western blots. Baicalein significantly increased viability and decreased rotenone-induced death of SH-SY5Y cells in a dose-dependent manner. Pre- and subsequent co-treatment with baicalein preserved the cell morphology and attenuated the nuclear apoptotic characteristics triggered by rotenone. Baicalein antagonized rotenone-induced overproduction of ROS, loss of ΔΨm, the increased expression of Bax, cleaved caspase-3 and phosphorylated ERK1/2 and the decreased expression of Bcl-2. The antioxidative effect, mitochondrial protection and modulation of anti-and pro-apoptotic proteins are related to the neuroprotective effects of baicalein against rotenone induced cell death in SH-SY5Y cells.

Mitochondria-derived reactive oxygen species drive GANT61-induced mesothelioma cell apoptosis.

PubMed

Lim, Chuan Bian; Prêle, Cecilia M; Baltic, Svetlana; Arthur, Peter G; Creaney, Jenette; Watkins, D Neil; Thompson, Philip J; Mutsaers, Steven E

2015-01-30

Gli transcription factors of the Hedgehog (Hh) pathway have been reported to be drivers of malignant mesothelioma (MMe) cell survival. The Gli inhibitor GANT61 induces apoptosis in various cancer cell models, and has been associated directly with Gli inhibition. However various chemotherapeutics can induce cell death through generation of reactive oxygen species (ROS) but whether ROS mediates GANT61-induced apoptosis is unknown. In this study human MMe cells were treated with GANT61 and the mechanisms regulating cell death investigated. Exposure of MMe cells to GANT61 led to G1 phase arrest and apoptosis, which involved ROS but not its purported targets, GLI1 or GLI2. GANT61 triggered ROS generation and quenching of ROS protected MMe cells from GANT61-induced apoptosis. Furthermore, we demonstrated that mitochondria are important in mediating GANT61 effects: (1) ROS production and apoptosis were blocked by mitochondrial inhibitor rotenone; (2) GANT61 promoted superoxide formation in mitochondria; and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide, and were more resistant to apoptosis induced by GANT61 than wild-type cells. Our data demonstrate for the first time that GANT61 induces apoptosis by promoting mitochondrial superoxide generation independent of Gli inhibition, and highlights the therapeutic potential of mitochondrial ROS-mediated anticancer drugs in MMe.

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

PubMed

Danylovych, H V

2016-01-01

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism for induction of apoptosis via the 18 kDa mitochondrial translocator protein.

PubMed

Veenman, Leo; Alten, Julia; Linnemannstöns, Karen; Shandalov, Yulia; Zeno, Sivan; Lakomek, Max; Gavish, Moshe; Kugler, Wilfried

2010-07-01

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

Mitochondrial Impairment in Cerebrovascular Endothelial Cells is Involved in the Correlation between Body Temperature and Stroke Severity

PubMed Central

Hu, Heng; Doll, Danielle N.; Sun, Jiahong; Lewis, Sara E.; Wimsatt, Jeffrey H.; Kessler, Matthew J.; Simpkins, James W.; Ren, Xuefang

2016-01-01

Stroke is the second leading cause of death worldwide. The prognostic influence of body temperature on acute stroke in patients has been recently reported; however, hypothermia has confounded experimental results in animal stroke models. This work aimed to investigate how body temperature could prognose stroke severity as well as reveal a possible mitochondrial mechanism in the association of body temperature and stroke severity. Lipopolysaccharide (LPS) compromises mitochondrial oxidative phosphorylation in cerebrovascular endothelial cells (CVECs) and worsens murine experimental stroke. In this study, we report that LPS (0.1 mg/kg) exacerbates stroke infarction and neurological deficits, in the mean time LPS causes temporary hypothermia in the hyperacute stage during 6 hours post-stroke. Lower body temperature is associated with worse infarction and higher neurological deficit score in the LPS-stroke study. However, warming of the LPS-stroke mice compromises animal survival. Furthermore, a high dose of LPS (2 mg/kg) worsens neurological deficits, but causes persistent severe hypothermia that conceals the LPS exacerbation of stroke infarction. Mitochondrial respiratory chain complex I inhibitor, rotenone, replicates the data profile of the LPS-stroke study. Moreover, we have confirmed that rotenone compromises mitochondrial oxidative phosphorylation in CVECs. Lastly, the pooled data analyses of a large sample size (n=353) demonstrate that stroke mice have lower body temperature compared to sham mice within 6 hours post-surgery; the body temperature is significantly correlated with stroke outcomes; linear regression shows that lower body temperature is significantly associated with higher neurological scores and larger infarct volume. We conclude that post-stroke body temperature predicts stroke severity and mitochondrial impairment in CVECs plays a pivotal role in this hypothermic response. These novel findings suggest that body temperature is prognostic for stroke severity in experimental stroke animal models and may have translational significance for clinical stroke patients - targeting endothelial mitochondria may be a clinically useful approach for stroke therapy. PMID:26816660

Mitochondrial genome-knockout cells demonstrate a dual mechanism of action for the electron transport complex I inhibitor mycothiazole.

PubMed

Meyer, Kirsten J; Singh, A Jonathan; Cameron, Alanna; Tan, An S; Leahy, Dora C; O'Sullivan, David; Joshi, Praneta; La Flamme, Anne C; Northcote, Peter T; Berridge, Michael V; Miller, John H

2012-04-01

Mycothiazole, a polyketide metabolite isolated from the marine sponge Cacospongia mycofijiensis, is a potent inhibitor of metabolic activity and mitochondrial electron transport chain complex I in sensitive cells, but other cells are relatively insensitive to the drug. Sensitive cell lines (IC(50) 0.36-13.8 nM) include HeLa, P815, RAW 264.7, MDCK, HeLa S3, 143B, 4T1, B16, and CD4/CD8 T cells. Insensitive cell lines (IC(50) 12.2-26.5 μM) include HL-60, LN18, and Jurkat. Thus, there is a 34,000-fold difference in sensitivity between HeLa and HL-60 cells. Some sensitive cell lines show a biphasic response, suggesting more than one mechanism of action. Mitochondrial genome-knockout ρ(0) cell lines are insensitive to mycothiazole, supporting a conditional mitochondrial site of action. Mycothiazole is cytostatic rather than cytotoxic in sensitive cells, has a long lag period of about 12 h, and unlike the complex I inhibitor, rotenone, does not cause G(2)/M cell cycle arrest. Mycothiazole decreases, rather than increases the levels of reactive oxygen species after 24 h. It is concluded that the cytostatic inhibitory effects of mycothiazole on mitochondrial electron transport function in sensitive cell lines may depend on a pre-activation step that is absent in insensitive cell lines with intact mitochondria, and that a second lower-affinity cytotoxic target may also be involved in the metabolic and growth inhibition of cells.

Mitochondrial Respiration Inhibitors Suppress Protein Translation and Hypoxic Signaling via the Hyperphosphorylation and Inactivation of Translation Initiation Factor eIF2α and Elongation Factor eEF2

PubMed Central

Li, Jun; Mahdi, Fakhri; Du, Lin; Datta, Sandipan; Nagle, Dale G.; Zhou, Yu-Dong

2011-01-01

Over 20000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 µM and 0.068 µM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods. PMID:21875114

Selective Inhibition of Deactivated Mitochondrial Complex I by Biguanides †

PubMed Central

Matsuzaki, Satoshi; Humphries, Kenneth M.

2015-01-01

Biguanides are widely used antihyperglycemic agents for diabetes mellitus and prediabetes treatment. Complex I is the rate limiting step of the mitochondrial electron transport chain (ETC), a major source of mitochondrial free radical production, and a known target of biguanides. Complex I has two reversible conformational states, active and de-active. The deactivated state is promoted in the absence of substrates, but is rapidly and fully reversed to the active state in the presence of NADH. The objective of this study was to determine the relative sensitivity of active/de-active complex I to biguanide-mediated inhibition and resulting superoxide radical (O2•−) production. Using isolated rat heart mitochondria, we show that deactivation of complex I sensitizes it to metformin and phenformin (4- and 3-fold, respectively), but not to other known complex I inhibitors, such as rotenone. Mitochondrial O2•− production by deactivated complex I was measured fluorescently by the NADH-dependent 2-hydroxyethidium formation at alkaline pH to impede reactivation. Superoxide production was 260.4% higher than in active complex I at pH 9.4. However, phenformin treatment of de-active complex I decreased O2•− production by 14.9% while rotenone increased production by 42.9%. Mitochondria isolated from rat hearts subjected to cardiac ischemia, a condition known to induce complex I deactivation, were sensitized to phenformin:mediated complex I inhibition. This supports that the effects of biguanides are likely to be influenced by the complex I state in vivo. These results demonstrate that the complex I active/de-active states are a determinant in biguanide-mediated inhibition. PMID:25719498

SNJ-1945, a calpain inhibitor, protects SH-SY5Y cells against MPP+ and rotenone

PubMed Central

Knaryan, Varduhi H.; Samantaray, Supriti; Sookyoung, Park; Azuma, Mitsuyoshi; Inoue, Jun; Banik, Naren L.

2014-01-01

Complex pathophysiology of Parkinson’s disease (PD) involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in PD and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. In order to unravel these mechanisms SH-SY5Y neuroblastoma cells were differentiated into dopaminergic and cholinergic phenotypes respectively and used as cell culture model following exposure to two parkinsonian neurotoxicants MPP+ and rotenone. SNJ-1945, a cell-permeable calpain inhibitor was tested for its neuroprotective efficacy. MPP+ and rotenone dose-dependently elevated the levels of intracellular free Ca2+ and induced a concomitant rise in the levels of active calpain. SNJ-1945 pre-treatment significantly protected cell viability and preserved cellular morphology following MPP+ and rotenone exposure. The neurotoxicants elevated the levels of reactive oxygen species (ROS) more profoundly in SH-SY5Y cells differentiated into dopaminergic phenotype, and this effect could be attenuated with SNJ-1945 pre-treatment. In contrast, significant levels of inflammatory mediators (cyclooxygenase-2, Cox-2 and cleaved p10 fragment of caspase-1) were upregulated in the cholinergic phenotype, which could be dose-dependently attenuated by the calpain inhibitor. Overall, SNJ-1945 was efficacious against MPP+ or rotenone-induced ROS generation, inflammatory mediators, and proteolysis. A post-treatment regimen of SNJ-1945 was also examined in cells and partial protection was attained with calpain inhibitor administration 1–3 h after exposure to MPP+ or rotenone. Taken together these results indicate that calpain inhibition is a valid target for protection against parkinsonian neurotoxicants, and SNJ-1945 is an efficacious calpain inhibitor in this context. PMID:24341912

Improved mitochondrial function underlies the protective effect of pirfenidone against tubulointerstitial fibrosis in 5/6 nephrectomized rats.

PubMed

Chen, Jun-Feng; Liu, Hong; Ni, Hai-Feng; Lv, Lin-Li; Zhang, Ming-Hui; Zhang, Ai-Hua; Tang, Ri-Ning; Chen, Ping-Sheng; Liu, Bi-Cheng

2013-01-01

Dysfunctional mitochondria participate in the progression of chronic kidney disease (CKD). Pirfenidone is a newly identified anti-fibrotic drug. However, its mechanism remains unclear. Mitochondrial dysfunction is an early event that occurs prior to the onset of renal fibrosis. In this context, we investigated the protective effect of pirfenidone on mitochondria and its relevance to apoptosis and oxidative stress in renal proximal tubular cells. A remnant kidney rat model was established. Human renal proximal tubular epithelial cells (HK2) using rotenone, a mitochondrial respiratory chain complex Ι inhibitor were further investigated in vitro to examine the mitochondrial protective effect of pirfenidone. Pirfenidone protected mitochondrial structures and functions by stabilizing the mitochondrial membrane potential, maintaining ATP production and improving the mitochondrial DNA (mtDNA) copy number. Pirfenidone decreased tubular cell apoptosis by inhibiting the mitochondrial apoptotic signaling pathway. Pirfenidone also reduced oxidative stress by enhancing manganese superoxide dismutase (Mn-SOD) and inhibiting intracellular reactive oxygen species (ROS) generation, which suggested that the anti-oxidant effects occurred at least partially via the mitochondrial pathway. Pirfenidone may be effective prior to the onset of renal fibrosis because this drug exerts its anti-fibrotic effect by protection of mitochondria in renal proximal tubular cells.

Improved Mitochondrial Function Underlies the Protective Effect of Pirfenidone against Tubulointerstitial Fibrosis in 5/6 Nephrectomized Rats

PubMed Central

Chen, Jun-Feng; Liu, Hong; Ni, Hai-Feng; Lv, Lin-Li; Zhang, Ming-Hui; Zhang, Ai-Hua; Tang, Ri-Ning; Chen, Ping-Sheng; Liu, Bi-Cheng

2013-01-01

Dysfunctional mitochondria participate in the progression of chronic kidney disease (CKD). Pirfenidone is a newly identified anti-fibrotic drug. However, its mechanism remains unclear. Mitochondrial dysfunction is an early event that occurs prior to the onset of renal fibrosis. In this context, we investigated the protective effect of pirfenidone on mitochondria and its relevance to apoptosis and oxidative stress in renal proximal tubular cells. A remnant kidney rat model was established. Human renal proximal tubular epithelial cells (HK2) using rotenone, a mitochondrial respiratory chain complex Ι inhibitor were further investigated in vitro to examine the mitochondrial protective effect of pirfenidone. Pirfenidone protected mitochondrial structures and functions by stabilizing the mitochondrial membrane potential, maintaining ATP production and improving the mitochondrial DNA (mtDNA) copy number. Pirfenidone decreased tubular cell apoptosis by inhibiting the mitochondrial apoptotic signaling pathway. Pirfenidone also reduced oxidative stress by enhancing manganese superoxide dismutase (Mn-SOD) and inhibiting intracellular reactive oxygen species (ROS) generation, which suggested that the anti-oxidant effects occurred at least partially via the mitochondrial pathway. Pirfenidone may be effective prior to the onset of renal fibrosis because this drug exerts its anti-fibrotic effect by protection of mitochondria in renal proximal tubular cells. PMID:24349535

Morinda citrifolia mitigates rotenone-induced striatal neuronal loss in male Sprague-Dawley rats by preventing mitochondrial pathway of intrinsic apoptosis.

PubMed

Kishore Kumar, S Narasimhan; Deepthy, Jayakumar; Saraswathi, Uthamaraman; Thangarajeswari, Mohan; Yogesh Kanna, Sathyamoorthy; Ezhil, Pannerselvam; Kalaiselvi, Periandavan

2017-11-01

Parkinson disease (PD) is a neurodegenerative disorder affecting mainly the motor system, as a result of death of dopaminergic neurons in the substantia nigra pars compacta. The present scenario of research in PD is directed to identify novel molecules that can be administered individually or co-administered with L-Dopa to prevent the L-Dopa-Induced Dyskinesia (LID) like states that arise during chronic L-Dopa administration. Hence, in this study, we investigated whether Morinda citrifolia has therapeutic effects in rotenone-induced Parkinson's disease (PD) with special reference to mitochondrial dysfunction mediated intrinsic apoptosis. Male Sprague-Dawley rats were stereotaxically infused with rotenone (3 µg in both SNPc and VTA) and co-treated with the ethyl acetate extract of Morinda citrifolia and levodopa. The results revealed that rotenone-induced cell death was reduced by MCE treatment as measured by decline in the levels of pro-apoptotic proteins. Moreover, MCE treatment significantly augmented the levels of anti-apoptotic Bcl2 and blocks the release of cytochrome c, thereby alleviating the rotenone-induced dopaminergic neuronal loss, as evidenced by tyrosine hydroxylase (TH) immunostaining in the striatum. Taken together, the results suggest that Morinda citrifolia may be beneficial for the treatment of neurodegenerative diseases like PD.

Baicalein antagonizes rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to Parkinsonism

PubMed Central

2012-01-01

Background Two active compounds, baicalein and its glycoside baicalin were found in the dried root of Scutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluate the protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to parkinsonism. Methods Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis was evaluated with a fluorescent DNA-binding probe Hoechst 33258. The production of reactive oxidative species (ROS) and loss of mitochondrial membrane potential (ΔΨm) were determined by fluorescent staining with DCFH-DA and Rhodanmine 123, respectively. The expression of Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 was determined by the Western blots. Results Baicalein significantly increased viability and decreased rotenone-induced death of SH-SY5Y cells in a dose-dependent manner. Pre- and subsequent co-treatment with baicalein preserved the cell morphology and attenuated the nuclear apoptotic characteristics triggered by rotenone. Baicalein antagonized rotenone-induced overproduction of ROS, loss of ΔΨm, the increased expression of Bax, cleaved caspase-3 and phosphorylated ERK1/2 and the decreased expression of Bcl-2. Conclusion The antioxidative effect, mitochondrial protection and modulation of anti-and pro-apoptotic proteins are related to the neuroprotective effects of baicalein against rotenone induced cell death in SH-SY5Y cells. PMID:22264378

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding.

PubMed

Plaza Dávila, M; Bucci, D; Galeati, G; Peña, F J; Mari, G; Giaretta, E; Tamanini, C; Spinaci, M

2015-12-01

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 μm) and EGCG (10, 20 and 60 μm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 μm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone. © 2015 Blackwell Verlag GmbH.

Guanosine protects human neuroblastoma SH-SY5Y cells against mitochondrial oxidative stress by inducing heme oxigenase-1 via PI3K/Akt/GSK-3β pathway.

PubMed

Dal-Cim, Tharine; Molz, Simone; Egea, Javier; Parada, Esther; Romero, Alejandro; Budni, Josiane; Martín de Saavedra, Maria D; del Barrio, Laura; Tasca, Carla I; López, Manuela G

2012-08-01

Mitochondrial perturbation and oxidative stress are key factors in neuronal vulnerability in several neurodegenerative diseases or during brain ischemia. Here we have investigated the protective mechanism of action of guanosine, the guanine nucleoside, in a human neuroblastoma cell line, SH-SY5Y, subjected to mitochondrial oxidative stress. Blockade of mitochondrial complexes I and V with rotenone plus oligomycin (Rot/oligo) caused a significant decrease in cell viability and an increase in ROS production. Guanosine that the protective effect of guanosine incubated concomitantly with Rot/oligo abolished Rot/oligo-induced cell death and ROS production in a concentration dependent manner; maximum protection was achieved at the concentration of 1mM. The cytoprotective effect afforded by guanosine was abolished by adenosine A(1) or A(2A) receptor antagonists (DPCPX or ZM241385, respectively), or by a large (big) conductance Ca(2+)-activated K(+) channel (BK) blocker (charybdotoxin). Evaluation of signaling pathways showed that the protective effect of guanosine was not abolished by a MEK inhibitor (PD98059), by a p38(MAPK) inhibitor (SB203580), or by a PKC inhibitor (cheleritrine). However, when blocking the PI3K/Akt pathway with LY294002, the neuroprotective effect of guanosine was abolished. Guanosine increased Akt and p-Ser-9-GSK-3β phosphorylation confirming this pathway plays a key role in guanosine's neuroprotective effect. Guanosine induced the antioxidant enzyme heme oxygenase-1 (HO-1) expression. The protective effects of guanosine were prevented by heme oxygenase-1 inhibitor, SnPP. Moreover, bilirubin, an antioxidant and physiologic product of HO-1, is protective against mitochondrial oxidative stress. In conclusion, our results show that guanosine can afford protection against mitochondrial oxidative stress by a signaling pathway that implicates PI3K/Akt/GSK-3β proteins and induction of the antioxidant enzyme HO-1. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mitochondrion-Derived Reactive Oxygen Species Lead to Enhanced Amyloid Beta Formation

PubMed Central

Schütt, Tanja; Kurz, Christopher; Eckert, Schamim H.; Schiller, Carola; Occhipinti, Angelo; Mai, Sören; Jendrach, Marina; Eckert, Gunter P.; Kruse, Shane E.; Palmiter, Richard D.; Brandt, Ulrich; Dröse, Stephan; Wittig, Ilka; Willem, Michael; Haass, Christian; Reichert, Andreas S.; Müller, Walter E.

2012-01-01

Abstract Aims: Intracellular amyloid beta (Aβ) oligomers and extracellular Aβ plaques are key players in the progression of sporadic Alzheimer's disease (AD). Still, the molecular signals triggering Aβ production are largely unclear. We asked whether mitochondrion-derived reactive oxygen species (ROS) are sufficient to increase Aβ generation and thereby initiate a vicious cycle further impairing mitochondrial function. Results: Complex I and III dysfunction was induced in a cell model using the respiratory inhibitors rotenone and antimycin, resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Aβ. Presence of an antioxidant rescued mitochondrial function and reduced formation of Aβ, demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Aβ showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Aβ was partly reduced by an antioxidant, indicating that Aβ formation was also ROS dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aβ levels in vivo. Innovation: We show for the first time that mitochondrion-derived ROS are sufficient to trigger Aβ production in vitro and in vivo. Conclusion: Several lines of evidence show that mitochondrion-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aβ itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD. Antioxid. Redox Signal. 16, 1421–1433. PMID:22229260

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)*

PubMed Central

Elguindy, Mahmoud M.; Nakamaru-Ogiso, Eiko

2015-01-01

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O2 activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC50 = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O2 activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O2 activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells. PMID:26063804

Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages.

PubMed

Mills, Evanna L; Kelly, Beth; Logan, Angela; Costa, Ana S H; Varma, Mukund; Bryant, Clare E; Tourlomousis, Panagiotis; Däbritz, J Henry M; Gottlieb, Eyal; Latorre, Isabel; Corr, Sinéad C; McManus, Gavin; Ryan, Dylan; Jacobs, Howard T; Szibor, Marten; Xavier, Ramnik J; Braun, Thomas; Frezza, Christian; Murphy, Michael P; O'Neill, Luke A

2016-10-06

Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Repurposing mitochondria from ATP production to ROS generation drives a pro-inflammatory phenotype in macrophages that depends on succinate oxidation by complex II

PubMed Central

Logan, A; Costa, A. S. H.; Varma, M.; Bryant, C. E.; Tourlomousis, P.; Däbritz, J. H. M.; Gottlieb, E.; Latorre, I.; Corr, S.C.; McManus, G.; Ryan, D.; Jacobs, H.T.; Szibor, M.; Xavier, R. J.; Braun, T.; Frezza, C.; Murphy, M. P.; O’Neill, L. A.

2018-01-01

Activated macrophages undergo metabolic reprogramming which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here we demonstrate that upon lipopolysaccharide (LPS) stimulation macrophages shift from producing ATP by oxidative phosphorylation to glycolysis, while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial ROS production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone, by uncoupling mitochondria, or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state. PMID:27667687

Sodium Butyrate Improves Locomotor Impairment and Early Mortality in a Rotenone-Induced Drosophila Model of Parkinson’s Disease

PubMed Central

St. Laurent, Robyn; O’Brien, Liam M.; Ahmad, S. Tariq

2013-01-01

Parkinson’s disease (PD) is a neurodegenerative disorder primarily affecting the dopaminergic neurons in the nigrastriatal pathway resulting in debilitating motor impairment in both familial and sporadic cases. Histone deacetylase (HDAC) inhibitors have been recently implicated as a therapeutic candidate because of their ability to correct the disrupted HDAC activity in PD and other neurodegenerative diseases. Sodium butyrate (SB), an HDAC inhibitor, reduces degeneration of dopaminergic neurons in a mutant alpha-synuclein Drosophila transgenic model of familial PD. Chronic exposure to the pesticide rotenone also causes selective degeneration of dopaminergic neurons and causes locomotor impairment and early mortality in a Drosophila model of chemically-induced PD. This study investigated the effects of sodium butyrate on locomotor impairment and early mortality in a rotenone-induced PD model. We show that treatment with 10 mM SB-supplemented food rescued the rotenone-induced locomotor impairment and early mortality in flies. Additionally, flies with the genetic knockdown of HDAC activity through Sin3A loss-of-function mutation (Sin3Alof) were resistant to rotenone-induced locomotor impairment and early mortality. Furthermore, SB-supplemented Sin3Alof flies had a modest additive effect for improving locomotor impairment. We also show SB-mediated improvement of rotenone-induced locomotor impairment was associated with elevated dopamine levels in the brain. However, the possibility of SB-mediated protective role through mechanisms independent from dopamine system is also discussed. These findings demonstrate that HDAC inhibitors like SB can ameliorate locomotor impairment in a rotenone-induced PD model. PMID:23623990

Mitochondrial Insult in a Parkinson's like symptoms model

NASA Astrophysics Data System (ADS)

Carrizales, Julio; Cantu, Manuel; Plas, Daniel; Daniel Plas Lab Team, Dr.

2014-03-01

Healthy cells require healthy mitochondria. If these organelles are damaged, many health consequences follow. For example, Parkinson's Disease (PD) is a major neurodegenerative disorder of unknown cause, but much evidence points to the mitochondrion as a key player in the onset of this disease. PD has been studied in animal models challenged with toxins that target the mitochondria. In our work, we have used the pesticide, Rotenone, a known inhibitor of protein Complex I in the mitochondrial electron transport chain. When this toxin is applied to the freshwater mollusk, Lymnaea stagnalis, or pond snail, severe motor deficits ensue. In this project, we are studying the direct effects of this toxin on mitochondrial structure and physiology. We expected that the morphology of the organelle may be altered. In addition, it is likely that the mitochondrial membrane potential necessary for normal function may decrease as the electron transport loses the ability to move protons from the matrix to the intermembrane space. we also are going to use Electrophysiology to compare and Identify the difference of the electrical signaling among healthy and unhealthy neurons. HHMI

Degradation of rotenone in yam bean seeds ( Pachyrhizus sp.) through food processing.

PubMed

Catteau, Lucy; Lautié, Emmanuelle; Koné, Oumou; Coppée, Marie; Hell, Kerstin; Pomalegni, Charles Bertrand; Quetin-Leclercq, Joëlle

2013-11-20

The purpose of this research is to screen different processes that could potentially decrease or even eliminate rotenone, a toxic isoflavonoid, from Pachyrhizus seeds. Yam bean seeds have very interesting nutritional characteristics, especially their high protein and lipid contents, and could potentially increase food security in under-nourished populations. However, they contain rotenone, a natural molecule previously used as an insecticide inhibiting the respiratory mitochondrial chain. It was also proven to be toxic to mammals as chronic exposure leads to the development of Parkinson-like symptoms in rats. As the thermosensitivity of rotenone had been reported, this study tested different processes (drying, roasting, boiling, frying, alcohol extraction), tegument removal, and traditional Beninese culinary recipes. Rotenone was then quantified in end-products by a validated method, associating microwave extraction, solid phase extraction (SPE), and HPLC-UV. With these processes a rotenone removal of up to 80% was obtained. The most effective methods were the drying and roasting of the seeds and the maceration of their flour in local alcohol. Rotenone degradation and elimination were confirmed by cytotoxic assays, effectively inducing a decrease in sample toxicity.

Distinct Effects of Rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on Cellular Bioenergetics and Cell Death

PubMed Central

Giordano, Samantha; Lee, Jisun; Darley-Usmar, Victor M.; Zhang, Jianhua

2012-01-01

Parkinson’s disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson’s like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP+), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson’s disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP+ exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP+, unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP+ at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics. PMID:22970265

MPP+ analogs acting on mitochondria and inducing neuro-degeneration.

PubMed

Kotake, Y; Ohta, S

2003-12-01

This review focuses on the mechanisms of action and the injurious effect of complex I inhibitors, of which 1-methyl-4-phenylpyridinium ion (MPP(+)) is a well studied example. These compounds can be divided into two groups, i.e. competitive inhibitors with respect to ubiquinone, such as piericidine A, and non-competitive inhibitors such as rotenone. Complex I inhibitors such as MPP(+) have been reported to induce anatomical, behavioral, and biochemical changes similar to those seen in Parkinson's disease, which is characterized by nigrostriatal dopaminergic neuro-degeneration. Spectroscopic analyses and structure-activity relationship studies have indicated that the V-shaped structure of the rotenone molecule is critical for binding to the rotenone binding site on complex I. Many isoquinoline derivatives, some of them endogenous, are also complex I inhibitors. Many lines of evidence show that complex I inhibitors elicit neuronal cell death. Recently, it was reported that chronic and systemic exposure to low-dose rotenone reproduces the features of Parkinson's disease. This work further focused attention on compounds acting on mitochondria, such as MPP(+). In Guadeloupe, the French West Indies, patients with atypical parkinsonism or progressive supranuclear palsy are frequently encountered. These diseases seem to be associated with ingestion of tropical herbal teas or tropical fruits of the Annonaceae family, which contain complex I inhibitors such as benzylisoquinoline derivatives and acetogenins. Complex I inhibitors may not simply result in reactive oxygen species generation or ATP exhaustion, but may influence complex downstream signal transduction processes. An understanding of these changes would throw light on the ways in which complex I inhibitors induce a wide range of abnormalities.

Oxidative stress-induced necrotic cell death via mitochondira-dependent burst of reactive oxygen species.

PubMed

Choi, Kyungsun; Kim, Jinho; Kim, Gyung W; Choi, Chulhee

2009-11-01

Oxidative stress is deeply involved in various brain diseases, including neurodegenerative diseases, stroke, and ischemia/reperfusion injury. Mitochondria are thought to be the target and source of oxidative stress. We investigated the role of mitochondria in oxidative stress-induced necrotic neuronal cell death in a neuroblastoma cell line and a mouse model of middle cerebral artery occlusion. The exogenous administration of hydrogen peroxide was used to study the role of oxidative stress on neuronal cell survival and mitochondrial function in vitro. Hydrogen peroxide induced non-apoptotic neuronal cell death in a c-Jun N-terminal kinase- and poly(ADP-ribosyl) polymerase-dependent manner. Unexpectedly, hydrogen peroxide treatment induced transient hyperpolarization of the mitochondrial membrane potential and a subsequent delayed burst of endogenous reactive oxygen species (ROS). The inhibition of mitochondrial hyperpolarization by diphenylene iodonium or rotenone, potent inhibitors of mitochondrial respiratory chain complex I, resulted in reduced ROS production and subsequent neuronal cell death in vitro and in vivo. The inhibition of mitochondrial hyperpolarization can protect neuronal cells from oxidative stress-induced necrotic cell death, suggesting a novel method of therapeutic intervention in oxidative stress-induced neurological disease.

Differential effects of temperature on reactive oxygen/nitrogen species production in rat pachytene spermatocytes and round spermatids.

PubMed

Pino, José A; Osses, Nelson; Oyarzún, Daniela; Farías, Jorge G; Moreno, Ricardo D; Reyes, Juan G

2013-02-01

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) like superoxide and nitric oxide are produced by testis and spermatogenic cells in response to heat stress. However, the magnitude and mechanisms of this production in spermatogenic cells have not been described. In this work, we evaluated ROS/RNS production, its pharmacology, mitochondrial oxidative metabolism, membrane potential and antioxidant capacity at different temperatures in isolated rat pachytene spermatocytes and round spermatids. Our results showed an increment in ROS/RNS production by pachytene spermatocytes when increasing the temperature to 40 °C. Instead, ROS/RNS production by round spermatids did not change at temperatures higher than 33 °C. ROS/RNS production was sensitive to NADPH oxidase inhibitor diphenylene iodonium or the mitochondrial complex I inhibitor rotenone. No additive effects were observed for these two compounds. Our results suggest an important mitochondrial ROS/RNS production in spermatogenic cells. Oligomycin-insensitive oxygen consumption (uncoupled oxygen consumption) increased with temperature and was significantly larger in round spermatids than pachytene spermatocytes, indicating a likely round spermatid mitochondrial uncoupling at high temperatures. A similar conclusion can be reached by measuring the mitochondrial membrane potential using rhodamine 123 fluorescence in permeabilized cells or JC-1 fluorescence in intact cells. The antioxidant capacity was higher in round spermatids than pachytene spermatocytes at 40 °C. Our results strongly suggest that at high temperatures (40 °C) pachytene spermatocytes are more susceptible to oxidative stress, but round spermatids are more protected because of a temperature-induced mitochondrial uncoupling together with a larger antioxidant capacity.

Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice

PubMed Central

Pan-Montojo, Francisco; Anichtchik, Oleg; Dening, Yanina; Knels, Lilla; Pursche, Stefan; Jung, Roland; Jackson, Sandra; Gille, Gabriele; Spillantini, Maria Grazia; Reichmann, Heinz; Funk, Richard H. W.

2010-01-01

In patients with Parkinson's disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system. PMID:20098733

Alternative mitochondrial electron transfer as a novel strategy for neuroprotection.

PubMed

Wen, Yi; Li, Wenjun; Poteet, Ethan C; Xie, Luokun; Tan, Cong; Yan, Liang-Jun; Ju, Xiaohua; Liu, Ran; Qian, Hai; Marvin, Marian A; Goldberg, Matthew S; She, Hua; Mao, Zixu; Simpkins, James W; Yang, Shao-Hua

2011-05-06

Neuroprotective strategies, including free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades for the treatment of neurological diseases. Unfortunately, none of the neuroprotectants has been proved effective in clinical trails. In the current study, we demonstrated that methylene blue (MB) functions as an alternative electron carrier, which accepts electrons from NADH and transfers them to cytochrome c and bypasses complex I/III blockage. A de novo synthesized MB derivative, with the redox center disabled by N-acetylation, had no effect on mitochondrial complex activities. MB increases cellular oxygen consumption rates and reduces anaerobic glycolysis in cultured neuronal cells. MB is protective against various insults in vitro at low nanomolar concentrations. Our data indicate that MB has a unique mechanism and is fundamentally different from traditional antioxidants. We examined the effects of MB in two animal models of neurological diseases. MB dramatically attenuates behavioral, neurochemical, and neuropathological impairment in a Parkinson disease model. Rotenone caused severe dopamine depletion in the striatum, which was almost completely rescued by MB. MB rescued the effects of rotenone on mitochondrial complex I-III inhibition and free radical overproduction. Rotenone induced a severe loss of nigral dopaminergic neurons, which was dramatically attenuated by MB. In addition, MB significantly reduced cerebral ischemia reperfusion damage in a transient focal cerebral ischemia model. The present study indicates that rerouting mitochondrial electron transfer by MB or similar molecules provides a novel strategy for neuroprotection against both chronic and acute neurological diseases involving mitochondrial dysfunction.

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity.

PubMed

Stelmashook, E V; Lozier, E R; Goryacheva, E S; Mergenthaler, P; Novikova, S V; Zorov, D B; Isaev, N K

2010-09-27

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca(2+)](i)) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca(2+)](i)). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca(2+)](i) increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca(2+)](i). Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca(2+)](i), demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca(2+)](i) and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca(2+)](i) and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

PubMed Central

Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

2015-01-01

We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

SIMILAR PATTERNS OF MITOCHONDRIAL VULNERABILITY AND RESCUE INDUCED BY GENETIC MODIFICATION OF α-SYNUCLEIN, PARKIN AND DJ-1 IN C. ELEGANS*

PubMed Central

Westlund, Beth; Perier, Celine; Burnam, Lucinda; Sluder, Anne; Hoener, Marius; Rodrigues, Cecilia MP; Alfonso, Aixa; Steer, Clifford; Liu, Leo; Przedborski, Serge; Wolozin, Benjamin

2014-01-01

How genetic and environmental factors interact in Parkinson’s disease is poorly understood. We have now compared the patterns of vulnerability and rescue of C. elegans with genetic modifications of three different genetic factors implicated in PD. We observed that expressing α-synuclein, deleting parkin (K08E3.7) or knocking down DJ-1 (B0432.2) or parkin, produces similar patterns of pharmacological vulnerability and rescue. C. elegans lines with these genetic changes were more vulnerable than non-transgenic nematodes to mitochondrial complex I inhibitors, including rotenone, fenperoximate, pyridaben or stigmatellin. In contrast, the genetic manipulations did not increase sensitivity to paraquat, sodium azide, divalent metal ions (FeII or CuII) or etoposide compared to non-transgenic nematodes. Each of the PD-related lines was also partially rescued by the anti-oxidant probucol, the mitochondrial complex II activator, D-β-hydroxybutyrate (DβHB) or the anti-apoptotic bile acid tauroursodeoxycholic acid (TUDCA). Complete protection in all lines was achieved by combining DβHB with TUDCA but not with probucol. These results show that diverse PD-related genetic modifications disrupt mitochondrial function in C. elegans, and they raise the possibility that mitochondrial disruption is a pathway shared in common by many types of familial PD. PMID:16239214

Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Cong; Sekine, Shuichi, E-mail: ssekine@facult

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and inmore » hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.« less

Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells*

PubMed Central

Sacoman, Juliana L.; Dagda, Raul Y.; Burnham-Marusich, Amanda R.; Dagda, Ruben K.; Berninsone, Patricia M.

2017-01-01

O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics. PMID:28100784

Neuroprotective effect of rotigotine against complex I inhibitors, MPP⁺ and rotenone, in primary mesencephalic cell culture.

PubMed

Radad, Khaled; Scheller, Dieter; Rausch, Wolf-Dieter; Reichmann, Heinz; Gille, Gabrielle

2014-01-01

Dopamine agonists are suggested to be more efficacious in treating Parkinson's disease (PD) as they have neuroprotective properties in addition to their receptor-related actions. The present study was designed to investigate the neuroprotective effects of rotigotine, a D3/D2/D1 dopamine receptor agonist, against the two powerful complex I inhibitors, 1-methyl-4-phenylpyridinium (MPP+) and rotenone, in primary mesencephalic cell culture relevant to PD. Primary mesencephalic cell cultures were prepared from embryonic mouse mesencephala at gestation day 14. Three sets of cultures were treated with rotigotine alone, rotigotine and MPP⁺, and rotigotine and rotenone to investigate the effect of rotigotine on the survival of dopaminergic neurons against age-, MPP⁺- and rotenone-induced cell death. At the end of each treatment, cultures were fixed and stained immunohistochemically against tyrosine hydroxylase (TH). The effect of rotigotine against rotenone-induced reactive oxygen species (ROS) production was measured using CH-H2DCFDA fluorescence dye. Rotigotine alone did not influence the survival of tyrosine hydroxylase immunoreactive (THir) neurons except at 10 µM, it significantly decreased the number of THir neurons by 40% compared to untreated controls. Treatment of cultures with 0.01 µM rotigotine rescued 10% of THir neurons against MPP⁺-induced cell death. Rotigotine was also found to significantly rescue 20% of THir neurons at 0.01 µM of rotenone-treated cultures. Using of CH-H2DCFDA fluorescence dye, it was found that rotigotine significantly attenuated ROS production compared to rotenone-treated cultures. Rotigotine provides minor protection against MPP⁺ and rescues a significant number of THir neurons against rotenone in primary mesencephalic cell cultures relevant to PD.

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2).

PubMed

Elguindy, Mahmoud M; Nakamaru-Ogiso, Eiko

2015-08-21

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O₂ activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC₅₀ = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O₂ activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O₂ activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The involvement of Eag1 potassium channels and miR-34a in rotenone-induced death of dopaminergic SH-SY5Y cells

PubMed Central

Horst, Camila Hillesheim; Titze-De-Almeida, Ricardo; Titze-De-Almeida, Simoneide Souza

2017-01-01

The loss of dopaminergic neurons and the resultant motor impairment are hallmarks of Parkinson's disease. The SH-SY5Y cell line is a model of dopaminergic neurons, and allows for the study of dopaminergic neuronal injury. Previous studies have revealed changes in Ether à go-go 1 (Eag1) potassium channel expression during p53-induced SH-SY5Y apoptosis, and the regulatory involvement of microRNA-34a (miR-34a) was demonstrated. In the present study, the involvement of Eag1 and miR-34a in rotenone-induced SH-SY5Y cell injury was investigated. Rotenone is a neurotoxin, which is often used to generate models of Parkinson's disease, since it causes the death of nigrostriatal neurons by inducing intracellular aggregation of alpha synuclein and ubiquitin. In the present study, rotenone resulted in a dose-dependent decrease in cell viability, as revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue cell counting assays. In addition, Eag1 was demonstrated to be constitutively expressed by SH-SY5Y cells, and involved in cell viability. Suppression of Eag1 with astemizole resulted in a dose-dependent decrease in cell viability, as revealed by MTT assay. Astemizole also enhanced the severity of rotenone-induced injury in SH-SY5Y cells. RNA interference against Eag1, using synthetic small interfering RNAs (siRNAs), corroborated this finding, as siRNAs potentiated rotenone-induced injury. Eag1-targeted siRNAs (kv10.1-3 or EAG1hum_287) resulted in a statistically significant 16.4–23.5% increase in vulnerability to rotenone. An increased number of apoptotic nuclei were observed in cells transfected with EAG1hum_287. Notably, this siRNA intensified rotenone-induced apoptosis, as revealed by an increase in caspase 3/7 activity. Conversely, a miR-34a inhibitor was demonstrated to exert neuroprotective effects. The viability of cells exposed to rotenone for 24 or 48 h and treated with miR-34a inhibitor was restored by 8.4–8.8%. In conclusion, Eag1 potassium channels and miR-34a are involved in the response to rotenone-induced injury in SH-SY5Y cells. The neuroprotective effect of mir-34a inhibitors merits further investigations in animal models of Parkinson's disease. PMID:28259991

[Effect of K-ATP channel opener-pinacidil on the liver mitochondria function in rats with different resistance to hypoxia during stress].

PubMed

Tkachenko, H M; Kurhaliuk, N M; Vovkanych, L S

2004-01-01

We have examined the influence of ATP-sensitive potassium (KATP) channel opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) on the changes of energy metabolism in the liver of rats under the stress conditions. The rats were divided in two groups with high and low resistance to hypoxia. The stress was modeled by placing the rats in a cage filled with water and closed with a net. The distance from water to the net was only 5 cm. The effects of KATP opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) on ADP-stimulating mitochondrial respiration by Chance, calcium capacity of organellas and processes of lipid peroxidation in the liver of rats with different resistance to hypoxia under the stress condition have been investigated. We have used the next substrates of oxidation: 0.35 mM succinate and 1 mM alpha-ketoglutarate. The additional analyses were conducted with the use of inhibitors: mitochondrial enzyme complex I 10 mM rotenone and succinate dehydrohenase 2 mM malonic acid. It was shown that the stress condition evoked the succinate oxidation and the decrease of alpha-ketoglutarate efficacy, the increase of calcium mitochondrial capacity and the intensification of lipid peroxidation processes. Under the presence of succinate, the increase of O2 uptake with simultaneous decrease of ADP/O ratio in rats with high resistance under stress was observed. Simultaneously, oxidation of alpha-ketoglutarate, a NAD-dependent substrate, was inhibited. Pinacidil caused the reorganization of mitochondrial energy metabolism in favour of NAD-dependent oxidation and the improvment of the protection against stress. The decrease of the efficacy of mitochondrial energy processes functioning was shown in animals with low resistance to hypoxia. KATP channel opener pinacidil has a protective effect on the processes of mitochondrial liver energy support under stress. These changes deal with the increase of alpha-ketoglutarate oxidation (respiratory rate and ADP/O) and the decrease of lipid peroxidation processes. We concluded about protective effect ofpinacidil on mitochondrial functioning under stress.

Tissue distribution and elimination of rotenone in rainbow trout

USGS Publications Warehouse

Gingerich, W.H.

1986-01-01

The fate of a single i.v. dose (120 μg/kg) of the piscicide [14C]rotenone was evaluated in rainbow trout for periods up to 72 h after dosing. Rotenone was rapidly cleared from the plasma; less than 2% of the dose remained in the plasma compartment after 20 min. The highest concentrations of rotenone residues (% dose/g tissue) were in the hepatobiliary system, bile, intestine, and in heart, lateral line swimming muscle, and posterior kidney; tissues that are highly dependent on oxidative metabolism. Although rotenone activity was present in all cell fractions examined, greater than 40% was associated with the mitochondrial fraction of liver, kidney, and muscle. More than 85% of the activity extracted from these tissues, except the liver, was parent rotenone. Elimination from whole body and major tissue depots conformed to simple first-order kinetics; the estimated half-life from whole body was 68.5 h. Branchial elimination accounted for 5% of the injected dose over a 4-h period, and urinary elimination was less than 2% over a 48-h period. Rotenone was eliminated essentially unchanged across the gills; however, parent rotenone was not found in either urine or bile. More than 80% of the activity in both urine and bile eluted from HPLC chromatographs as a highly polar fraction that was not hydrolyzed by incubation with either β-glucuronidase or sulfatase. The results imply that hepatobiliary excretion is the major route of elimination for rotenone residues in the trout and that metabolism to a more polar form is a prerequisite for elimination in both the bile and the urine

The role of mitochondrial reactive oxygen species in pH regulation in articular chondrocytes.

PubMed

Milner, P I; Wilkins, R J; Gibson, J S

2007-07-01

To examine the effect of O(2) and the role, and source, of reactive oxygen species (ROS) on pH regulation in articular chondrocytes. Cartilage from equine metacarpo/tarsophalangeal joints was digested (collagenase) to isolate chondrocytes and loaded with 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein, a pH-sensitive fluorophore. O(2) tension was maintained using Eschweiler tonometers and a Wosthoff gas mixer. Cells were exposed to agents which alter ROS levels, mitochondrial inhibitors and/or inhibitors of protein phosphorylation. ROS levels were determined by dichlorofluorescein and mitochondrial membrane potential measured using JC-1. pH homeostasis was dependent on ROS. Na(+)/H(+) exchanger (NHE) activity was inhibited at low O(2) tension (acid efflux reducing from 2.30+/-0.05 to 1.27+/-0.11mMmin(-1) at 1%). NHE activity correlated with ROS levels (r(2)=0.65). ROS levels were increased by antimycin A (with levels at 1% O(2) tension increasing from 59+/-9% of the value at 20% to 87+/-7%), but reduced by rotenone, myxothiazol and diphenyleneiodonium. Hypoxia induced depolarisation of the mitochondrial membrane potential (with JC-1 red-green fluorescence ratio at 1% O(2) tension decreasing to 40+/-10% of the value at 20%). The response to changes in O(2) and to antimycin A was inhibited by staurosporine, wortmanin and calyculin A. The fall in ROS levels in hypoxia reduces the ability of articular chondrocytes to regulate pH, inhibiting NHE activity via changes in protein phosphorylation. The site of ROS generation is likely to be mitochondrial electron transport chain complex III. These effects are important to understanding normal chondrocyte function and response to altered O(2) tension.

Rotenone Activates Phagocyte NADPH Oxidase through Binding to Its Membrane Subunit gp91phox

PubMed Central

Zhou, Hui; Zhang, Feng; Chen, Shih-heng; Zhang, Dan; Wilson, Belinda; Hong, Jau-shyong; Gao, Hui-Ming

2011-01-01

Rotenone, a widely used pesticide, reproduces Parkinsonism in rodents and associates with increased risk for Parkinson’s disease. We previously reported rotenone increased superoxide production through stimulating microglial phagocyte NADPH oxidase (PHOX). The present study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91phox. Functional studies showed both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91phox/p22phox) and cytosolic subunits (p67phox and p47phox). Rotenone-elicited extracellular superoxide release in p47phox-deficient macrophages suggested rotenone enabled to activate PHOX through a p47phox-independent mechanism. Increased membrane translocation of p67phox, elevated binding of p67phox to rotenone-treated membrane fractions, and co-immunoprecipitation of p67phox and gp91phox in rotenone-treated wild-type and p47phox-deficient macrophages indicated p67phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91phox. Rac1, a Rho-like small GTPase, enhanced p67phox-gp91phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91phox; such an interaction triggered membrane translocation of p67phox, leading to PHOX activation and superoxide production. PMID:22094225

Neuroprotective effect of Demethoxycurcumin, a natural derivative of Curcumin on rotenone induced neurotoxicity in SH-SY 5Y Neuroblastoma cells.

PubMed

Ramkumar, Muthu; Rajasankar, Srinivasagam; Gobi, Veerappan Venkatesh; Dhanalakshmi, Chinnasamy; Manivasagam, Thamilarasan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed; Kalandar, Ameer; Chidambaram, Ranganathan

2017-04-18

Mitochondrial dysfunction and oxidative stress are the main toxic events leading to dopaminergic neuronal death in Parkinson's disease (PD) and identified as vital objective for therapeutic intercession. This study investigated the neuro-protective effects of the demethoxycurcumin (DMC), a derivative of curcumin against rotenone induced neurotoxicity. SH-SY5Y neuroblastoma cells are divided into four experimental groups: untreated cells, cells incubated with rotenone (100 nM), cells treated with DMC (50 nM) + rotenone (100 nM) and DMC alone treated. 24 h after treatment with rotenone and 28 h after treatment with DMC, cell viability was assessed using the MTT assay, and levels of ROS and MMP, plus expression of apoptotic protein were analysed. Rotenone induced cell death in SH-SY5Y cells was significantly reduced by DMC pretreatment in a dose-dependent manner, indicating the potent neuroprotective effects of DMC. Rotenone treatment significantly increases the levels of ROS, loss of MMP, release of Cyt-c and expression of pro-apoptotic markers and decreases the expression of anti-apoptotic markers. Even though the results of the present study indicated that the DMC may serve as a potent therapeutic agent particularly for the treatment of neurodegenerative diseases like PD, further pre-clinical and clinical studies are required.

[Involvement of hydrogen peroxide in the regulation of coexpression of alternative oxidase and rotenone-insensitive NADH dehydrogenase in tomato leaves and calluses].

PubMed

Eprintsev, A T; Mal'tseva, E V; Shatskikh, A S; Popov, V N

2011-01-01

The involvement of active oxygen forms in the regulation of the expression of mitochondrial respiratory chain components, which are not related to energy storing, has been in vitro and in vivo studied in Lycopersicum esculentum L. The highest level of transcription of genes encoding alternative oxidase and NADH dehydrogenase has been observed in green tomato leaves. It has been shown that even low H2O2 concentrations activate both aoxlalpha and ndb1 genes, encoding alternative oxidase and external mitochondrial rotenone-insensitive NADH dehydrogenase, respectively. According to our results, in the case of an oxidative stress, alternative oxidase and NADH dehydrogenase are coexpressed in tomato plant tissues, and active oxygen forms serve as the secondary messengers of their coexpression.

Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs After Exposure to Hyperoxia

PubMed Central

Audi, Said H.; Staniszewski, Kevin S.; Haworth, Steven T.; Jacobs, Elizabeth R.; Ranji, Mahsa; Zablocki, Clement J.

2013-01-01

Recently, we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes nicotinamide adenine dinucleotide (NADH) and oxidized form of flavin adenine dinucleotide \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$({\\rm FADH}_{2})$\\end{document} (FAD), as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this paper was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${>}{95\\%}~{\\rm O}_{2}$\\end{document} for 48 h) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, when compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change in mitochondrial redox state of hyperoxic lungs prior to histological changes characteristic of hyperoxia. PMID:25379360

Opposite and tissue-specific effects of coenzyme Q2 on mPTP opening and ROS production between heart and liver mitochondria: role of complex I.

PubMed

Gharib, Abdallah; De Paulis, Damien; Li, Bo; Augeul, Lionel; Couture-Lepetit, Elisabeth; Gomez, Ludovic; Angoulvant, Denis; Ovize, Michel

2012-05-01

Coenzyme Q(2) (CoQ(2)) is known to inhibit mitochondrial permeability transition pore (mPTP) opening in isolated rat liver mitochondria. In this study, we investigated and compared the effects of CoQ(2) on mPTP opening and ROS production in isolated rabbit heart and rat liver mitochondria. Mitochondria were isolated from New Zealand White rabbit hearts and Wistar rat livers. Oxygen consumption, Ca(2+)-induced mPTP opening, ROS production and NADH DUb-reductase activity were measured. Rotenone was used to investigate the effect of CoQ(2) on respiratory complex I activity. CoQ(2) (23 μM) reduced the respiratory control index by 32% and 57% (p<0.01) in heart and liver mitochondria respectively, mainly through an increased oxygen consumption in state 4. CoQ(2) induced a 60% (p<0.05) decrease of calcium retention capacity (CRC) in heart mitochondria and inversely a 46% (p<0.05) increase in liver mitochondria. In basal condition, CoQ(2) induced a 170% (p<0.05) increase of H(2)O(2) production in heart mitochondria and 21% (ns) decrease of H(2)O(2) production in liver mitochondria. Because rotenone, a complex I inhibitor, increases H(2)O(2) production in heart but not in liver mitochondria we investigated the CoQ(2) effect in a dose-response assay of complex I inhibition by rotenone in both mitochondria. CoQ(2) antagonized the effect of rotenone on respiratory complex I activity in liver but not in heart mitochondria. CoQ(2) significantly reduced NADH DUb-reductase activity in liver (-47%) and heart (-37%) mitochondria. In conclusion, our data showed that on the contrary to what was observed in liver mitochondria, CoQ(2) favors mPTP opening and ROS production in heart mitochondria through an opposite effect on respiratory complex I activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Vanillin Attenuated Behavioural Impairments, Neurochemical Deficts, Oxidative Stress and Apoptosis Against Rotenone Induced Rat Model of Parkinson's Disease.

PubMed

Dhanalakshmi, Chinnasamy; Janakiraman, Udaiyappan; Manivasagam, Thamilarasan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed; Kalandar, Ameer; Khan, Mohammed Abdul Sattar; Guillemin, Gilles J

2016-08-01

Vanillin (4-hydroxy-3-methoxybenzaldehyde), a pleasant smelling organic aromatic compound, is widely used as a flavoring additive in food, beverage, cosmetic and drug industries. It is reported to cross the blood brain barrier and also displayed antioxidant and neuroprotective activities. We previously reported the neuroprotective effect of vanillin against rotenone induced in in vitro model of PD. The present experiment was aimed to analyze the neuroprotective effect of vanillin on the motor and non-motor deficits, neurochemical variables, oxidative, anti-oxidative indices and the expression of apoptotic markers against rotenone induced rat model of Parkinson's disease (PD). Rotenone treatment exhibited motor and non-motor impairments, neurochemical deficits, oxidative stress and apoptosis, whereas oral administration of vanillin attenuated the above-said indices. However further studies are needed to explore the mitochondrial protective and anti-inflammatory properties of vanillin, as these processes play a vital role in the cause and progression of PD.

Effects of mitochondrial poisons on glutathione redox potential and carotid body chemoreceptor activity.

PubMed

Gomez-Niño, A; Agapito, M T; Obeso, A; Gonzalez, C

2009-01-01

Low oxygen sensing in chemoreceptor cells involves the inhibition of specific plasma membrane K(+) channels, suggesting that mitochondria-derived reactive oxygen species (ROS) link hypoxia to K(+) channel inhibition, subsequent cell depolarization and activation of neurotransmitter release. We have used several mitochondrial poisons, alone and in combination with the antioxidant N-acetylcysteine (NAC), and quantify their capacity to alter GSH/GSSG levels and glutathione redox potential (E(GSH)) in rat diaphragm. Selected concentrations of mitochondrial poisons with or without NAC were tested for their capacity to activate neurotransmitter release in chemoreceptor cells and to alter ATP levels in intact rat carotid body (CB). We found that rotenone (1 microM), antimycin A (0.2 microg/ml) and sodium azide (5mM) decreased E(GSH); NAC restored E(GSH) to control values. At those concentrations mitochondrial poisons activated neurotransmitter release from CB chemoreceptor cells and decreased CB ATP levels, NAC being ineffective to modify these responses. Additional experiments with 3-nitroprionate (5mM), lower concentrations of rotenone and dinitrophenol revealed variable relationships between E(GSH) and chemoreceptor cell neurotransmitter release responses and ATP levels. These findings indicate a lack of correlation between mitochondrial-generated modifications of E(GSH) and chemoreceptor cells activity. This lack of correlation renders unlikely that alteration of mitochondrial production of ROS is the physiological pathway chemoreceptor cells use to signal hypoxia.

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage.

PubMed

Indo, Hiroko P; Davidson, Mercy; Yen, Hsiu-Chuan; Suenaga, Shigeaki; Tomita, Kazuo; Nishii, Takeshi; Higuchi, Masahiro; Koga, Yasutoshi; Ozawa, Toshihiko; Majima, Hideyuki J

2007-01-01

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.

Down-regulation of HSP60 Suppresses the Proliferation of Glioblastoma Cells via the ROS/AMPK/mTOR Pathway

PubMed Central

Tang, Haiping; Li, Jin; Liu, Xiaohui; Wang, Guihuai; Luo, Minkui; Deng, Haiteng

2016-01-01

Glioblastoma is a fatal and incurable cancer with the hyper-activated mTOR pathway. HSP60, a major chaperone for maintenance of mitochondrial proteostasis, is highly expressed in glioblastoma patients. To understand the effects of HSP60 on glioblastoma tumorigenesis and progression, we characterized the HSP60-knockdowned glioblastoma cells and revealed that HSP60 silencing markedly suppressed cell proliferation and promoted cell to undergo the epithelial-mesenchymal transition (EMT). Proteomic analysis showed that ribosomal proteins were significantly downregulated whereas EMT-associated proteins were up-regulated in HSP60-knockdowned U87 cells as confirmed by a distinct enrichment pattern in newly synthesized proteins with azido-homoalanine labeling. Biochemical analysis revealed that HSP60 knockdown increased reactive oxygen species (ROS) production that led to AMPK activation, similarly to the complex I inhibitor rotenone-induced AMPK activation. Activated AMPK suppressed mTORC1 mediated S6K and 4EBP1 phosphorylation to decrease protein translation, which slowed down cell growth and proliferation. On the other hand, high levels of ROS in HSP60 knockdowned or rotenone-treated U87 cells contributed to EMT. These results indicate that HSP60 silencing deactivates the mTOR pathway to suppress glioblastoma progression, suggesting that HSP60 is a potential therapeutic target for glioblastoma treatment. PMID:27325206

Transgenic mice expressing cyan fluorescent protein as a reporter strain to detect the effects of rotenone toxicity on retinal ganglion cells.

PubMed

Hayworth, C R; Rojas, J C; Gonzalez-Lima, F

2008-01-01

This is the first study using a reporter transgenic model to investigate the effects of an environmental toxin on the retina. Rotenone is a widely used pesticide that inhibits mitochondrial complex I and produces neurotoxicity. Previous studies demonstrated the time course and dose response of rotenone toxicity on retinal ganglion cells (RGC). However, previous analyses of rotenone-induced retinotoxicity provided little detail of the optic nerve axons and cellular pathology. These limitations were successfully surmounted by using a transgenic mouse line shown to express cyan fluorescent protein (CFP) in neurons, including RGC, under regulatory elements of the human the thy1.1 promoter (thy-CFP). Data showed that CFP expression is limited to RGC and their processes in the retina of thy-CFP mice. Eyes exposed to the pesticide rotenone displayed marked alterations in RGC morphology, inner plexiform layer, optic disc, and optic nerves. After 24 h, the number of CFP-labeled RGC was reduced 50%. Correlated with a loss of RGC bodies was an approximate 50% reduction in CFP fluorescence intensity at the optic disc. The findings showed that rotenone-induced degeneration of RGC and their processes can be visualized with exquisite detail in thy-CFP mice, and that this approach may provide a novel and effective way to monitor the association between environmental toxins and neurodegeneration in living animals.

Neuroprotective Effects of Proanthocyanidins, Natural Flavonoids Derived From Plants, on Rotenone-Induced Oxidative Stress and Apoptotic Cell Death in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Ma, Jian; Gao, Shan-Shan; Yang, Hai-Jie; Wang, Mian; Cheng, Bin-Feng; Feng, Zhi-Wei; Wang, Lei

2018-01-01

Proanthocyanidins (PA) are natural flavonoids widely present in many vegetables, fruits, nuts and seeds, and especially in grape seed. In the present study, we examined the neuroprotective effects of PA and the underlying molecular mechanism in rotenone model of Parkinson's disease (PD). We found that pretreatment with PA significantly reduced rotenone-induced oxidative stress in human neuroblastoma SH-SY5Y dopaminergic cells. In addition, PA markedly enhanced cell viability against rotenone neurotoxicity and considerably blocked rotenone-induced activation of caspase-9, caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP), biochemical features of apoptosis. Further study demonstrated that the anti-apoptotic effect of PA was mediated by suppressing p38, JNK, and ERK signaling, and inhibitors of these three signaling pathways reproduced the protective effect of PA separately. In summary, our results demonstrated that PA mitigated rotenone-induced ROS generation and antagonized apoptosis in SH-SY5Y cells by inhibiting p38, JNK, and ERK signaling pathways, and it may provide a new insight of PA in PD therapy.

Effects of metabolic modifiers such as carnitines, coenzyme Q10, and PUFAs against different forms of neurotoxic insults: metabolic inhibitors, MPTP, and methamphetamine.

PubMed

Virmani, Ashraf; Gaetani, Franco; Binienda, Zbigniew

2005-08-01

A number of strategies using the nutritional approach are emerging for the protection of the brain from damage caused by metabolic toxins, age, or disease. Neural dysfunction and metabolic imbalances underlie many diseases, and the inclusion of metabolic modifiers may provide an alternative and early intervention approach that may prevent further damage. Various models have been developed to study the impact of metabolism on brain function. These have also proven useful in expanding our understanding of neurodegeneration processes. For example, the metabolic compromise induced by inhibitors such as 3-nitropropionic acid (3-NPA), rotenone, and 1-methyl-4-phenylpyridinium (MPP+) can cause neurodegeneration in animal models and these models are thought to simulate the processes that may lead to diseases such as Huntington's and Parkinson's diseases. These inhibitors of metabolism are thought to selectively kill neurons by inhibiting various mitochondrial enzymes. However, the eventual cell death is attributed to oxidative stress damage of selectively vulnerable cells, especially highly differentiated neurons. Various studies indicate that the neurotoxicity resulting from these types of metabolic compromise is related to mitochondrial dysfunction and may be ameliorated by metabolic modifiers such as L-carnitine (L-C), creatine, and coenzyme Q10, as well as by antioxidants such as lipoic acid, vitamin E, and resveratrol. Mitochondrial function and cellular metabolism are also affected by the dietary intake of essential polyunsaturated fatty acids (PUFAs), which may regulate membrane composition and influence cellular processes, especially the inflammatory pathways. Cellular metabolic function may also be ameliorated by caloric restriction diets. L-C is a naturally occurring quaternary ammonium compound that is a vital cofactor for the mitochondrial entry and oxidation of fatty acids. Any factors affecting L-C levels may also affect ATP levels. This endogenous compound, L-C, together with its acetyl ester, acetyl-L-carnitine (ALC), also participates in the control of the mitochondrial acyl-CoA/CoA ratio, peroxisomal oxidation of fatty acids, and production of ketone bodies. A deficiency of carnitine is known to have major deleterious effects on the CNS. We have examined L-C and its acetylated derivative, ALC, as potential neuroprotective compounds using various known metabolic inhibitors, as well as against drugs of abuse such as methamphetamine.

Association between ROS production, swelling and the respirasome integrity in cardiac mitochondria.

PubMed

Jang, Sehwan; Javadov, Sabzali

2017-09-15

Although mitochondrial Ca 2+ overload and ROS production play a critical role in mitochondria-mediated cell death, a cause-effect relationship between them remains elusive. This study elucidated the crosstalk between mitochondrial swelling, ROS production, and electron transfer chain (ETC) supercomplexes in rat heart mitochondria in response to Ca 2+ and tert-butyl hydroperoxide (TBH), a lipid-soluble organic peroxide. Results showed that ROS production induced by TBH was significantly increased in the presence of Ca 2+ in a dose-dependent manner. TBH markedly inhibited the state 3 respiration rate with no effect on the mitochondrial swelling. Ca 2+ exerted a slight effect on mitochondrial respiration that was greatly aggravated by TBH. Analysis of supercomplexes revealed a minor difference in the presence of TBH and/or Ca 2+ . However, incubation of mitochondria in the presence of high Ca 2+ (1 mM) or inhibitors of ETC complexes (rotenone and antimycin A) induced disintegration of the main supercomplex, respirasome. Thus, PTP-dependent swelling of mitochondria solely depends on Ca 2+ but not ROS. TBH has no effect on the respirasome while Ca 2+ induces disintegration of the supercomplex only at a high concentration. Intactness of individual ETC complexes I and III is important for maintenance of the structural integrity of the respirasome. Copyright © 2017 Elsevier Inc. All rights reserved.

Mitochondrial redox cycling of mitoquinone leads to superoxide production and cellular apoptosis.

PubMed

Doughan, Abdulrahman K; Dikalov, Sergey I

2007-11-01

The mitochondria-targeted drug mitoquinone (MitoQ) has been used as an antioxidant that may selectively block mitochondrial oxidative damage; however, it has been recently suggested to increase reactive oxygen species (ROS) generation in malate- and glutamate-fueled mitochondria. To address this controversy, we studied the effects of MitoQ on endothelial and mitochondrial ROS production. We found that in a cell-free system with flavin-containing enzyme cytochrome P-450 reductase, MitoQ is a very efficient redox cycling agent and produced more superoxide compared with equal concentrations of menadione (10-1,000 nM). Treatment of endothelial cells with MitoQ resulted in a dramatic increase in superoxide production. In isolated mitochondria, MitoQ increased complex I-driven mitochondrial ROS production, whereas supplementation with ubiquinone-10 had no effect on ROS production. Similar results were observed in mitochondria isolated from endothelial cells incubated for 1 h with MitoQ. Inhibitor analysis suggested that the redox cycling of MitoQ occurred at two sites on complex I, proximal and distal to the rotenone-binding site. This was confirmed by demonstrating the redox cycling of MitoQ on purified mitochondrial complex I as well as NADH-fueled submitochondrial particles. Mitoquinone time- and dose-dependently increased endothelial cell apoptosis. These findings demonstrate that MitoQ may be prooxidant and proapoptotic because its quinone group can participate in redox cycling and superoxide production. In light of these results, studies using mitoquinone as an antioxidant should be interpreted with caution.

OxLDL enhances L-type Ca2+ currents via lysophosphatidylcholine-induced mitochondrial reactive oxygen species (ROS) production.

PubMed

Fearon, Ian M

2006-03-01

To examine the mechanisms underlying oxidised LDL- (oxLDL)-induced alterations in Ca(2+) currents, an effect which underlies altered vascular contractility and cardiac myocyte function. Ca(2+) currents (I(Ca)) were recorded by whole-cell patch-clamp in HEK293 cells expressing L-type Ca(2+) channel alpha(1C) subunits or isolated rat ventricular myocytes. oxLDL (but not native LDL) significantly enhanced recombinant I(Ca), an effect mimicked by 1 microM lysophosphatidylcholine (LPC). LPC failed to enhance I(Ca) either in mitochondrial electron transport chain-depleted rho(0) cells, or in the presence of rotenone (1 microM), or MPP(+) (10 microM). The LPC response was similarly ablated by ascorbate (200 microM) or TROLOX (500 microM) and by the mitochondria-targeted antioxidant, MitoQ (250 nM). In myocytes, enhancement of I(Ca) due to LPC was similarly abrogated with rotenone and MitoQ. These data suggest that LPC enhanced recombinant Ca(2+) currents due to increased mitochondrial ROS production. In support with this, LPC enhanced fluorescence in HEK293 cells and cardiac myocytes loaded with a ROS-sensitive mitochondrial dye, reduced mitotracker red. LPC up-regulates L-type Ca(2+) currents due to altered mitochondrial ROS production, an effect which mediates the response of the native I(Ca) in cardiac myocytes to oxLDL.

Chronic systemic pesticide exposure reproduces features of Parkinson's disease.

PubMed

Betarbet, R; Sherer, T B; MacKenzie, G; Garcia-Osuna, M; Panov, A V; Greenamyre, J T

2000-12-01

The cause of Parkinson's disease (PD) is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate a systemic defect in mitochondrial complex I. We report that chronic, systemic inhibition of complex I by the lipophilic pesticide, rotenone, causes highly selective nigrostriatal dopaminergic degeneration that is associated behaviorally with hypokinesia and rigidity. Nigral neurons in rotenone-treated rats accumulate fibrillar cytoplasmic inclusions that contain ubiquitin and alpha-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.

Effect of hexavalent chromium on electron leakage of respiratory chain in mitochondria isolated from rat liver.

PubMed

Xie, Ying; Zhong, Caigao; Zeng, Ming; Guan, Lan; Luo, Lei

2013-01-01

In the present study, we explored reactive axygen species (ROS) production in mitochondria, the mechanism of hexavalent chromium (Cr(VI)) hepatotoxicity, and the role of protection by GSH. Intact mitochondria were isolated from rat liver tissues and mitochondrial basal respiratory rates of NADH and FADH2 respiratory chains were determined. Mitochondria were treated with Cr(VI), GSH and several complex inhibitors. Mitochondria energized by glutamate/malate were separately or jointly treated with Rotenone (Rot), diphenyleneiodonium (DPI) and antimycinA (Ant), while mitochondria energized by succinate were separately or jointly treated with Rot, DPI ' thenoyltrifluoroacetone (TTFA) and Ant. Cr(VI) concentration-dependently induced ROS production in the NADH and FADH2 respiratory chain in liver mitochondria. Basal respiratory rate of the mitochondrial FADH2 respiratory chain was significantly higher than that of NADH respiratory chain. Hepatic mitochondrial electron leakage induced by Cr(VI) from NADH respiratory chain were mainly from ubiquinone binding sites of complex I and complex III. Treatment with 50µM Cr(VI) enhances forward movement of electrons through FADH2 respiratory chain and leaking through the ubiquinone binding site of complex III. Moreover, the protective effect of GSH on liver mitochondria electron leakage is through removing excess H2O2 and reducing total ROS. Copyright © 2013 S. Karger AG, Basel.

Strain-Dependent Oxidant Release in Articular Cartilage Originates from Mitochondria

PubMed Central

J, Brouillette M; S, Ramakrishnan P; M, Wagner V; E, Sauter E; J, Journot B; O, McKinley T; A, Martin J

2013-01-01

Mechanical loading is essential for articular cartilage homeostasis and plays a central role in the cartilage pathology, yet the mechanotransduction processes that underlie these effects remain unclear. Previously we showed that lethal amounts of reactive oxygen species (ROS) were liberated from the mitochondria in response to mechanical insult, and that chondrocyte deformation may be a source of ROS. To this end, we hypothesized that mechanically-induced mitochondrial ROS is related to the magnitude of cartilage deformation. To test this, we measured axial tissue strains in cartilage explants subjected to semi-confined compressive stresses of 0, 0.05, 0.1, 0.25, 0.5, or 1.0 MPa. The presence of ROS was then determined by confocal imaging with dihydroethidium (DHE), an oxidant sensitive fluorescent probe. Our results indicated that ROS levels increased linearly relative to the magnitude of axial strains (r2 = 0.83, p < 0.05), and significant cell death was observed at strains > 40%. By contrast, hydrostatic stress, which causes minimal tissue strain, had no significant effect. Cell permeable superoxide dismutase mimetic Mn(III)tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) significantly decreased ROS levels at 0.5 and 0.25 MPa. Electron transport chain inhibitor, rotenone, and cytoskeletal inhibitor, cytochalasin B, significantly decreased ROS levels at 0.25 MPa. Our findings strongly suggest that ROS and mitochondrial oxidants contribute to cartilage mechanobiology. PMID:23896937

Neuroprotective Effects of Proanthocyanidins, Natural Flavonoids Derived From Plants, on Rotenone-Induced Oxidative Stress and Apoptotic Cell Death in Human Neuroblastoma SH-SY5Y Cells

PubMed Central

Ma, Jian; Gao, Shan-Shan; Yang, Hai-Jie; Wang, Mian; Cheng, Bin-Feng; Feng, Zhi-Wei; Wang, Lei

2018-01-01

Proanthocyanidins (PA) are natural flavonoids widely present in many vegetables, fruits, nuts and seeds, and especially in grape seed. In the present study, we examined the neuroprotective effects of PA and the underlying molecular mechanism in rotenone model of Parkinson's disease (PD). We found that pretreatment with PA significantly reduced rotenone-induced oxidative stress in human neuroblastoma SH-SY5Y dopaminergic cells. In addition, PA markedly enhanced cell viability against rotenone neurotoxicity and considerably blocked rotenone-induced activation of caspase-9, caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP), biochemical features of apoptosis. Further study demonstrated that the anti-apoptotic effect of PA was mediated by suppressing p38, JNK, and ERK signaling, and inhibitors of these three signaling pathways reproduced the protective effect of PA separately. In summary, our results demonstrated that PA mitigated rotenone-induced ROS generation and antagonized apoptosis in SH-SY5Y cells by inhibiting p38, JNK, and ERK signaling pathways, and it may provide a new insight of PA in PD therapy. PMID:29904339

CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

PubMed

Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing

2016-09-01

A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p < 0.05), (ii) elevation in ΔΨm (p < 0.05), (iii) increased OCR and ATP formation (p < 0.05), (iv) increased intracellular NO levels (p < 0.05), (v) increased mitochondrial ROS production (p < 0.05), and (vi) increased susceptibility to rotenone (p < 0.05). Treatment with isradipine was able to partially rescue these negative effects of CNTF-ACM (p < 0.05). CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells

PubMed Central

Di Rita, Anthea; D’Acunzo, Pasquale; Simula, Luca; Campello, Silvia; Strappazzon, Flavie; Cecconi, Francesco

2018-01-01

Therapeutic strategies are needed to protect dopaminergic neurons in Parkinson’s disease (PD) patients. Oxidative stress caused by dopamine may play an important role in PD pathogenesis. Selective autophagy of mitochondria (mitophagy), mainly regulated by PINK1 and PARKIN, plays an important role in the maintenance of cell homeostasis. Mutations in those genes cause accumulation of damaged mitochondria, leading to nigral degeneration and early-onset PD. AMBRA1ActA is a fusion protein specifically expressed at the mitochondria, and whose expression has been shown to induce a powerful mitophagy in mammalian cells. Most importantly, the pro-autophagy factor AMBRA1 is sufficient to restore mitophagy in fibroblasts of PD patients carrying PINK1 and PARKIN mutations. In this study, we investigated the potential neuroprotective effect of AMBRA1-induced mitophagy against 6-hydroxydopamine (6-OHDA)- and rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We demonstrated that AMBRA1ActA overexpression was sufficient to induce mitochondrial clearance in SH-SY5Y cells. We found that apoptosis induced by 6-OHDA and rotenone was reversed by AMBRA1-induced mitophagy. Finally, transfection of SH-SY5Y cells with a vector encoding AMBRA1ActA significantly reduced 6-OHDA and rotenone-induced generation of reactive oxygen species (ROS). Altogether, our results indicate that AMBRA1ActA is able to induce mitophagy in SH-SY5Y cells in order to suppress oxidative stress and apoptosis induced by both 6-OHDA and rotenone. These results strongly suggest that AMBRA1 may have promising neuroprotective properties with an important role in limiting ROS-induced dopaminergic cell death, and the utmost potential to prevent PD or other neurodegenerative diseases associated with mitochondrial oxidative stress. PMID:29755319

AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Di Rita, Anthea; D'Acunzo, Pasquale; Simula, Luca; Campello, Silvia; Strappazzon, Flavie; Cecconi, Francesco

2018-01-01

Therapeutic strategies are needed to protect dopaminergic neurons in Parkinson's disease (PD) patients. Oxidative stress caused by dopamine may play an important role in PD pathogenesis. Selective autophagy of mitochondria (mitophagy), mainly regulated by PINK1 and PARKIN, plays an important role in the maintenance of cell homeostasis. Mutations in those genes cause accumulation of damaged mitochondria, leading to nigral degeneration and early-onset PD. AMBRA1 ActA is a fusion protein specifically expressed at the mitochondria, and whose expression has been shown to induce a powerful mitophagy in mammalian cells. Most importantly, the pro-autophagy factor AMBRA1 is sufficient to restore mitophagy in fibroblasts of PD patients carrying PINK1 and PARKIN mutations. In this study, we investigated the potential neuroprotective effect of AMBRA1-induced mitophagy against 6-hydroxydopamine (6-OHDA)- and rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We demonstrated that AMBRA1 ActA overexpression was sufficient to induce mitochondrial clearance in SH-SY5Y cells. We found that apoptosis induced by 6-OHDA and rotenone was reversed by AMBRA1-induced mitophagy. Finally, transfection of SH-SY5Y cells with a vector encoding AMBRA1 ActA significantly reduced 6-OHDA and rotenone-induced generation of reactive oxygen species (ROS). Altogether, our results indicate that AMBRA1 ActA is able to induce mitophagy in SH-SY5Y cells in order to suppress oxidative stress and apoptosis induced by both 6-OHDA and rotenone. These results strongly suggest that AMBRA1 may have promising neuroprotective properties with an important role in limiting ROS-induced dopaminergic cell death, and the utmost potential to prevent PD or other neurodegenerative diseases associated with mitochondrial oxidative stress.

Ethanol production improvement driven by genome-scale metabolic modeling and sensitivity analysis in Scheffersomyces stipitis

PubMed Central

2017-01-01

The yeast Scheffersomyces stipitis naturally produces ethanol from xylose, however reaching high ethanol yields is strongly dependent on aeration conditions. It has been reported that changes in the availability of NAD(H/+) cofactors can improve fermentation in some microorganisms. In this work genome-scale metabolic modeling and phenotypic phase plane analysis were used to characterize metabolic response on a range of uptake rates. Sensitivity analysis was used to assess the effect of ARC on ethanol production indicating that modifying ARC by inhibiting the respiratory chain ethanol production can be improved. It was shown experimentally in batch culture using Rotenone as an inhibitor of the mitochondrial NADH dehydrogenase complex I (CINADH), increasing ethanol yield by 18%. Furthermore, trajectories for uptakes rates, specific productivity and specific growth rate were determined by modeling the batch culture, to calculate ARC associated to the addition of CINADH inhibitor. Results showed that the increment in ethanol production via respiratory inhibition is due to excess in ARC, which generates an increase in ethanol production. Thus ethanol production improvement could be predicted by a change in ARC. PMID:28658270

Evidence for the role of oxidative stress in the acetylation of histone H3 by ethanol in rat hepatocytes

PubMed Central

Choudhury, Mahua; Park, Pil-Hoon; Jackson, Daniel; Shukla, Shivendra D.

2010-01-01

The relationship between ethanol induced oxidative stress and acetylation of histone H3 at lysine 9 (H3AcK9) remains unknown and was therefore investigated in primary cultures of rat hepatocytes. Cells were treated with ethanol and a select group of pharmacological agents and the status of H3AcK9 and reactive oxygen species (ROS) were monitored. When hepatocytes were exposed to ethanol (50 mM, 24 hr) in the presence of N-acetyl cystein (ROS reducer) or dietary antioxidants (quercetin, resveratrol), or NADPH oxidase inhibitor apocynin, ethanol induced increases in ROS and H3AcK9, both were significantly reduced. On the other hand, l-buthionine-sulfoximine (ROS inducer) and inhibitor of mitochondrial complex I (rotenone) and III (antimycin) increased ethanol induced H3AcK9 (p<0.01). Oxidative stress also affected ethanol induced alcohol dehydrogenase 1 (ADH1) mRNA expression. These results demonstrate for the first time that oxidative stress is involved in the ethanol induced histone H3 acetylation in hepatocytes. PMID:20705415

Evidence for the role of oxidative stress in the acetylation of histone H3 by ethanol in rat hepatocytes.

PubMed

Choudhury, Mahua; Park, Pil-Hoon; Jackson, Daniel; Shukla, Shivendra D

2010-09-01

The relationship between ethanol-induced oxidative stress and acetylation of histone H3 at lysine 9 (H3AcK9) remains unknown and was therefore investigated in primary cultures of rat hepatocytes. Cells were treated with ethanol, and a select group of pharmacological agents and the status of H3AcK9 and reactive oxygen species (ROS) were monitored. Pretreatment of hepatocytes with N-acetyl cystein (ROS reducer), or dietary antioxidants (quercetin, reserveratrol), or NADPH (reduced nicotinamide adenine dinucleotide phosphate) oxidase inhibitor apocynin, significantly reduced ethanol (50 mM, 24 h) induced increases in ROS and H3AcK9. In contrast, l-buthionine sulfoximine (ROS inducer) and inhibitor of mitochondrial complexes I (rotenone) and III (antimycin) increased ethanol-induced H3AcK9 (P<.01). Oxidative stress also affected ethanol-induced alcohol dehydrogenase 1 mRNA expression. These results demonstrate for the first time that oxidative stress is involved in the ethanol-induced histone H3 acetylation in hepatocytes. Copyright © 2010 Elsevier Inc. All rights reserved.

Ethanol production improvement driven by genome-scale metabolic modeling and sensitivity analysis in Scheffersomyces stipitis.

PubMed

Acevedo, Alejandro; Conejeros, Raúl; Aroca, Germán

2017-01-01

The yeast Scheffersomyces stipitis naturally produces ethanol from xylose, however reaching high ethanol yields is strongly dependent on aeration conditions. It has been reported that changes in the availability of NAD(H/+) cofactors can improve fermentation in some microorganisms. In this work genome-scale metabolic modeling and phenotypic phase plane analysis were used to characterize metabolic response on a range of uptake rates. Sensitivity analysis was used to assess the effect of ARC on ethanol production indicating that modifying ARC by inhibiting the respiratory chain ethanol production can be improved. It was shown experimentally in batch culture using Rotenone as an inhibitor of the mitochondrial NADH dehydrogenase complex I (CINADH), increasing ethanol yield by 18%. Furthermore, trajectories for uptakes rates, specific productivity and specific growth rate were determined by modeling the batch culture, to calculate ARC associated to the addition of CINADH inhibitor. Results showed that the increment in ethanol production via respiratory inhibition is due to excess in ARC, which generates an increase in ethanol production. Thus ethanol production improvement could be predicted by a change in ARC.

A ketogenic diet as a potential novel therapeutic intervention in amyotrophic lateral sclerosis.

PubMed

Zhao, Zhong; Lange, Dale J; Voustianiouk, Andrei; MacGrogan, Donal; Ho, Lap; Suh, Jason; Humala, Nelson; Thiyagarajan, Meenakshisundaram; Wang, Jun; Pasinetti, Giulio M

2006-04-03

The cause of neuronal death in amyotrophic lateral sclerosis (ALS) is uncertain but mitochondrial dysfunction may play an important role. Ketones promote mitochondrial energy production and membrane stabilization. SOD1-G93A transgenic ALS mice were fed a ketogenic diet (KD) based on known formulations for humans. Motor performance, longevity, and motor neuron counts were measured in treated and disease controls. Because mitochondrial dysfunction plays a central role in neuronal cell death in ALS, we also studied the effect that the principal ketone body, D-beta-3 hydroxybutyrate (DBH), has on mitochondrial ATP generation and neuroprotection. Blood ketones were > 3.5 times higher in KD fed animals compared to controls. KD fed mice lost 50% of baseline motor performance 25 days later than disease controls. KD animals weighed 4.6 g more than disease control animals at study endpoint; the interaction between diet and change in weight was significant (p = 0.047). In spinal cord sections obtained at the study endpoint, there were more motor neurons in KD fed animals (p = 0.030). DBH prevented rotenone mediated inhibition of mitochondrial complex I but not malonate inhibition of complex II. Rotenone neurotoxicity in SMI-32 immunopositive motor neurons was also inhibited by DBH. This is the first study showing that diet, specifically a KD, alters the progression of the clinical and biological manifestations of the G93A SOD1 transgenic mouse model of ALS. These effects may be due to the ability of ketone bodies to promote ATP synthesis and bypass inhibition of complex I in the mitochondrial respiratory chain.

Krebs cycle metabolites and preferential succinate oxidation following neonatal hypoxic-ischemic brain injury in mice.

PubMed

Sahni, Prateek V; Zhang, Jimmy; Sosunov, Sergey; Galkin, Alexander; Niatsetskaya, Zoya; Starkov, Anatoly; Brookes, Paul S; Ten, Vadim S

2018-02-01

BackgroundReverse electron transport (RET) driven by the oxidation of succinate has been proposed as the mechanism of accelerated production of reactive oxygen species (ROS) in post-ischemic mitochondria. However, it remains unclear whether upon reperfusion, mitochondria preferentially oxidase succinate.MethodsNeonatal mice were subjected to Rice-Vannucci model of hypoxic-ischemic brain injury (HI) followed by assessment of Krebs cycle metabolites, mitochondrial substrate preference, and H 2 O 2 generation rate in the ischemic brain.ResultsWhile brain mitochondria from control mice exhibited a rotenone-sensitive complex-I-dependent respiration, HI-brain mitochondria, at the initiation of reperfusion, demonstrated complex-II-dependent respiration, as rotenone minimally affected, but inhibition of complex-II ceased respiration. This was associated with a 30-fold increase of cerebral succinate concentration and significantly elevated H 2 O 2 emission rate in HI-mice compared to controls. At 60 min of reperfusion, cerebral succinate content and the mitochondrial response to rotenone did not differ from that in controls.ConclusionThese data are the first ex vivo evidence, that at the initiation of reperfusion, brain mitochondria transiently shift their metabolism from complex-I-dependent oxidation of NADH toward complex II-linked oxidation of succinate. Our study provides a critical piece of support for existence of the RET-dependent mechanism of elevated ROS production in reperfusion.

Mdivi-1 ameliorates early brain injury after subarachnoid hemorrhage via the suppression of inflammation-related blood-brain barrier disruption and endoplasmic reticulum stress-based apoptosis.

PubMed

Fan, Lin-Feng; He, Ping-You; Peng, Yu-Cong; Du, Qing-Hua; Ma, Yi-Jun; Jin, Jian-Xiang; Xu, Hang-Zhe; Li, Jian-Ru; Wang, Zhi-Jiang; Cao, Sheng-Long; Li, Tao; Yan, Feng; Gu, Chi; Wang, Lin; Chen, Gao

2017-11-01

Aberrant modulation of mitochondrial dynamic network, which shifts the balance of fusion and fission towards fission, is involved in brain damage of various neurodegenerative diseases including Parkinson's disease, Huntington's disease and Alzheimer's disease. A recent research has shown that the inhibition of mitochondrial fission alleviates early brain injury after experimental subarachnoid hemorrhage, however, the underlying molecular mechanisms have remained to be elucidated. This study was undertaken to characterize the effects of the inhibition of dynamin-related protein-1 (Drp1, a dominator of mitochondrial fission) on blood-brain barrier (BBB) disruption and neuronal apoptosis following SAH and the potential mechanisms. The endovascular perforation model of SAH was performed in adult male Sprague Dawley rats. The results indicated Mdivi-1(a selective Drp1 inhibitor) reversed the morphologic changes of mitochondria and Drp1 translocation, reduced ROS levels, ameliorated the BBB disruption and brain edema remarkably, decreased the expression of MMP-9 and prevented degradation of tight junction proteins-occludin, claudin-5 and ZO-1. Mdivi-1 administration also inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB), leading to decreased expressions of TNF-ɑ, IL-6 and IL-1ß. Moreover, Mdivi-1 treatment attenuated neuronal cell death and improved neurological outcome. To investigate the underlying mechanisms further, we determined that Mdivi-1 reduced p-PERK, p-eIF2α, CHOP, cleaved caspase-3 and Bax expression as well as increased Bcl-2 expression. Rotenone (a selective inhibitor of mitochondrial complexes I) abolished both the anti-BBB disruption and anti-apoptosis effects of Mdivi-1. In conclusion, these data implied that excessive mitochondrial fission might inhibit mitochondrial complex I to become a cause of oxidative stress in SAH, and the inhibition of Drp1 by Mdivi-1 attenuated early brain injury after SAH probably via the suppression of inflammation-related blood-brain barrier disruption and endoplasmic reticulum stress-based apoptosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential programming of p53-deficient embryonic cells during rotenone block

EPA Science Inventory

Mitochondrial dysfunction has been implicated in chemical toxicities. The present study used an in vitro model to investigate the differential expression of metabolic pathways during cellular stress in p53- efficient embryonic fibroblasts compared to p53-deficient cells. These c...

Iron-sulfur protein in mitochondrial complexes of Spodoptera litura as potential site for ROS generation.

PubMed

Li, Liangde; Dong, Xiaolin; Shu, Benshui; Wang, Zheng; Hu, Qiongbo; Zhong, Guohua

2014-12-01

Mitochondrial complex I is the main source of reactive oxygen species (ROS) production, but the exact site of superoxide generation or their relative contribution is not clear. This study aims to determine the function of iron-sulfur clusters (ISCU) in the initiation of ROS generation. ISCU2 and ISCU8 were cloned from Spodoptera litura which shared the conserved amino acid sequence with other insects. The expressions of the two genes were ubiquitous throughout the whole development stages and tissues. Knockdown of ISCU2 and ISCU8 resulted in the decline of the ROS, whereas rotenone and azadirachtin treatment up-regulated ROS levels by increasing mRNA expression. Furthermore, antioxidant enzyme activity of SOD and POD were up-regulated by rotenone and azadirachtin treatment and then declined after ISCU was silenced. Our results suggest the possibility that the molecules of ISCU2 and ISCU8 in complex I may serve as potential sites in the initiation of ROS generation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rotenone, Paraquat, and Parkinson’s Disease

PubMed Central

Tanner, Caroline M.; Kamel, Freya; Ross, G. Webster; Hoppin, Jane A.; Goldman, Samuel M.; Korell, Monica; Marras, Connie; Bhudhikanok, Grace S.; Kasten, Meike; Chade, Anabel R.; Comyns, Kathleen; Richards, Marie Barber; Meng, Cheryl; Priestley, Benjamin; Fernandez, Hubert H.; Cambi, Franca; Umbach, David M.; Blair, Aaron; Sandler, Dale P.; Langston, J. William

2011-01-01

Background Mitochondrial dysfunction and oxidative stress are pathophysiologic mechanisms implicated in experimental models and genetic forms of Parkinson’s disease (PD). Certain pesticides may affect these mechanisms, but no pesticide has been definitively associated with PD in humans. Objectives Our goal was to determine whether pesticides that cause mitochondrial dysfunction or oxidative stress are associated with PD or clinical features of parkinsonism in humans. Methods We assessed lifetime use of pesticides selected by mechanism in a case–control study nested in the Agricultural Health Study (AHS). PD was diagnosed by movement disorders specialists. Controls were a stratified random sample of all AHS participants frequency-matched to cases by age, sex, and state at approximately three controls: one case. Results In 110 PD cases and 358 controls, PD was associated with use of a group of pesticides that inhibit mitochondrial complex I [odds ratio (OR) = 1.7; 95% confidence interval (CI), 1.0–2.8] including rotenone (OR = 2.5; 95% CI, 1.3–4.7) and with use of a group of pesticides that cause oxidative stress (OR = 2.0; 95% CI, 1.2–3.6), including paraquat (OR = 2.5; 95% CI, 1.4–4.7). Conclusions PD was positively associated with two groups of pesticides defined by mechanisms implicated experimentally—those that impair mitochondrial function and those that increase oxidative stress—supporting a role for these mechanisms in PD pathophysiology. PMID:21269927

LRRK2 modulates vulnerability to mitochondrial dysfunction in C. elegans

PubMed Central

Saha, Shamol; Guillily, Maria; Ferree, Andrew; Lanceta, Joel; Chan, Diane; Ghosh, Joy; Hsu, Cindy H.; Segal, Lilach; Raghavan, Kesav; Matsumoto, Kunihiro; Hisamoto, Naoki; Kuwahara, Tomoki; Iwatsubo, Takeshi; Moore, Landon; Goldstein, Lee; Cookson, Mark; Wolozin, Benjamin

2009-01-01

Summary Mutations in leucine rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson’s disease. We generated lines of C. elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 expression increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C or kinase dead LRRK2 was less than protection by wild type LRRK2. Knockdown of lrk-1, the endogenous orthologue of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT∷GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the WT line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing WT LRRK2; however loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different than that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition, and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson’s disease. PMID:19625511

Induction of Mitochondrial Reactive Oxygen Species Production by Itraconazole, Terbinafine, and Amphotericin B as a Mode of Action against Aspergillus fumigatus

PubMed Central

Shekhova, Elena

2017-01-01

ABSTRACT Drug resistance in fungal pathogens is of incredible importance to global health, yet the mechanisms of drug action remain only loosely defined. Antifungal compounds have been shown to trigger the intracellular accumulation of reactive oxygen species (ROS) in human-pathogenic yeasts, but the source of those ROS remained unknown. In the present study, we examined the role of endogenous ROS for the antifungal activity of the three different antifungal substances itraconazole, terbinafine, and amphotericin B, which all target the fungal cell membrane. All three antifungals had an impact on fungal redox homeostasis by causing increased intracellular ROS production. Interestingly, the elevated ROS levels induced by antifungals were abolished by inhibition of the mitochondrial respiratory complex I with rotenone. Further, evaluation of lipid peroxidation using the thiobarbituric acid assay revealed that rotenone pretreatment decreased ROS-induced lipid peroxidation during incubation of Aspergillus fumigatus with itraconazole and terbinafine. By applying the mitochondrion-specific lipid peroxidation probe MitoPerOx, we also confirmed that ROS are induced in mitochondria and subsequently cause significant oxidation of mitochondrial membrane in the presence of terbinafine and amphotericin B. To summarize, our study suggests that the induction of ROS production contributes to the ability of antifungal compounds to inhibit fungal growth. Moreover, mitochondrial complex I is the main source of deleterious ROS production in A. fumigatus challenged with antifungal compounds. PMID:28848005

Neuroprotective Effects of Açaí (Euterpe oleracea Mart.) against Rotenone In Vitro Exposure.

PubMed

Machado, Alencar Kolinski; Andreazza, Ana Cristina; da Silva, Tatiane Morgana; Boligon, Aline Augusti; do Nascimento, Vanusa; Scola, Gustavo; Duong, Angela; Cadoná, Francine Carla; Ribeiro, Euler Esteves; da Cruz, Ivana Beatrice Mânica

2016-01-01

Neuropsychiatric diseases, such as bipolar disorder (BD) and schizophrenia (SCZ), have a very complex pathophysiology. Several current studies describe an association between psychiatric illness and mitochondrial dysfunction and consequent cellular modifications, including lipid, protein, and DNA damage, caused by cellular oxidative stress. Euterpe oleracea (açaí) is a powerful antioxidant fruit. Açaí is an Amazonian palm fruit primarily found in the lowlands of the Amazonian rainforest, particularly in the floodplains of the Amazon River. Given this proposed association, this study analyzed the potential in vitro neuropharmacological effect of Euterpe oleracea (açaí) extract in the modulation of mitochondrial function and oxidative metabolism. SH-SY5Y cells were treated with rotenone to induce mitochondrial complex I dysfunction and before and after we exposed the cells to açaí extract at 5  μ g/mL. Treated and untreated cells were then analyzed by spectrophotometric, fluorescent, immunological, and molecular assays. The results showed that açaí extract can potentially increase protein amount and enzyme activity of mitochondrial complex I, mainly through NDUFS7 and NDUFS8 overexpression. Açaí extract was also able to decrease cell reactive oxygen species levels and lipid peroxidation. We thus suggest açaí as a potential candidate for drug development and a possible alternative BD therapy.

Neuroprotective Effects of Açaí (Euterpe oleracea Mart.) against Rotenone In Vitro Exposure

PubMed Central

Andreazza, Ana Cristina; da Silva, Tatiane Morgana; do Nascimento, Vanusa; Duong, Angela; Ribeiro, Euler Esteves

2016-01-01

Neuropsychiatric diseases, such as bipolar disorder (BD) and schizophrenia (SCZ), have a very complex pathophysiology. Several current studies describe an association between psychiatric illness and mitochondrial dysfunction and consequent cellular modifications, including lipid, protein, and DNA damage, caused by cellular oxidative stress. Euterpe oleracea (açaí) is a powerful antioxidant fruit. Açaí is an Amazonian palm fruit primarily found in the lowlands of the Amazonian rainforest, particularly in the floodplains of the Amazon River. Given this proposed association, this study analyzed the potential in vitro neuropharmacological effect of Euterpe oleracea (açaí) extract in the modulation of mitochondrial function and oxidative metabolism. SH-SY5Y cells were treated with rotenone to induce mitochondrial complex I dysfunction and before and after we exposed the cells to açaí extract at 5 μg/mL. Treated and untreated cells were then analyzed by spectrophotometric, fluorescent, immunological, and molecular assays. The results showed that açaí extract can potentially increase protein amount and enzyme activity of mitochondrial complex I, mainly through NDUFS7 and NDUFS8 overexpression. Açaí extract was also able to decrease cell reactive oxygen species levels and lipid peroxidation. We thus suggest açaí as a potential candidate for drug development and a possible alternative BD therapy. PMID:27781077

Glucose, Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals.

PubMed

Hohnholt, Michaela C; Andersen, Vibe H; Bak, Lasse K; Waagepetersen, Helle S

2017-01-01

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.

[F-18]Fluorodihydrorotenone: Synthesis and evaluation of a mitochondrial electron transport chain (ETC) complex I probe for PET

DOE Office of Scientific and Technical Information (OSTI.GOV)

VanBrocklin, H.F.; Enas, J.D.; Hanrahan, S.M.

1994-05-01

The mitochondrial electron transport chain (ETC) consists of five enzyme complexes (I-V) which participate in the transfer of electrons to oxygen and phosphorylation of ADP (oxidative phosphorylation). ETC dysfunction has been linked to several genetic neurological diseases as well as implicated in Parkinson`s (complex I) and Huntington`s (complex I) disease and normal aging processes. Dihydrorotenone (DHR) is a specific high affinity inhibitor of complex I. In order to develop a PET tracer for complex I, we have labeled DHR with fluorine-18. The tosylate precursor was produced in three steps from commercially available rotenone. Fluorine-18 was introduced by nucleophilic displacement ofmore » the tosylate using tetrabutyl-ammonium fluoride. Subsequent oxidation with MnO{sub 2} and HPLC purification gave the desired [{sup 18}F]fluoro-DHR. Initial biodistribution studies were carried out in {approximately}200 g male Sprague-Dawley rats. The tracer was taken up rapidly in the heart, an organ highly enriched with mitochondria, (5.5-6% injected dose (ID)/g at 30 minutes) and in the brain ({approximately}1.5% ID/g at 1 hour).« less

Neuroprotective effects of vildagliptin in rat rotenone Parkinson's disease model: role of RAGE-NFκB and Nrf2-antioxidant signaling pathways.

PubMed

Abdelsalam, Rania M; Safar, Marwa M

2015-06-01

Gliptins have been recently shown to conquer neuronal degeneration in cell cultures via modulating glucagon-like peptide (GLP)-1. This peptide produced in the gut not only crosses the blood-brain barrier but is also synthesized in the brain and acts on GLP-1R exerting central anti-inflammatory and antiapoptotic effects, thus impeding neuronal damage. This study investigated the antiparkinsonian effect of vildagliptin, a dipeptidyl peptidase (DPP)-4 inhibitor in a rat rotenone model targeting mainly the RAGE-NFκB/Nrf2-signaling pathways, to judge the potential anti-inflammatory/antioxidant effects of the drug. Vildagliptin markedly improved the motor performance in the open field and rotarod tests, effects that were emphasized by the accompanied reduction in striatal dopamine content. It modified the striatal energy level (ADP/ATP) associated with partial antagonism of body weight reduction. This incretin enhancer suppressed nuclear factor (NF)κB and, consequently, the downstream inflammatory mediator tumor necrosis factor-α. Normalization of receptor for advanced glycated end product (RAGE) is a main finding which justifies the anti-inflammatory effects of vildagliptin, together with hampering striatal inducible nitric oxide synthase, intracellular adhesion molecule-1 as well as myeloperoxidase. The antioxidant potential of vildagliptin was depicted as entailing reduction in thiobarbituric acid-reactive substances and the transcriptional factor Nrf-2 level. Vildagliptin guarded against neuronal demise through an antiapoptotic effect as reflected by the reduction in the mitochondrial matrix component cytochrome c and the key downstream executioner caspase-3. In conclusion, vildagliptin is endowed with various neuroprotective effects and thus can be a promising candidate for the management of Parkinson's disease. In the rat rotenone model of Parkinson's disease (PD), striatal RAGE/NFκB signaling was up-regulated associated with elevated levels of inflammatory, oxidative stress, and apoptotic mediators resulting in dopaminergic neurons death and hence motor impairment. Vildagliptin, a dipeptidyl peptidase (DPP)-4 inhibitor, blocked the RAGE/NFκB cascade exerting a potential antiparkinsonian effect. RAGE, receptor for advanced glycation end product; NFκB, nuclear factor κB; TNFα, tumor necrosis factor alpha; ICAM, intracellular adhesion molecule; iNOS, inducible nitric oxide synthase; MPO, myeloperoxidase. © 2015 International Society for Neurochemistry.

Occurrence of the malate-aspartate shuttle in various tumor types.

PubMed

Greenhouse, W V; Lehninger, A L

1976-04-01

The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm.

Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

PubMed

Hrynevich, Sviatlana V; Pekun, Tatyana G; Waseem, Tatyana V; Fedorovich, Sergei V

2015-06-01

Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

Anacardic Acids from Cashew Nuts Prevent Behavioral Changes and Oxidative Stress Induced by Rotenone in a Rat Model of Parkinson's Disease.

PubMed

Medeiros-Linard, Cybelle Façanha Barreto; Andrade-da-Costa, Belmira Lara da Silveira; Augusto, Ricielle Lopes; Sereniki, Adriana; Trevisan, Maria Teresa Sales; Perreira, Renata de Cássia Ribas; de Souza, Francisco Thiago Correia; Braz, Glauber Ruda Feitoza; Lagranha, Claudia Jacques; de Souza, Ivone Antônia; Wanderley, Almir Gonçalves; Smailli, Soraya S; Lafayette, Simone Sette Lopes

2018-03-08

Anacardic acids (AAs) are alkyl phenols mainly presenting in cashew nuts. The antioxidant effects of these compounds have been an area of interest in recent research, with findings suggesting potential therapeutic use for certain diseases. Nevertheless, none of these studies were performed in order to test the hypothesis of whether anacardic acids are capable of preventing behavioral changes and oxidative stress induced by the pesticide rotenone in experimental model of Parkinson's disease. In our research, adult male rats were treated orally with AAs (1, 3, 10, 25, 50, or 100 mg/kg/day) 1 h before rotenone (3 mg/kg; s.c.) for five consecutive days. The behavioral testing strategies, including tests for general locomotor activity (open field), motor coordination (rotarod), and spatial memory performance (elevated T-maze), were carried out. Lipoperoxidation levels and total superoxide dismutase (t-SOD) activity, as well as cytoplasmic and mitochondrial SOD gene expression, were assessed in the substantia nigra (SN), striatum, and cerebral cortex. The results showed that AAs dose-dependently prevented the rotenone-induced learning and motor impairment from 10 mg/kg/day. AAs also precluded rotenone-induced lipoperoxidation in all doses, acting directly on the mitochondria, and improved the t-SOD activity in the doses 25-100 mg/kg/day. AAs per se (100 mg/kg/day) increased SOD gene expression and t-SOD activity. Our findings indicate that the oral administration of AAs prevents rotenone-induced behavioral changes and oxidative stress, in part due to a modulatory action on the mitochondria and SOD gene expression. These data suggest that AAs have promising neuroprotective action against degenerative changes in Parkinson's disease.

Progesterone impairs cell respiration and suppresses a compensatory increase in glucose transport in isolated rat skeletal muscle: a non-genomic mechanism contributing to metabolic adaptation to late pregnancy?

PubMed

Gras, F; Brunmair, B; Quarré, L; Szöcs, Z; Waldhäusl, W; Fürnsinn, C

2007-12-01

The aim of the study was to gain better insight into the mechanisms responsible for impaired glucose metabolism during late pregnancy. We explored the direct effects of progesterone on glucose metabolism of skeletal muscle. Specimens of skeletal muscle from untreated rats were incubated with progesterone and rates of substrate fluxes through the various pathways of glucose metabolism were analysed. Progesterone dose-dependently reduced the rates of glucose and pyruvate oxidation (insulin-stimulated rates after 5 h of exposure to 1 and 10 mumol/l progesterone: glucose oxidation, -6 +/- 4%, NS, and -39 +/- 4%, p < 0.001; pyruvate oxidation, -28 +/- 2% and -55 +/- 4%, p < 0.001 each) and increased lactate release (+28 +/- 4% and +58 +/- 9%, p < 0.005 each), which indicated inhibition of mitochondrial respiratory function. Impairment of cell respiration, e.g. by the specific inhibitor rotenone, is known to trigger a compensatory increase in glucose transport, but this response was blunted in the case of progesterone (change of glucose transport in response to 10 mumol/l progesterone vs 60 nmol/l rotenone, both causing a reduction in glucose oxidation by -39%: progesterone, +14 +/- 8% vs rotenone, +84 +/- 23%, p < 0.03). Further experiments dealt with the underlying mechanisms and revealed a rapid mode of action (50 mumol/l progesterone, reduction in insulin-stimulated glucose oxidation after 30 min: -29 +/- 7%, p < 0.01) not affected by blockers of gene expression or the nuclear progesterone receptor. Progesterone inhibits cell respiration and at the same time suppresses a compensatory increase in glucose transport, causing cellular carbohydrate deficiency in isolated rat skeletal muscle. This effect is mediated by a direct, rapid and non-genomic mechanism and could contribute to pregnancy-associated changes in glucose homeostasis.

α-Synuclein transgenic mice reveal compensatory increases in Parkinson's disease-associated proteins DJ-1 and parkin and have enhanced α-synuclein and PINK1 levels after rotenone treatment.

PubMed

George, Sonia; Mok, Su San; Nurjono, Milawaty; Ayton, Scott; Finkelstein, David I; Masters, Colin L; Li, Qiao-Xin; Culvenor, Janetta G

2010-10-01

Parkinson's disease (PD) is a severe neurodegenerative disorder characterised by loss of dopaminergic neurons of the substantia nigra. The pathological hallmarks are cytoplasmic inclusions termed Lewy bodies consisting primarily of aggregated alpha-synuclein (alphaSN). Different lines of transgenic mice have been developed to model PD but have failed to recapitulate the hallmarks of this disease. Since treatment of rodents with the pesticide rotenone can reproduce nigrostriatal cell loss and other features of PD, we aimed to test chronic oral administration of rotenone to transgenic mice over-expressing human alphaSN with the A53T mutation. Initial assessment of this transgenic line for compensatory molecular changes indicated decreased brain beta-synuclein expression and significantly increased levels of the PD-associated oxidative stress response protein, DJ-1, and the E3 ubiquitin ligase enzyme, Parkin. Rotenone treatment of 30 mg/kg for 25 doses over a 35-day period was tolerated in the transgenic mice and resulted in decreased spontaneous locomotor movement and increased cytoplasmic alphaSN expression. The mitochondrial Parkinson's-associated PTEN-induced kinase 1 protein levels were also increased in transgenic mouse brain after rotenone treatment; there was no change in brain dopamine levels or nigrostriatal cell loss. These hA53T alphaSN transgenic mice provide a useful model for presymptomatic Parkinson's features and are valuable for study of associated compensatory changes in early Parkinson's disease stages.

[Assessment of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H].

PubMed

Cheng, Ying; Ren, Mingming; Niu, Yanyan; Qiao, Jianhua; Aneba, S; Chorvat, D; Chorvatova, A

2009-12-01

The primary function of cardiac mitochondria is the production of ATP to support heart contraction. Examination of the mitochondrial redox state is therefore crucially important to sensitively detect early signs of mitochondrial function in pathophysiological conditions, such as ischemia, diabetes and heart failure. We study fingerprinting of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply. Here NAD(P)H is studied as a marker for non-invasive fluorescent probing of the mitochondrial function. NAD(P) H fluorescence is recorded in cardiac cells following excitation with 375nm UV-light and detection by spectrally-resolved time-correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and fluorescence lifetimes. Modulation of NADH production and/or mitochondrial respiration is tested to study dynamic characteristics of NAD(P) H fluorescence decay. Our results show that at least a 3-exponential decay model, with 0.4-0.7ns, 1.2-1.9ns and 8.0-13. Ons lifetime pools is necessary to describe cardiomyocyte autofluorescence (AF) within 420-560nm spectral range. Increased mitochondrial NADH production by ketone bodies enhanced the fluorescence intensity, without significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF intensity and shortened the average fluorescence lifetime. Dinitrophenol (DNP), an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF intensity, broadened the spectral shoulder at 520 nm and increased the average fluorescence lifetime. These effects are comparable to the study of NADH fluorescence decay in vitro. In the present contribution we demonstrated that spectrally-resolved fluorescence lifetime technique provides promising new tool for analysis of mitochondrial NAD(P) H fluorescence with good reproducibility in living cardiomyocytes. This approach will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level. In the future, this approach can prove helpful in the clinical diagnosis and treatment of mitochondrial disorder.

Induction of Mitochondrial Reactive Oxygen Species Production by Itraconazole, Terbinafine, and Amphotericin B as a Mode of Action against Aspergillus fumigatus.

PubMed

Shekhova, Elena; Kniemeyer, Olaf; Brakhage, Axel A

2017-11-01

Drug resistance in fungal pathogens is of incredible importance to global health, yet the mechanisms of drug action remain only loosely defined. Antifungal compounds have been shown to trigger the intracellular accumulation of reactive oxygen species (ROS) in human-pathogenic yeasts, but the source of those ROS remained unknown. In the present study, we examined the role of endogenous ROS for the antifungal activity of the three different antifungal substances itraconazole, terbinafine, and amphotericin B, which all target the fungal cell membrane. All three antifungals had an impact on fungal redox homeostasis by causing increased intracellular ROS production. Interestingly, the elevated ROS levels induced by antifungals were abolished by inhibition of the mitochondrial respiratory complex I with rotenone. Further, evaluation of lipid peroxidation using the thiobarbituric acid assay revealed that rotenone pretreatment decreased ROS-induced lipid peroxidation during incubation of Aspergillus fumigatus with itraconazole and terbinafine. By applying the mitochondrion-specific lipid peroxidation probe MitoPerOx, we also confirmed that ROS are induced in mitochondria and subsequently cause significant oxidation of mitochondrial membrane in the presence of terbinafine and amphotericin B. To summarize, our study suggests that the induction of ROS production contributes to the ability of antifungal compounds to inhibit fungal growth. Moreover, mitochondrial complex I is the main source of deleterious ROS production in A. fumigatus challenged with antifungal compounds. Copyright © 2017 American Society for Microbiology.

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets.

PubMed

Qureshi, Farhan M; Dejene, Eden A; Corbin, Kathryn L; Nunemaker, Craig S

2015-05-01

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca(2+)]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca(2+)]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48h to a variety of stressors: cytokines (low-grade inflammation), 28mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca(2+)]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca(2+)]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3-11mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca(2+)]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca(2+)]i using 30mM KCl+250μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3-11mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca(2+)]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca(2+)]i but not conventional insulin secretion and 'metabolic' stressors (FFAs, 28G, rotenone) impacted insulin secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mitochondrial Respiratory Function Induces Endogenous Hypoxia

PubMed Central

Prior, Sara; Kim, Ara; Yoshihara, Toshitada; Tobita, Seiji; Takeuchi, Toshiyuki; Higuchi, Masahiro

2014-01-01

Hypoxia influences many key biological functions. In cancer, it is generally believed that hypoxic condition is generated deep inside the tumor because of the lack of oxygen supply. However, consumption of oxygen by cancer should be one of the key means of regulating oxygen concentration to induce hypoxia but has not been well studied. Here, we provide direct evidence of the mitochondrial role in the induction of intracellular hypoxia. We used Acetylacetonatobis [2-(2′-benzothienyl) pyridinato-kN, kC3’] iridium (III) (BTP), a novel oxygen sensor, to detect intracellular hypoxia in living cells via microscopy. The well-differentiated cancer cell lines, LNCaP and MCF-7, showed intracellular hypoxia without exogenous hypoxia in an open environment. This may be caused by high oxygen consumption, low oxygen diffusion in water, and low oxygen incorporation to the cells. In contrast, the poorly-differentiated cancer cell lines: PC-3 and MDAMB231 exhibited intracellular normoxia by low oxygen consumption. The specific complex I inhibitor, rotenone, and the reduction of mitochondrial DNA (mtDNA) content reduced intracellular hypoxia, indicating that intracellular oxygen concentration is regulated by the consumption of oxygen by mitochondria. HIF-1α was activated in endogenously hypoxic LNCaP and the activation was dependent on mitochondrial respiratory function. Intracellular hypoxic status is regulated by glucose by parabolic dose response. The low concentration of glucose (0.045 mg/ml) induced strongest intracellular hypoxia possibly because of the Crabtree effect. Addition of FCS to the media induced intracellular hypoxia in LNCaP, and this effect was partially mimicked by an androgen analog, R1881, and inhibited by the anti-androgen, flutamide. These results indicate that mitochondrial respiratory function determines intracellular hypoxic status and may regulate oxygen-dependent biological functions. PMID:24586439

Lactate oxidation coupled to energy production in mitochondria like particles from Setaria digitata, a filarial parasite.

PubMed

Sivan, V M; Raj, R K

1994-10-14

In the filarial parasite, Setaria digitata, the mitochondria like particles (MLP) show NAD reduction with sodium lactate. The MLP also reduces dye and ferricyanide with lactate. The ferricyanide reduction by lactate is found to be sensitive to the cytochrome o inhibitor orthohydroxy diphenyl (OHD) and complex I inhibitor rotenone, modulated by ADP (+) and ATP (-) and inhibited by pyruvate and oxaloacetate. MLP shows lactate oxidation sensitive to OHD, rotenone and sodium malonate. Thus, the lactate utilizing complex system, consisting of an NADH generating MLP bound lactate dehydrogenase and a lactate flavocytochrome reductase tightly linked to complex I and cytochrome o, produces ATP in functional association with fumarate reductase complex and other enzyme systems. Hence, this study provides new dimensions to the study of metabolism in filarial parasites.

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

PubMed

Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

2017-09-01

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Coenzyme Q1 redox metabolism during passage through the rat pulmonary circulation and the effect of hyperoxia

PubMed Central

Audi, Said H.; Merker, Marilyn P.; Krenz, Gary S.; Ahuja, Taniya; Roerig, David L.; Bongard, Robert D.

2008-01-01

The objective was to evaluate the pulmonary disposition of the ubiquinone homolog coenzyme Q1 (CoQ1) on passage through lungs of normoxic (exposed to room air) and hyperoxic (exposed to 85% O2 for 48 h) rats. CoQ1 or its hydroquinone (CoQ1H2) was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of CoQ1H2 and CoQ1 were measured. CoQ1H2 appeared in the venous effluent when CoQ1 was infused, and CoQ1 appeared when CoQ1H2 was infused. In normoxic lungs, CoQ1H2 efflux rates when CoQ1 was infused decreased by 58 and 33% in the presence of rotenone (mitochondrial complex I inhibitor) and dicumarol [NAD(P)H-quinone oxidoreductase 1 (NQO1) inhibitor], respectively. Inhibitor studies also revealed that lung CoQ1H2 oxidation was via mitochondrial complex III. In hyperoxic lungs, CoQ1H2 efflux rates when CoQ1 was infused decreased by 23% compared with normoxic lungs. Based on inhibitor effects and a kinetic model, the effect of hyperoxia could be attributed predominantly to 47% decrease in the capacity of complex I-mediated CoQ1 reduction, with no change in the other redox processes. Complex I activity in lung homogenates was also lower for hyperoxic than for normoxic lungs. These studies reveal that lung complexes I and III and NQO1 play a dominant role in determining the vascular concentration and redox status of CoQ1 during passage through the pulmonary circulation, and that exposure to hyperoxia decreases the overall capacity of the lung to reduce CoQ1 to CoQ1H2 due to a depression in complex I activity. PMID:18703762

Coenzyme Q1 redox metabolism during passage through the rat pulmonary circulation and the effect of hyperoxia.

PubMed

Audi, Said H; Merker, Marilyn P; Krenz, Gary S; Ahuja, Taniya; Roerig, David L; Bongard, Robert D

2008-10-01

The objective was to evaluate the pulmonary disposition of the ubiquinone homolog coenzyme Q(1) (CoQ(1)) on passage through lungs of normoxic (exposed to room air) and hyperoxic (exposed to 85% O(2) for 48 h) rats. CoQ(1) or its hydroquinone (CoQ(1)H(2)) was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of CoQ(1)H(2) and CoQ(1) were measured. CoQ(1)H(2) appeared in the venous effluent when CoQ(1) was infused, and CoQ(1) appeared when CoQ(1)H(2) was infused. In normoxic lungs, CoQ(1)H(2) efflux rates when CoQ(1) was infused decreased by 58 and 33% in the presence of rotenone (mitochondrial complex I inhibitor) and dicumarol [NAD(P)H-quinone oxidoreductase 1 (NQO1) inhibitor], respectively. Inhibitor studies also revealed that lung CoQ(1)H(2) oxidation was via mitochondrial complex III. In hyperoxic lungs, CoQ(1)H(2) efflux rates when CoQ(1) was infused decreased by 23% compared with normoxic lungs. Based on inhibitor effects and a kinetic model, the effect of hyperoxia could be attributed predominantly to 47% decrease in the capacity of complex I-mediated CoQ(1) reduction, with no change in the other redox processes. Complex I activity in lung homogenates was also lower for hyperoxic than for normoxic lungs. These studies reveal that lung complexes I and III and NQO1 play a dominant role in determining the vascular concentration and redox status of CoQ(1) during passage through the pulmonary circulation, and that exposure to hyperoxia decreases the overall capacity of the lung to reduce CoQ(1) to CoQ(1)H(2) due to a depression in complex I activity.

Overexpression of Endogenous Anti-Oxidants with Selenium Supplementation Protects Trophoblast Cells from Reactive Oxygen Species-Induced Apoptosis in a Bcl-2-Dependent Manner.

PubMed

Khera, Alisha; Vanderlelie, Jessica J; Holland, Olivia; Perkins, Anthony V

2017-06-01

The human placenta provides life support for the developing foetus, and a healthy placenta is a prerequisite to a healthy start to life. Placental tissue is subject to oxidative stress which can lead to pathological conditions of pregnancy such as preeclampsia, preterm labour and intrauterine growth restriction. Up-regulation of endogenous anti-oxidants may alleviate placental oxidative stress and provide a therapy for these complications of pregnancy. In this study, selenium supplementation, as inorganic sodium selenite (NaSel) or organic selenomethionine (SeMet), was used to increase the protein production and cellular activity of the important redox active proteins glutathione peroxidase (GPx) and thioredoxin reductase (Thx-Red). Placental trophoblast cell lines, BeWo, JEG-3 and Swan-71, were cultured in various concentrations of NaSel or SeMet for 24 h and cell extracts prepared for western blots and enzyme assays. Rotenone and antimycin were used to stimulate mitochondrial reactive oxygen species (ROS) production and induce apoptosis. Trophoblast cells supplemented with 100 nM NaSel and 500 nM SeMet exhibited significantly enhanced expression and activity of both GPx and Thx-Red. Antimycin and rotenone were found to generate ROS when measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, and selenium supplementation was shown to reduce ROS production in a dose-dependent manner. Rotenone, 100 μM treatment for 4 h, caused trophoblast cell apoptosis as evidenced by increased Annexin V binding and decreased expression of Bcl-2. In both assays of apoptosis, selenium supplementation was able to prevent apoptosis, preserve Bcl-2 expression and protect trophoblast cells from mitochondrial oxidative stress. This data suggests that selenoproteins such as GPx and Thx-Red have an important role in protecting trophoblast cells from mitochondrial oxidative stress and that selenium supplementation may be important in treating some placental pathologies.

Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

PubMed

Kim, Chiho; Lee, Juhyung; Ko, Yeon Uk; Oh, Young J

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIP S20A , but not CHIP WT , attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Sonic hedgehog pathway activation increases mitochondrial abundance and activity in hippocampal neurons

PubMed Central

Yao, Pamela J.; Manor, Uri; Petralia, Ronald S.; Brose, Rebecca D.; Wu, Ryan T. Y.; Ott, Carolyn; Wang, Ya-Xian; Charnoff, Ari; Lippincott-Schwartz, Jennifer; Mattson, Mark P.

2017-01-01

Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. We present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway affects multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense, as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons. PMID:27932496

Coenzyme Q(1) as a probe for mitochondrial complex I activity in the intact perfused hyperoxia-exposed wild-type and Nqo1-null mouse lung.

PubMed

Bongard, Robert D; Myers, Charles R; Lindemer, Brian J; Baumgardt, Shelley; Gonzalez, Frank J; Merker, Marilyn P

2012-05-01

Previous studies showed that coenzyme Q(1) (CoQ(1)) reduction on passage through the rat pulmonary circulation was catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex I, but that NQO1 genotype was not a factor in CoQ(1) reduction on passage through the mouse lung. The aim of the present study was to evaluate the complex I contribution to CoQ(1) reduction in the isolated perfused wild-type (NQO1(+/+)) and Nqo1-null (NQO1(-)/(-)) mouse lung. CoQ(1) reduction was measured as the steady-state pulmonary venous CoQ(1) hydroquinone (CoQ(1)H(2)) efflux rate during infusion of CoQ(1) into the pulmonary arterial inflow. CoQ(1)H(2) efflux rates during infusion of 50 μM CoQ(1) were not significantly different for NQO1(+/+) and NQO1(-/-) lungs (0.80 ± 0.03 and 0.68 ± 0.07 μmol·min(-1)·g lung dry wt(-1), respectively, P > 0.05). The mitochondrial complex I inhibitor rotenone depressed CoQ(1)H(2) efflux rates for both genotypes (0.19 ± 0.08 and 0.08 ± 0.04 μmol·min(-1)·g lung dry wt(-1) for NQO1(+/+) and NQO1(-/-), respectively, P < 0.05). Exposure of mice to 100% O(2) for 48 h also depressed CoQ(1)H(2) efflux rates in NQO1(+/+) and NQO1(-/-) lungs (0.43 ± 0.03 and 0.11 ± 0.04 μmol·min(-1)·g lung dry wt(-1), respectively, P < 0.05 by ANOVA). The impact of rotenone or hyperoxia on CoQ(1) redox metabolism could not be attributed to effects on lung wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total venous effluent CoQ(1) recoveries, the latter measured by spectrophotometry or mass spectrometry. Complex I activity in mitochondria-enriched lung fractions was depressed in hyperoxia-exposed lungs for both genotypes. This study provides new evidence for the potential utility of CoQ(1) as a nondestructive indicator of the impact of pharmacological or pathological exposures on complex I activity in the intact perfused mouse lung.

Nitric oxide protects the mitochondria of anterior pituitary cells and prevents cadmium-induced cell death by reducing oxidative stress.

PubMed

Poliandri, Ariel H B; Machiavelli, Leticia I; Quinteros, Alnilan F; Cabilla, Jimena P; Duvilanski, Beatriz H

2006-02-15

Cadmium (Cd2+) is a highly toxic metal that affects the endocrine system. We have previously shown that Cd2+ induces caspase-3 activation and apoptosis of anterior pituitary cells and that endogenous nitric oxide (NO) protects these cells from Cd2+. Here we investigate the mechanisms by which NO exerts this protective role. Cd2+ (25 microM) reduced the mitochondrial membrane potential (MMP) as measured by flow cytometry. Cd2+-induced apoptosis was mitochondrial dependent since cyclosporin A protected the cells from this metal. Inhibition of NO synthesis with 0.5 mM L-NAME increased the effect of Cd2+ on MMP, whereas the NO donor DETANONOate (0.1 mM) reduced it. Cd2+ increased the production of reactive oxygen species (ROS) as measured by flow cytometry. This effect was electron-transfer-chain-dependent since it was inhibited by rotenone. In fact, rotenone reduced the cytotoxic effect of the metal. The action of Cd2+ on mitochondrial integrity was ROS dependent. Trolox, an antioxidant, inhibited the effect of the metal on the MMP. Cd2+-induced increase in ROS generation was reduced by DETANONOate. There are discrepancies concerning the role of NO in Cd2+ toxicity. Here we show that NO reduces Cd2+ toxicity by protecting the mitochondria from oxidative stress in a system where NO plays a regulatory role.

Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells

PubMed Central

Bacsi, Attila; Woodberry, Mitchell; Widger, William; Papaconstantinou, John; Mitra, Sankar; Peterson, Johnny W.; Boldogh, Istvan

2011-01-01

3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2•− generation in mitochondria respiring on the complex I substrates pyruvate + malate, an effect fully inhibited by rotenone. Antimycin A increased O2•− production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2•− production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2•− formation driven with the complex II substrate succinate. At 0.6 μM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2•− formation; however, at 40 μM myxothiazol (which completely inhibits both complexes I and III) eliminated O2•− production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2•− from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II. PMID:17011837

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

PubMed Central

Parry, Robert C.; Wang, Qingqing; Gillespie, Kevin P.; Saillant, Noelle N.; Sims, Carrie; Mesaros, Clementina; Snyder, Nathaniel W.; Blair, Ian A.

2016-01-01

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism. PMID:27077278

Chrysotoxine, a novel bibenzyl compound selectively antagonizes MPP⁺, but not rotenone, neurotoxicity in dopaminergic SH-SY5Y cells.

PubMed

Song, Ju-Xian; Shaw, Pang-Chui; Wong, Ngok-Shun; Sze, Cho-Wing; Yao, Xin-Sheng; Tang, Chi-Wai; Tong, Yao; Zhang, Yan-Bo

2012-07-11

Chrysotoxine is a naturally occurring bibenzyl compound found in medicinal Dendrobium species. We previously reported that chrysotoxine structure-specifically suppressed 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell death. Whether chrysotoxine and other structurally similar bibenzyl compounds could also inhibit the neurotoxicity of 1-methyl-4-phenyl pyridinium (MPP(+)) and rotenone has not been investigated. We showed herein that chrysotoxine inhibited MPP(+), but not rotenone, induced dopaminergic cell death in SH-SY5Y cells. The overproduction of reactive oxygen species (ROS), mitochondrial dysfunction as indexed by the decrease in membrane potential, increase in calcium concentration and NF-κB activation triggered by MPP(+) were blocked by chrysotoxine pretreatment. The imbalance between the pro-apoptotic signals (Bax, caspase-3, ERK and p38 MAPK) and the pro-survival signals (Akt/PI3K/GSK-3β) induced by MPP(+) was partially or totally rectified by chrysotoxine. The results indicated that ROS inhibition, mitochondria protection, NF-κB modulation and regulation of multiple signals determining cell survival and cell death were involved in the protective effects of chrysotoxine against MPP(+) toxicity in SH-SY5Y cells. Given the different toxic profiles of 6-OHDA and MPP(+) as compared to rotenone, our results also indicated that DAT inhibition may partially account for the neuroprotective effects of chrysotoxine. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Neuroprotective efficacy of Bacopa monnieri against rotenone induced oxidative stress and neurotoxicity in Drosophila melanogaster.

PubMed

Hosamani, Ravikumar; Muralidhara

2009-11-01

Bacopa monnieri, Linn. (Brahmi, BM), traditionally used to improve mental health in Indian ayurvedic system of medicine is known to possess various neuropharmacolgical properties. In the recent past, Drosophila has been widely used as a model to study various neurodegenerative diseases. Environmental toxins like rotenone, a specific inhibitor of complex I is employed to increase oxidative stress mediated neuropathology and sporadic Parkinson's disease. In this study, we examined the neuroprotective properties of BM against rotenone induced oxidative damage and neurotoxicity. Flies (Oregon K strain, adult males) exposed to a standardized BM powder for 7 days in the diet exhibited significant diminution in the levels of endogenous oxidative markers viz., malondialdehyde, hydroperoxide and protein carbonyl content. Further, BM offered complete protection against rotenone (500 microM) induced oxidative stress and markedly inhibited dopamine depletion (head region, 33%; body region, 44%) in flies. Flies exposed to rotenone+BM exhibited a lower incidence of mortality (40-66% protection) and performed better in a negative geotaxis assay (45-65%) both suggesting the neuroprotective potential of BM. Interestingly, BM also conferred significant resistance (43-54% protection) in a paraquat oxidative stress bioassay. The neuroprotective effects of BM were highly comparable to those of a commercially available Brahmi preparation. Although the precise mechanism/s underlying the neuroprotective efficacy of BM are not clear, it is hypothesized that it is wholly or in part related to its ability to mitigate rotenone induced oxidative stress. Further, our approach confirms the utility of the Drosophila model in screening putative neuroprotective phytomedicines prior to their use in mammalian models.

Astrocyte-specific DJ-1 overexpression protects against rotenone-induced neurotoxicity in a rat model of Parkinson's disease.

PubMed

De Miranda, Briana R; Rocha, Emily M; Bai, Qing; El Ayadi, Amina; Hinkle, David; Burton, Edward A; Timothy Greenamyre, J

2018-07-01

DJ-1 is a redox-sensitive protein with several putative functions important in mitochondrial physiology, protein transcription, proteasome regulation, and chaperone activity. High levels of DJ-1 immunoreactivity are reported in astrocytes surrounding pathology associated with idiopathic Parkinson's disease, possibly reflecting the glial response to oxidative damage. Previous studies showed that astrocytic over-expression of DJ-1 in vitro prevented oxidative stress and mitochondrial dysfunction in primary neurons. Based on these observations, we developed a pseudotyped lentiviral gene transfer vector with specific tropism for CNS astrocytes in vivo to overexpress human DJ-1 protein in astroglial cells. Following vector delivery to the substantia nigra and striatum of adult Lewis rats, the DJ-1 transgene was expressed robustly and specifically within astrocytes. There was no observable transgene expression in neurons or other glial cell types. Three weeks after vector infusion, animals were exposed to rotenone to induce Parkinson's disease-like pathology, including loss of dopaminergic neurons, accumulation of endogenous α-synuclein, and neuroinflammation. Animals over-expressing hDJ-1 in astrocytes were protected from rotenone-induced neurodegeneration, and displayed a marked reduction in neuronal oxidative stress and microglial activation. In addition, α-synuclein accumulation and phosphorylation were decreased within substantia nigra dopaminergic neurons in DJ-1-transduced animals, and expression of LAMP-2A, a marker of chaperone mediated autophagy, was increased. Together, these data indicate that astrocyte-specific overexpression of hDJ-1 protects neighboring neurons against multiple pathologic features of Parkinson's disease and provides the first direct evidence in vivo of a cell non-autonomous neuroprotective function of astroglial DJ-1. Copyright © 2018 Elsevier Inc. All rights reserved.

Effectiveness of γ-oryzanol in reducing neuromotor deficits, dopamine depletion and oxidative stress in a Drosophila melanogaster model of Parkinson's disease induced by rotenone.

PubMed

Araujo, Stífani Machado; de Paula, Mariane Trindade; Poetini, Marcia Rósula; Meichtry, Luana; Bortolotto, Vandreza Cardoso; Zarzecki, Micheli Stefani; Jesse, Cristiano Ricardo; Prigol, Marina

2015-12-01

The γ-orizanol present in rice bran oil contains a mix of steryl triterpenyl esters of ferulic acid, which is believed to be linked to its antioxidant potential. In this study we investigated the neuroprotective actions of γ-orizanol (ORY) against the toxicity induced by rotenone (ROT) in Drosophila melanogaster. The flies (both genders) aged between 1 and 5 days old were divided into four groups of 50 flies each: (1) control, (2) ORY 25 μM, (3) ROT 500 μM, (4) ORY 25 μM+ROT 500 μM. Flies were concomitantly exposed to a diet containing ROT and ORY for 7 days according to their respective groups. Survival and behavior analyses were carried out in vivo, and ex vivo analyses involved acetylcholinesterase activity (AChE), determination of dopaminergic levels, cellular viability and mitochondrial viability, activities of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), reactive species levels (RS), lipid peroxidation (TBARS) and contents of total thiols and non-proteic thiols (NPSH). Our results show for the first time that ORY not only acts as an endogenous activator of the cellular antioxidant defenses, but it also ameliorates rotenone induced mortality, oxidative stress and mitochondrial dysfunction. Our salient findings regarded the restoration of cholinergic deficits, dopamine levels and improved motor function provided by ORY. These results demonstrate the neuroprotective potential of ORY and that this effect can be potentially due to its antioxidant action. In conclusion, the present results show that ORY is effective in reducing the ROT induced toxicity in D. melanogaster, which showed a neuroprotective action, possibly due to the presence of the antioxidant constituents such as the ferulic acid. Copyright © 2015. Published by Elsevier B.V.

Homeostasis of the protonmotive force in phosphorylating mitochondria.

PubMed

Duszyński, J; Bogucka, K; Wojtczak, L

1984-12-18

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (delta mu H+) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and delta mu H+ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of delta mu H+, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on delta mu H+; for example, the excess of oligomycin produced a 90% inhibition of the respiration while delta mu H+ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing delta mu H+ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of delta mu H+, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and delta mu H+.

Human Atg8-cardiolipin interactions in mitophagy: Specific properties of LC3B, GABARAPL2 and GABARAP.

PubMed

Antón, Zuriñe; Landajuela, Ane; Hervás, Javier H; Montes, L Ruth; Hernández-Tiedra, Sonia; Velasco, Guillermo; Goñi, Felix M; Alonso, Alicia

2016-12-01

The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria. We have applied quantitative biophysical techniques to the study of CL interaction with various Atg8 human orthologs, namely LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts preferentially with CL over other di-anionic lipids, that CL-LC3B binding occurs with positive cooperativity, and that the CL-LC3B interaction relies only partially on electrostatic forces. CL-induced increased membrane fluidity appears also as an important factor helping LC3B to bind CL. The LC3B C terminus remains exposed to the hydrophilic environment after protein binding to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria and subsequent delivery of mitochondria to lysosomes. We have also observed that GABARAP, but not GABARAPL2, interacts with CL in vitro. However neither GABARAP nor GABARAPL2 were translocated to mitochondria in rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might play specific roles in different autophagic processes.

Flavonoid-induced glutathione depletion: Potential implications for cancer treatment☆

PubMed Central

Kachadourian, Remy; Day, Brian J.

2014-01-01

The ability of a number of flavonoids to induce glutathione (GSH) depletion was measured in lung (A549), myeloid (HL-60), and prostate (PC-3) human tumor cells. The hydroxychalcone (2′-HC) and the dihydroxychalcones (2′,2-, 2′,3-, 2′,4-, and 2′,5′-DHC) were the most effective in A549 and HL-60 cells, depleting more than 50% of intracellular GSH within 4 h of exposure at 25 µM. In contrast, the flavones chrysin and apigenin were the most effective in PC-3 cells, depleting 50–70% of intracellular GSH within 24 h of exposure at 25 µM. In general, these flavonoids were more effective than three classical substrates of multidrug resistance protein 1 (MK-571, indomethacin, and verapamil). Prototypic flavonoids (2′,5′-DHC and chrysin) were subsequently tested for their abilities to potentiate the toxicities of prooxidants (etoposide, rotenone, 2-methoxyestradiol, and curcumin). In A549 cells, 2′,5′-DHC potentiated the cytotoxicities of rotenone, 2-methoxyestradiol, and curcumin, but not etoposide. In HL-60 and PC-3 cells, chrysin potentiated the cytotoxicity of curcumin, cytotoxicity that was attenuated by the catalytic antioxidant manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP). Assessments of mitochondrial GSH levels mitochondrial membrane potential and cytochrome c release showed that the potentiation effects induced by 2′,5′-DHC and chrysin involve mitochondrial dysfunction. PMID:16781454

Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

NASA Technical Reports Server (NTRS)

Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

1999-01-01

Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

Effects of Commonly Used Pesticides in China on the Mitochondria and Ubiquitin-Proteasome System in Parkinson’s Disease

PubMed Central

Chen, Tingting; Tan, Jieqiong; Wan, Zhengqing; Zou, Yongyi; Kessete Afewerky, Henok; Zhang, Zhuohua

2017-01-01

Evidence continues to accumulate that pesticides are the leading candidates of environmental toxins that may contribute to the pathogenesis of Parkinson’s disease. The mechanisms, however, remain largely unclear. According to epidemiological studies, we selected nine representative pesticides (paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate, tebufenpyrad, trichlorphon and carbaryl) which are commonly used in China and detected the effects of the pesticides on mitochondria and ubiquitin-proteasome system (UPS) function. Our results reveal that all the nine studied pesticides induce morphological changes of mitochondria at low concentrations. Paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate and tebufenpyrad induced mitochondria fragmentation. Furthermore, some of them (paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad) caused a significant dose-dependent decrease of intracellular ATP. Interestingly, these pesticides which induce mitochondria dysfunction also inhibit 26S and 20S proteasome activity. However, two out of the nine pesticides, namely trichlorphon and carbaryl, were found not to cause mitochondrial fragmentation or functional damage, nor inhibit the activity of the proteasome, which provides significant guidance for selection of pesticides in China. Moreover, our results demonstrate a potential link between inhibition of mitochondria and the UPS, and pesticide-induced Parkinsonism. PMID:29168786

Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Babu, Dinesh, E-mail: dinesh.babu@ugent.be; Leclercq, Georges; Goossens, Vera

2015-10-15

Targeting excessive production of reactive oxygen species (ROS) could be an effective therapeutic strategy to prevent oxidative stress-associated gastrointestinal inflammation. NADPH oxidase (NOX) and mitochondrial complexes (I and II) are the major sources of ROS production contributing to TNF-α/cycloheximide (CHX)-induced apoptosis in the mouse intestinal epithelial cell line, MODE-K. In the current study, the influence of a polyphenolic compound (resveratrol) and a water-soluble carbon monoxide (CO)-releasing molecule (CORM-A1) on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells was assessed. This was compared with H{sub 2}O{sub 2}-, rotenone- or antimycin-A-induced ROS-generating systems. Intracellular total ROS, mitochondrial-derived ROS and mitochondrialmore » superoxide anion (O{sub 2}·{sup −}) production levels were assessed. Additionally, the influence on TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψ{sub m}) and mitochondrial function was studied. In basal conditions, CORM-A1 did not affect intracellular total or mitochondrial ROS levels, while resveratrol increased intracellular total ROS but reduced mitochondrial ROS production. TNF-α/CHX- and H{sub 2}O{sub 2}-mediated increase in intracellular total ROS production was reduced by both resveratrol and CORM-A1, whereas only resveratrol attenuated the increase in mitochondrial ROS triggered by TNF-α/CHX. CORM-A1 decreased antimycin-A-induced mitochondrial O{sub 2}·{sup −} production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O{sub 2}·{sup −} levels, while resveratrol abolished all three effects. Finally, resveratrol greatly reduced and abolished TNF-α/CHX-induced mitochondrial depolarization and mitochondrial dysfunction, while CORM-A1 only mildly affected these parameters. These data indicate that the cytoprotective effect of resveratrol is predominantly due to mitigation of mitochondrial ROS, while CORM-A1 acts solely on NOX-derived ROS to protect MODE-K cells from TNF-α/CHX-induced cell death. This might explain the more pronounced cytoprotective effect of resveratrol. - Highlights: • In MODE-K IECs, TNF-α/CHX induces correlating ROS, mitochondrial O{sub 2}·{sup −} and cell death. • CORM-A1 does not influence basal intracellular ROS and mitochondrial O{sub 2}·{sup −} levels. • Resveratrol increases basal intracellular ROS but decreases mitochondrial O{sub 2}·{sup −} levels. • CORM-A1 acts solely on NOX-derived ROS to protect from cell death by TNF-α/CHX. • Cytoprotection by resveratrol is predominantly due to reduction of mitochondrial O{sub 2}·{sup −}.« less

Glucose deprivation increases monocarboxylate transporter 1 (MCT1) expression and MCT1-dependent tumor cell migration.

PubMed

De Saedeleer, C J; Porporato, P E; Copetti, T; Pérez-Escuredo, J; Payen, V L; Brisson, L; Feron, O; Sonveaux, P

2014-07-31

The glycolytic end-product lactate is a pleiotropic tumor growth-promoting factor. Its activities primarily depend on its uptake, a process facilitated by the lactate-proton symporter monocarboxylate transporter 1 (MCT1). Therefore, targeting the transporter or its chaperon protein CD147/basigin, itself involved in the aggressive malignant phenotype, is an attractive therapeutic option for cancer, but basic information is still lacking regarding the regulation of the expression, interaction and activities of both proteins. In this study, we found that glucose deprivation dose-dependently upregulates MCT1 and CD147 protein expression and their interaction in oxidative tumor cells. While this posttranslational induction could be recapitulated using glycolysis inhibition, hypoxia, oxidative phosphorylation (OXPHOS) inhibitor rotenone or hydrogen peroxide, it was blocked with alternative oxidative substrates and specific antioxidants, pointing out at a mitochondrial control. Indeed, we found that the stabilization of MCT1 and CD147 proteins upon glucose removal depends on mitochondrial impairment and the associated generation of reactive oxygen species. When glucose was a limited resource (a situation occurring naturally or during the treatment of many tumors), MCT1-CD147 heterocomplexes accumulated, including in cell protrusions of the plasma membrane. It endowed oxidative tumor cells with increased migratory capacities towards glucose. Migration increased in cells overexpressing MCT1 and CD147, but it was inhibited in glucose-starved cells provided with an alternative oxidative fuel, treated with an antioxidant, lacking MCT1 expression, or submitted to pharmacological MCT1 inhibition. While our study identifies the mitochondrion as a glucose sensor promoting tumor cell migration, MCT1 is also revealed as a transducer of this response, providing a new rationale for the use of MCT1 inhibitors in cancer.

A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preta, Giulio; Klark, Rainier de; Glas, Rickard, E-mail: rickard.glas@ki.se

2009-11-27

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to {gamma}-irradiation, and that nuclear expression of TPPII was present in most {gamma}-irradiated transformed cell lines. We used amore » panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after {gamma}-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following {gamma}-irradiation (at 1-4 h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in {gamma}-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.« less

A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses.

PubMed

Preta, Giulio; de Klark, Rainier; Glas, Rickard

2009-11-27

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to gamma-irradiation, and that nuclear expression of TPPII was present in most gamma-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after gamma-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following gamma-irradiation (at 1-4h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in gamma-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.

Physical Fitness and Mitochondrial Respiratory Capacity in Horse Skeletal Muscle

PubMed Central

Lemieux, Hélène; Mouithys-Mickalad, Ange; Serteyn, Didier

2012-01-01

Background Within the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general. Methodology/Principal Findings We applied high-resolution respirometry and multiple substrate-uncoupler-inhibitor titration protocols to study mitochondrial physiology in small (1.0–2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses. Oxidative phosphorylation (OXPHOS) capacity (pmol O2•s−1•mg−1 wet weight) with combined Complex I and II (CI+II) substrate supply (malate+glutamate+succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained and competitive horses (N = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 (N = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the CI+II substrate cocktail. Relative to this maximum OXPHOS capacity, Complex I (CI)-linked OXPHOS capacities were only 50% with glutamate+malate, 64% with pyruvate+malate, and 68% with pyruvate+malate+glutamate, and ∼78% with CII-linked succinate+rotenone. OXPHOS capacity with glutamate+malate increased with fitness relative to CI+II-supported ETS capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the CII/CI+II substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the CI- over CII-linked pathway capacity was reduced with physical fitness. Conclusions/Significance The scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of CI-linked respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology. PMID:22529950

Dermal fibroblasts from long-lived Ames dwarf mice maintain their in vivo resistance to mitochondrial generated reactive oxygen species (ROS)

PubMed Central

Hsieh, Ching-Chyuan; Papaconstantinou, John

2009-01-01

Activation of p38 MAPK by ROS involves dissociation of an inactive, reduced thioredoxin-ASK1 complex [(SH)2Trx-ASK1]. Release of ASK1 activates its kinase activity thus stimulating the p38 MAPK pathway. The level of p38 MAPK activity is, therefore, regulated by the balance of free vs. bound ASK1. Longevity of Ames dwarf mice is attributed to their resistance to oxidative stress. The levels of (SH)2 Trx-ASK1 are more abundant in young and old dwarf mice compared to their age-matched controls suggesting that the levels of this complex may play a role in their resistance to oxidative stress. In these studies we demonstrate that dermal fibroblasts from these long-lived mice exhibit (a) higher levels of (SH)2Trx-ASK1 that correlate with their resistance to ROS generated by inhibitors of electron transport chain complexes CI (rotenone), CII (3-nitropropionic acid), CIII, (antimycin A), and H2O2-mediated activation of p38 MAPK, and (b) maintain their in vivo resistance to ROS generated by 3NPA. We propose that elevated levels of (SH)2Trx-ASK1 play a role in conferring resistance to mitochondrial generated oxidative stress and decreased endogenous ROS which are characteristics of longevity determination. PMID:20157567

A Comparative Proteome Profile of Female Mouse Gonads Suggests a Tight Link between the Electron Transport Chain and Meiosis Initiation.

PubMed

Shen, Cong; Li, Mingrui; Zhang, Pan; Guo, Yueshuai; Zhang, Hao; Zheng, Bo; Teng, Hui; Zhou, Tao; Guo, Xuejiang; Huo, Ran

2018-01-01

Generation of haploid gametes by meiosis is a unique property of germ cells and is critical for sexual reproduction. Leaving mitosis and entering meiosis is a key step in germ cell development. Several inducers or intrinsic genes are known to be important for meiotic initiation, but the regulation of meiotic initiation, especially at the protein level, is still not well understood. We constructed a comparative proteome profile of female mouse fetal gonads at specific time points (11.5, 12.5, and 13.5 days post coitum), spanning a critical window for initiation of meiosis in female germ cells. We identified 3666 proteins, of which 473 were differentially expressed. Further bioinformatics analysis showed that these differentially expressed proteins were enriched in the mitochondria, especially in the electron transport chain and, notably, 9 proteins in electron transport chain Complex I were differentially expressed. We disrupted the mitochondrial electron transport chain function by adding the complex I inhibitor, rotenone to 11.5 days post coitum female gonads cultured in vitro. This treatment resulted in a decreased proportion of meiotic germ cells, as assessed by staining for histone γH2AX. Rotenone treatment also caused decreased ATP levels, increased reactive oxygen species levels and failure of the germ cells to undergo premeiotic DNA replication. These effects were partially rescued by adding Coenzyme Q10. Taken together, our results suggested that a functional electron transport chain is important for meiosis initiation. Our characterization of the quantitative proteome of female gonads provides an inventory of proteins, useful for understanding the mechanisms of meiosis initiation and female fertility. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Measurement of Mitochondrial Mass by Flow Cytometry during Oxidative Stress.

PubMed

Doherty, Edward; Perl, Andras

2017-07-01

Properly assessing mitochondrial health is crucial to understand their role in disease. MitoTracker green (MTG) and nonylacridine orange (NAO) are fluorescent probes which have been commonly used to assess mitochondrial mass. This is based on the assumption that both MTG and NAO accumulate in mitochondria regardless of the mitochondrial transmembrane potential (ΔΨ m ). Here, we utilized flow cytometry to evaluate the performance of these probes for assessment of mitochondrial mass relative to forward (FSC) and side scatter (SSC) in human peripheral blood lymphocytes (PBL). In isolated mitochondria, two subpopulations were identified by FSC and SSC measurements which were matched to subpopulations stained by MTG and NAO. The performance of these dyes was examined under oxidative and nitrosative stress induced by rotenone and NOC-18 while N -acetylcysteine (NAC) was employed as an antioxidant. Production of reactive oxygen species (ROS) and ΔΨ m were monitored in parallel. With respect to representation of mitochondrial mass, neither MTG nor NAO was affected by ΔΨ m . However, MTG showed significant correlation with cytosolic and mitochondrial ROS production and nitrosative stress. Our data suggest that NAO may be more suitable than MTG for assessment of mitochondrial mass by flow cytometry during oxidative stress.

Evaluation of the rotenone-induced activation of the Nrf2 pathway in a neuronal model derived from human induced pluripotent stem cells.

PubMed

Zagoura, Dimitra; Canovas-Jorda, David; Pistollato, Francesca; Bremer-Hoffmann, Susanne; Bal-Price, Anna

2017-06-01

Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology, including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore, in the current study, we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM), as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes, NQO1 and SRXN1. Interestingly, exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover, rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH + ) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Long-term microfluidic glucose and lactate monitoring in hepatic cell culture

PubMed Central

Prill, Sebastian; Jaeger, Magnus S.; Duschl, Claus

2014-01-01

Monitoring cellular bioenergetic pathways provides the basis for a detailed understanding of the physiological state of a cell culture. Therefore, it is widely used as a tool amongst others in the field of in vitro toxicology. The resulting metabolic information allows for performing in vitro toxicology assays for assessing drug-induced toxicity. In this study, we demonstrate the value of a microsystem for the fully automated detection of drug-induced changes in cellular viability by continuous monitoring of the metabolic activity over several days. To this end, glucose consumption and lactate secretion of a hepatic tumor cell line were continuously measured using microfluidically addressed electrochemical sensors. Adapting enzyme-based electrochemical flat-plate sensors, originally designed for human whole-blood samples, to their use with cell culture medium supersedes the common manual and laborious colorimetric assays and off-line operated external measurement systems. The cells were exposed to different concentrations of the mitochondrial inhibitor rotenone and the cellular response was analyzed by detecting changes in the rates of the glucose and lactate metabolism. Thus, the system provides real-time information on drug-induced liver injury in vitro. PMID:24926387

Identification of small molecules that improve ATP synthesis defects conferred by Leber’s hereditary optic neuropathy mutations

PubMed Central

Datta, Sandipan; Tomilov, Alexey; Cortopassi, Gino

2016-01-01

Inherited mitochondrial complex I mutations cause blinding Leber's hereditary Optic Neuropathy (LHON), for which no curative therapy exists. A specific biochemical consequence of LHON mutations in the presence of trace rotenone was observed: deficient complex I-dependent ATP synthesis (CIDAS) and mitochondrial O2 consumption, proportional to the clinical severity of the three primary LHON mutations. We optimized a high-throughput assay of CIDAS to screen 1600 drugs to 2, papaverine and zolpidem, which protected CIDAS in LHON cells concentration-dependently. TSPO and cAMP were investigated as protective mechanisms, but a conclusive mechanism remains to be elucidated; next steps include testing in animal models. PMID:27497748

Real-time monitoring of metabolic function in liver-on-chip microdevices tracks the dynamics of mitochondrial dysfunction

PubMed Central

Bavli, Danny; Prill, Sebastian; Ezra, Elishai; Levy, Gahl; Cohen, Merav; Vinken, Mathieu; Vanfleteren, Jan; Jaeger, Magnus; Nahmias, Yaakov

2016-01-01

Microfluidic organ-on-a-chip technology aims to replace animal toxicity testing, but thus far has demonstrated few advantages over traditional methods. Mitochondrial dysfunction plays a critical role in the development of chemical and pharmaceutical toxicity, as well as pluripotency and disease processes. However, current methods to evaluate mitochondrial activity still rely on end-point assays, resulting in limited kinetic and prognostic information. Here, we present a liver-on-chip device capable of maintaining human tissue for over a month in vitro under physiological conditions. Mitochondrial respiration was monitored in real time using two-frequency phase modulation of tissue-embedded phosphorescent microprobes. A computer-controlled microfluidic switchboard allowed contiguous electrochemical measurements of glucose and lactate, providing real-time analysis of minute shifts from oxidative phosphorylation to anaerobic glycolysis, an early indication of mitochondrial stress. We quantify the dynamics of cellular adaptation to mitochondrial damage and the resulting redistribution of ATP production during rotenone-induced mitochondrial dysfunction and troglitazone (Rezulin)-induced mitochondrial stress. We show troglitazone shifts metabolic fluxes at concentrations previously regarded as safe, suggesting a mechanism for its observed idiosyncratic effect. Our microfluidic platform reveals the dynamics and strategies of cellular adaptation to mitochondrial damage, a unique advantage of organ-on-chip technology. PMID:27044092

Neuroprotective Effects of Protein Tyrosine Phosphatase 1B Inhibition against ER Stress-Induced Toxicity

PubMed Central

Jeon, Yu-Mi; Lee, Shinrye; Kim, Seyeon; Kwon, Younghwi; Kim, Kiyoung; Chung, Chang Geon; Lee, Seongsoo; Lee, Sung Bae; Kim, Hyung-Jun

2017-01-01

Several lines of evidence suggest that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Protein tyrosine phosphatase 1B (PTP1B) is known to regulate the ER stress signaling pathway, but its role in neuronal systems in terms of ER stress remains largely unknown. Here, we showed that rotenone-induced toxicity in human neuroblastoma cell lines and mouse primary cortical neurons was ameliorated by PTP1B inhibition. Moreover, the increase in the level of ER stress markers (eIF2α phosphorylation and PERK phosphorylation) induced by rotenone treatment was obviously suppressed by concomitant PTP1B inhibition. However, the rotenone-induced production of reactive oxygen species (ROS) was not affected by PTP1B inhibition, suggesting that the neuroprotective effect of the PTP1B inhibitor is not associated with ROS production. Moreover, we found that MG132-induced toxicity involving proteasome inhibition was also ameliorated by PTP1B inhibition in a human neuroblastoma cell line and mouse primary cortical neurons. Consistently, downregulation of the PTP1B homologue gene in Drosophila mitigated rotenone- and MG132-induced toxicity. Taken together, these findings indicate that PTP1B inhibition may represent a novel therapeutic approach for ER stress-mediated neurodegenerative diseases. PMID:28359145

The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.

PubMed

Halestrap, A P

1975-04-01

1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of pyruvate transport at higher temperatures to be made. The activation energy of mitochondrial pyruvate transport was found to be 113 kJ (27 kcal)/mol and by extrapolation the rate of transport of pyruvate at 37 degrees C to be 42 nmol/min per mg of protein. The possibility that pyruvate transport into mitochondria may be rate limiting and involved in the regulation of gluconegenesis is discussed. 6. The transport of various monocarboxylic acids into mitochondria was studied by monitoring proton influx. The transport of dichloroacetate, difluoroacetate and oxamate appeared to be largely dependent on the pyruvate carrier and could be inhibited by pyruvate-transport inhibitors. However, many other halogenated and 2-oxo acids which could exchange with pyruvate on the carrier entered freely even in the presence of inhibitor.

Critical role of mitochondrial ROS is dependent on their site of production on the electron transport chain in ischemic heart.

PubMed

Madungwe, Ngonidzashe B; Zilberstein, Netanel F; Feng, Yansheng; Bopassa, Jean C

2016-01-01

Reactive oxygen species (ROS) generation has been implicated in many pathologies including ischemia/reperfusion (I/R) injury. This led to multiple studies on antioxidant therapies to treat cardiovascular diseases but paradoxically, results have so far been mixed as ROS production can be beneficial as a signaling mechanism and in cardiac protection via preconditioning interventions. We investigated whether the differential impact of increased ROS in injury as well as in protection could be explained by their site of production on the mitochondrial electron transport chain. Using amplex red to measure ROS production, we found that mitochondria isolated from hearts after I/R produced more ROS than non-ischemic when complex I substrate (glutamate/malate) was used. Interestingly, the substrates of complex II (succinate) and ubiquinone (sn-glycerol 3-phosphate, G3P) produced less ROS in mitochondria from I/R hearts compared to normal healthy hearts. The inhibitors of complex I (rotenone) and complex III (antimycin A) increased ROS production when glutamate/malate and G3P were used; in contrast, they reduced ROS production when the complex II substrate was used. Mitochondrial calcium retention capacity required to induce mitochondrial permeability transition pore (mPTP) opening was measured using calcium green fluorescence and was found to be higher when mitochondria were treated with G3P and succinate compared to glutamate/malate. Furthermore, Langendorff hearts treated with glutamate/malate exhibited reduced cardiac functional recovery and increased myocardial infarct size compared to hearts treated with G3P. Thus, ROS production by the stimulated respiratory chain complexes I and III has opposite roles: cardio-deleterious when produced in complex I and cardio-protective when produced in complex III. The mechanism of these ROS involves the inhibition of the mPTP opening, a key event in cell death following ischemia/reperfusion injury.

Biomolecular characterization of exosomes released from cancer stem cells: Possible implications for biomarker and treatment of cancer.

PubMed

Kumar, Dhruv; Gupta, Dwijendra; Shankar, Sharmila; Srivastava, Rakesh K

2015-02-20

Cancer recognized as one of the leading irrepressible health issues is contributing to increasing mortality-rate day-by-day. The tumor microenvironment is an important field of cancer to understand the detection, treatment and prevention of cancer. Recently, cancer stem cell (CSC) research has shown promising results aiming towards cancer diagnostics and treatment. Here, we found that prostate and breast cancer stem cells secreted vesicles of endosomal origin, called exosomes showed strong connection between autophagy and exosomes released from CSCs. Exosomes may serve as vesicles to communicate with neoplastic cells (autocrine and paracrine manner) and normal cells (paracrine and endocrine manner) and thereby suppress immune systems and regulate neoplastic growth, and metastasis. They can also be used as biomarkers for various cancers. We detected tetraspanin proteins (CD9, CD63, CD81), Alix and tumor susceptibility gene-101 (TSG101) of exosomal markers from rotenone treated CSCs. We have also detected the induction of autophagy genes, Atg7 and conversion of autophagy marker (LC3-I to LC3-II), and tetraspanin proteins (CD9, CD63, CD81) in rotenone treated CSCs by western blotting. The mRNA expression of CD9, CD63, CD81 and TSG101 analyzed by qRT-PCR showed that the rotenone induced the expression of CD9, CD63, CD81 and TSG101 in CSCs. Electron microscopy of rotenone treated CSCs showed the mitochondrial damage of CSCs as confirmed by the release of exosomes from CSCs. The constituents of exosomes may be useful to understand the mechanism of exosomes formation, release and function, and also serve as a useful biomarker and provide novel therapeutic strategies for the treatment and prevention of cancer.

Krebs cycle metabolites and preferential succinate oxidation following neonatal hypoxic-ischemic brain injury in mice

PubMed Central

Sahni, Prateek V.; Zhang, Jimmy; Sosunov, Sergey; Galkin, Alexander; Niatsetskaya, Zoya; Starkov, Anatoly; Brookes, Paul S.; Ten, Vadim S.

2017-01-01

Background Reverse electron transport (RET) driven by the oxidation of succinate has been proposed as the mechanism of accelerated production of reactive oxygen species (ROS) in post-ischemic mitochondria. However, it remains unclear whether upon reperfusion, mitochondria preferentially oxidase succinate. Methods Neonatal mice were subjected to Rice-Vannucci model of hypoxicischemic brain injury (HI) followed by assessment of Krebs cycle metabolites, mitochondrial substrate preference, and H2O2 generation rate in the ischemic brain. Results While brain mitochondria from control mice exhibited a rotenonesensitive complex-I-dependent respiration, HI-brain mitochondria, at the initiation of reperfusion, demonstrated complex-II-dependent respiration, as rotenone minimally affected, but inhibition of complex-II ceased respiration. This was associated with a 30-fold increase of cerebral succinate concentration and significantly elevated H2O2 emission rate in HI-mice compared to controls. At sixty minutes of reperfusion, cerebral succinate content and the mitochondrial response to rotenone did not differ from that in controls. Conclusion These data are the first ex-vivo evidence, that at the initiation of reperfusion, brain mitochondria transiently shift their metabolism from complex-I-dependent oxidation of NADH toward complex II-linked oxidation of succinate. Our study provides a critical piece of support for existence of the RET-dependent mechanism of elevated ROS production in reperfusion. PMID:29211056

Activation of PPARα by Oral Clofibrate Increases Renal Fatty Acid Oxidation in Developing Pigs.

PubMed

He, Yonghui; Khan, Imad; Bai, Xiumei; Odle, Jack; Xi, Lin

2017-12-08

The objective of this study was to evaluate the effects of peroxisome proliferator-activated receptor α (PPARα) activation by clofibrate on both mitochondrial and peroxisomal fatty acid oxidation in the developing kidney. Ten newborn pigs from 5 litters were randomly assigned to two groups and fed either 5 mL of a control vehicle (2% Tween 80) or a vehicle containing clofibrate (75 mg/kg body weight, treatment). The pigs received oral gavage daily for three days. In vitro fatty acid oxidation was then measured in kidneys with and without mitochondria inhibitors (antimycin A and rotenone) using [1- 14 C]-labeled oleic acid (C18:1) and erucic acid (C22:1) as substrates. Clofibrate significantly stimulated C18:1 and C22:1 oxidation in mitochondria ( p < 0.001) but not in peroxisomes. In addition, the oxidation rate of C18:1 was greater in mitochondria than peroxisomes, while the oxidation of C22:1 was higher in peroxisomes than mitochondria ( p < 0.001). Consistent with the increase in fatty acid oxidation, the mRNA abundance and enzyme activity of carnitine palmitoyltransferase I (CPT I) in mitochondria were increased. Although mRNA of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase (mHMGCS) was increased, the β-hydroxybutyrate concentration measured in kidneys did not increase in pigs treated with clofibrate. These findings indicate that PPARα activation stimulates renal fatty acid oxidation but not ketogenesis.

Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

PubMed

Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

2014-04-01

Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy. Copyright © 2014. Published by Elsevier B.V.

Delivery of Dual Drug Loaded Lipid Based Nanoparticles across the Blood-Brain Barrier Impart Enhanced Neuroprotection in a Rotenone Induced Mouse Model of Parkinson's Disease.

PubMed

Kundu, Paromita; Das, Manasi; Tripathy, Kalpalata; Sahoo, Sanjeeb K

2016-12-21

Parkinson's disease (PD) is the most widespread form of dementia where there is an age related degeneration of dopaminergic neurons in the substantia nigra region of the brain. Accumulation of α-synuclein (αS) protein aggregate, mitochondrial dysfunction, oxidative stress, and neuronal cell death are the pathological hallmarks of PD. In this context, amalgamation of curcumin and piperine having profound cognitive properties, and antioxidant activity seems beneficial. However, the blood-brain barrier (BBB) is the major impediment for delivery of neurotherapeutics to the brain. The present study involves formulation of curcumin and piperine coloaded glyceryl monooleate (GMO) nanoparticles coated with various surfactants with a view to enhance the bioavailability of curcumin and penetration of both drugs to the brain tissue crossing the BBB and to enhance the anti-parkinsonism effect of both drugs in a single platform. In vitro results demonstrated augmented inhibition of αS protein into oligomers and fibrils, reduced rotenone induced toxicity, oxidative stress, and apoptosis, and activation of autophagic pathway by dual drug loaded NPs compared to native counterpart. Further, in vivo studies revealed that our formulated dual drug loaded NPs were able to cross BBB, rescued the rotenone induced motor coordination impairment, and restrained dopaminergic neuronal degeneration in a PD mouse model.

Site of mitochondrial reactive oxygen species production in skeletal muscle of chronic obstructive pulmonary disease and its relationship with exercise oxidative stress.

PubMed

Puente-Maestu, Luis; Tejedor, Alberto; Lázaro, Alberto; de Miguel, Javier; Alvarez-Sala, Luis; González-Aragoneses, Federico; Simón, Carlos; Agustí, Alvar

2012-09-01

Exercise triggers skeletal muscle oxidative stress in patients with chronic obstructive pulmonary disease (COPD). The objective of this research was to study the specific sites of reactive oxygen species (ROS) production in mitochondria isolated from skeletal muscle of patients with COPD and its relationship with local oxidative stress induced by exercise. Vastus lateralis biopsies were obtained in 16 patients with COPD (66 ± 10 yr; FEV(1), 54 ± 12% ref) and in 14 control subjects with normal lung function who required surgery because of lung cancer (65 ± 7 yr; FEV(1), 91 ± 14% ref) at rest and after exercise. In these biopsies we isolated mitochondria and mitochondrial membrane fragments and determined in vitro mitochondrial oxygen consumption (Mit$$\\stackrel{.}{\\hbox{ V }}$$o(2)) and ROS production before and after inhibition of complex I (rotenone), complex II (stigmatellin), and complex III (antimycin-A). We related the in vitro ROS production during state 3 respiration), which mostly corresponds to the mitochondria respiratory state during exercise, with skeletal muscle oxidative stress after exercise, as measured by thiobarbituric acid reactive substances.State 3 Mit$$\\stackrel{.}{\\hbox{ V }}$$o(2) was similar in patients with COPD and control subjects (191 ± 27 versus 229 ± 46 nmol/min/mg; P = 0.058), whereas H(2)O(2) production was higher in the former (147 ± 39 versus 51 ± 8 pmol/mg/h; P < 0.001). The addition of complexI, II, and III inhibitors identify complex III as the main site of H(2)O(2) release by mitochondria in patients with COPD and in control subjects. The mitochondrial production of H(2)O(2) in state 3 respiration was related (r = 0.69; P < 0.001) to postexercise muscle thiobarbituric acid reactive substance levels. Our results show that complex III is the main site of the enhanced mitochondrial H(2)O(2) production that occurs in skeletal muscle of patients with COPD, and the latter appears to contribute to muscle oxidative damage.

Reactive oxygen species are involved in regulating alpha1-adrenoceptor-activated vascular smooth muscle contraction.

PubMed

Tsai, Ming-Ho; Jiang, Meei Jyh

2010-08-23

Reactive oxygen species (ROS) were shown to mediate aberrant contractility in hypertension, yet the physiological roles of ROS in vascular smooth muscle contraction have remained elusive. This study aimed to examine whether ROS regulate alpha1-adrenoceptor-activated contraction by altering myosin phosphatase activities. Using endothelium-denuded rat tail artery (RTA) strips, effects of anti-oxidants on isometric force, ROS production, phosphorylation of the 20-kDa myosin light chain (MLC20), and myosin phosphatase stimulated by alpha1-adrenoceptor agonist phenylephrine were examined. An antioxidant, N-acetyl-L-cysteine (NAC), and two NADPH oxidase inhibitors, apocynin and VAS2870, dose-dependently inhibited contraction activated by phenylephrine. Phenylephrine stimulated superoxide anion production that was diminished by the pretreatment of apocynin, VAS2870, superoxide scavenger tiron or mitochondria inhibitor rotenone, but not by xanthine oxidase inhibitor allopurinol or cyclooxygenase inhibitor indomethacin. Concurrently, NADPH oxidase activity in RTA homogenates increased within 1 min upon phenylephrine stimulation, sustained for 10 min, and was abolished by the co-treatment with apocynin, but not allopurinol or rotenone. Phenylephrine-induced MLC20 phosphorylation was dose-dependently decreased by apocynin. Furthermore, apocynin inhibited phenylephrine-stimulated RhoA translocation to plasma membrane and phosphorylation of both myosin phosphatase regulatory subunit MYPT1Thr855 and myosin phosphatase inhibitor CPI-17Thr38. ROS, probably derived from NADPH oxidase and mitochondria, partially regulate alpha1-adrenoceptor-activated smooth muscle contraction by altering myosin phosphatase-mediated MLC20 phosphorylation through both RhoA/Rho kinase- and CPI-17-dependent pathways.

Mitochondrial transfer of mesenchymal stem cells effectively protects corneal epithelial cells from mitochondrial damage.

PubMed

Jiang, Dan; Gao, Fei; Zhang, Yuelin; Wong, David Sai Hung; Li, Qing; Tse, Hung-Fat; Xu, Goufeng; Yu, Zhendong; Lian, Qizhou

2016-11-10

Recent studies have demonstrated that mesenchymal stem cells (MSCs) can donate mitochondria to airway epithelial cells and rescue mitochondrial damage in lung injury. We sought to determine whether MSCs could donate mitochondria and protect against oxidative stress-induced mitochondrial dysfunction in the cornea. Co-culturing of MSCs and corneal epithelial cells (CECs) indicated that the efficiency of mitochondrial transfer from MSCs to CECs was enhanced by Rotenone (Rot)-induced oxidative stress. The efficient mitochondrial transfer was associated with increased formation of tunneling nanotubes (TNTs) between MSCs and CECs, tubular connections that allowed direct intercellular communication. Separation of MSCs and CECs by a transwell culture system revealed no mitochiondrial transfer from MSCs to CECs and mitochondrial function was impaired when CECs were exposed to Rot challenge. CECs with or without mitochondrial transfer from MSCs displayed a distinct survival capacity and mitochondrial oxygen consumption rate. Mechanistically, increased filopodia outgrowth in CECs for TNT formation was associated with oxidative inflammation-activated NFκB/TNFαip2 signaling pathways that could be attenuated by reactive oxygen species scavenger N-acetylcysteine (NAC) treatment. Furthermore, MSCs grown on a decellularized porcine corneal scaffold were transplanted onto an alkali-injured eye in a rabbit model. Enhanced corneal wound healing was evident following healthy MSC scaffold transplantation. And transferred mitochondria was detected in corneal epithelium. In conclusion, mitochondrial transfer from MSCs provides novel protection for the cornea against oxidative stress-induced mitochondrial damage. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.

Betaine is a positive regulator of mitochondrial respiration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Icksoo, E-mail: icksoolee@dankook.ac.kr

2015-01-09

Highlights: • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration. • Betaine increases mitochondrial membrane potential and cellular energy levels. • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect. - Abstract: Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro.more » Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.« less

Role of mitochondrial electron transport complex I in coenzyme Q1 reduction by intact pulmonary arterial endothelial cells and the effect of hyperoxia.

PubMed

Merker, Marilyn P; Audi, Said H; Lindemer, Brian J; Krenz, Gary S; Bongard, Robert D

2007-09-01

The objective was to determine the impact of intact normoxic and hyperoxia-exposed (95% O(2) for 48 h) bovine pulmonary arterial endothelial cells in culture on the redox status of the coenzyme Q(10) homolog coenzyme Q(1) (CoQ(1)). When CoQ(1) (50 microM) was incubated with the cells for 30 min, its concentration in the medium decreased over time, reaching a lower level for normoxic than hyperoxia-exposed cells. The decreases in CoQ(1) concentration were associated with generation of CoQ(1) hydroquinone (CoQ(1)H(2)), wherein 3.4 times more CoQ(1)H(2) was produced in the normoxic than hyperoxia-exposed cell medium (8.2 +/- 0.3 and 2.4 +/- 0.4 microM, means +/- SE, respectively) after 30 min. The maximum CoQ(1) reduction rate for the hyperoxia-exposed cells, measured using the cell membrane-impermeant redox indicator potassium ferricyanide, was about one-half that of normoxic cells (11.4 and 24.1 nmol x min(-1) x mg(-1) cell protein, respectively). The mitochondrial electron transport complex I inhibitor rotenone decreased the CoQ(1) reduction rate by 85% in the normoxic cells and 44% in the hyperoxia-exposed cells. There was little or no inhibitory effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors on CoQ(1) reduction. Intact cell oxygen consumption rates and complex I activities in mitochondria-enriched fractions were also lower for hyperoxia-exposed than normoxic cells. The implication is that intact pulmonary endothelial cells influence the redox status of CoQ(1) via complex I-mediated reduction to CoQ(1)H(2), which appears in the extracellular medium, and that the hyperoxic exposure decreases the overall CoQ(1) reduction capacity via a depression in complex I activity.

Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jason Z.; Ke, Yuebin; Misra, Hara P.

Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone,more » a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with low NQO1 expression, mitochondria play a critical role in beta-Lp redox activation. • In cancer cells with high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1.« less

Estrogen regulates spatially distinct cardiac mitochondrial subpopulations.

PubMed

Ribeiro Junior, Rogério Faustino; Rodrigues, Paula Lopes; Morra, Elis Aguiar; Ronconi, Karoline Sousa; Do Val Lima, Patrícia Ribeiro; Porto, Marcella Leite; Simões, Maylla Ronacher; Vassallo, Dalton Valentim; Figueiredo, Suely Gomes; Stefanon, Ivanita

2017-07-01

Increased susceptibility to permeability transition pore (mPTP) is a significant concern to decreased cardiac performance in postmenopausal females. The goal of this study was to assess the effects of estrogen deficiency on the two spatially distinct mitochondrial subpopulations from left ventricle: subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM) based on: morphology, membrane potential, oxidative phosphorylation, mPTP and reactive oxygen species production. Female rats (8weeks old) that underwent bilateral ovariectomy were randomly assigned to receive daily treatment with placebo (OVX), estrogen replacement (OVX+E2) and Sham for 60days. The yield for IFM was found higher in the OVX group and lower in the SSM. SSM internal complexity and size were higher in the OVX group, although membrane potential was not different. The maximal rate of mitochondrial respiration, states 3 and 4, using glutamate+malate as substrate, were higher in IFM and SSM from the OVX group. The respiratory control ratio (RCR - state3/state 4), was not different in both SSM and IFM with glutamate+malate. The ADP:O ratio was found lower in IFM and SSM from OVX compared to Sham. When pyruvate was used, state 3 was found unchanged in both IFM and SSM, state 4 was greater in IFM from OVX rats compared to Sham and the ADP/O ratio was decreased. The RCR was unchanged in both subpopulations. The IFM from OVX rats presented a lower Ca 2+ retention capacity compared to Sham, however, the SSM remained unchanged. Hydrogen peroxide formation was found increased in the IFM from OVX animals with glutamate+malate and rotenone+succinate as substrates. The SSM showed increased ROS production only with rotenone+succinate. Western blot analyzes showed decreased levels of PGC-1α and NRF-1 in the OVX group. Estrogen replacement was able to restore most of the alterations induced by ovariectomy. In conclusion, our data shows that estrogen deficiency has distinct effects on the two spatially distinct mitochondrial subpopulations in oxidative phosphorylation, morphology, calcium retention capacity and ROS production. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

An Evaluation of ToxCast Angiogenic Disruptors for Effects on ...

EPA Pesticide Factsheets

Angiogenesis is a critical developmental process and a potential target for chemical teratogenesis. Over one-tenth of the Tox21 library of 10,000 compounds have been shown to disrupt mitochondrial function [Attene-Ramos et al., 2015]. Previous studies utilizing ToxCast chemicals have shown a correlation between vascular disruption in Tg(kdrl:EGFP)mitfab692 zebrafish embryos and mitochondrial disruption reported in literature [McCollum et al., submitted]. To more closely examine this correlation, we culled ToxCast data for mitochondrial translocator protein (TSPO; NovaScreen) and mitochondrial membrane potential (MMP) and biomass (Tox21 and Apredica) for a total of 192 chemicals tested for adverse effects on vascular development in transgenic zebrafish embryos [McCollum et al., submitted; Tal et al., submitted]. This set included 40 compounds that disrupted vascular development in zebrafish embryos (zVDC) and 152 compounds that did not. The zVDC set displayed consistent in vitro bioactivity on mitochondrial membrane potential (with a Pearson Chi-Square value of 16.92, p < 0.0001), but did not have consistent effects on mitochondrial biomass (0.4; p = 0.527) or translocator protein ligand binding (0.05; p = 0.823). The effect on MMP is consistent with the hypothesis that disruption of the mitochondrial respiratory complexes is a potential mode of action of angiogenic disruptors (complex I for pyridaben, fenpyroxymate, tebufenpyrad, and rotenone; complex III for py

The chalcone compound isosalipurposide (ISPP) exerts a cytoprotective effect against oxidative injury via Nrf2 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Jae Yun; Cho, Seung Sik; Yang, Ji Hye

2015-08-15

The chalcone compound isosalipurposide (ISPP) has been successfully isolated from the native Korean plant species Corylopsis coreana Uyeki (Korean winter hazel). However, the therapeutic efficacy of ISPP remains poorly understood. This study investigated whether ISPP has the capacity to activate NF-E2-related factor (Nrf2)-antioxidant response element (ARE) signaling and induce its target gene expression, and to determined the protective role of ISPP against oxidative injury of hepatocytes. In HepG2 cells, nuclear translocation of Nrf2 is augmented by ISPP treatment. Consistently, ISPP increased ARE reporter gene activity and the protein levels of glutamate cysteine ligase (GCL) and hemeoxygenase (HO-1), resulting in increasedmore » intracellular glutathione levels. Cells pretreated with ISPP were rescued from tert-butylhydroperoxide-induced reactive oxygen species (ROS) production and glutathione depletion and consequently, apoptotic cell death. Moreover, ISPP ameliorated the mitochondrial dysfunction and apoptosis induced by rotenone which is an inhibitor of complex 1 of the mitochondrial respiratory chain. The specific role of Nrf2 activation by ISPP was demonstrated using an ARE-deletion mutant plasmid and Nrf2-knockout cells. Finally, we observed that extracellular signal-regulated kinase (ERK) and AMP-activated protein kinase (AMPK), but not protein kinase C (PKC)-δ or other mitogen-activated protein kinases (MAPKs), are involved in the activation of Nrf2 by ISPP. Taken together, our results demonstrate that ISPP has a cytoprotective effect against oxidative damage mediated through Nrf2 activation and induction of its target gene expression in hepatocytes. - Highlights: • We investigated the effect of ISPP on Nrf2 activation. • ISPP increased Nrf2 activity and its target gene expression. • ISPP inhibited the mitochondrial dysfunction and ROS production. • Nrf2 activation by ISPP is dependent on ERK1/2 and AMPK phosphorylation. • ISPP may be a promising therapeutics for the oxidative stress-mediated liver disease.« less

Role of reactive oxygen species and sulfide-quinone oxoreductase in hydrogen sulfide-induced contraction of rat pulmonary arteries

PubMed Central

Prieto-Lloret, Jesus; Snetkov, Vladimir A.; Shaifta, Yasin; Docio, Inmaculada; Connolly, Michelle J.; MacKay, Charles E.; Knock, Greg A.

2018-01-01

Application of H2S (“sulfide”) elicits a complex contraction in rat pulmonary arteries (PAs) comprising a small transient contraction (phase 1; Ph1) followed by relaxation and then a second, larger, and more sustained contraction (phase 2; Ph2). We investigated the mechanisms causing this response using isometric myography in rat second-order PAs, with Na2S as a sulfide donor. Both phases of contraction to 1,000 μM Na2S were attenuated by the pan-PKC inhibitor Gö6983 (3 μM) and by 50 μM ryanodine; the Ca2+ channel blocker nifedipine (1 μM) was without effect. Ph2 was attenuated by the mitochondrial complex III blocker myxothiazol (1 μM), the NADPH oxidase (NOX) blocker VAS2870 (10 μM), and the antioxidant TEMPOL (3 mM) but was unaffected by the complex I blocker rotenone (1 μM). The bath sulfide concentration, measured using an amperometric sensor, decreased rapidly following Na2S application, and the peak of Ph2 occurred when this had fallen to ~50 μM. Sulfide caused a transient increase in NAD(P)H autofluorescence, the offset of which coincided with development of the Ph2 contraction. Sulfide also caused a brief mitochondrial hyperpolarization (assessed using tetramethylrhodamine ethyl ester), followed immediately by depolarization and then a second more prolonged hyperpolarization, the onset of which was temporally correlated with the Ph2 contraction. Sulfide application to cultured PA smooth muscle cells increased reactive oxygen species (ROS) production (recorded using L012); this was absent when the mitochondrial flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using small interfering RNA. We propose that the Ph2 contraction is largely caused by SQR-mediated sulfide metabolism, which, by donating electrons to ubiquinone, increases electron production by complex III and thereby ROS production. PMID:29351439

Intermittent Fasting Applied in Combination with Rotenone Treatment Exacerbates Dopamine Neurons Degeneration in Mice

PubMed Central

Tatulli, Giuseppe; Mitro, Nico; Cannata, Stefano M.; Audano, Matteo; Caruso, Donatella; D’Arcangelo, Giovanna; Lettieri-Barbato, Daniele; Aquilano, Katia

2018-01-01

Intermittent fasting (IF) was suggested to be a powerful nutritional strategy to prevent the onset of age-related neurodegenerative diseases associated with compromised brain bioenergetics. Whether the application of IF in combination with a mitochondrial insult could buffer the neurodegenerative process has never been explored yet. Herein, we defined the effects of IF in C57BL/6J mice treated once per 24 h with rotenone (Rot) for 28 days. Rot is a neurotoxin that inhibits the mitochondrial complex I and causes dopamine neurons degeneration, thus reproducing the neurodegenerative process observed in Parkinson’s disease (PD). IF (24 h alternate-day fasting) was applied alone or in concomitance with Rot treatment (Rot/IF). IF and Rot/IF groups showed the same degree of weight loss when compared to control and Rot groups. An accelerating rotarod test revealed that only Rot/IF mice have a decreased ability to sustain the test at the higher speeds. Rot/IF group showed a more marked decrease of dopaminergic neurons and increase in alpha-synuclein (α-syn) accumulation with respect to Rot group in the substantia nigra (SN). Through lipidomics and metabolomics analyses, we found that in the SN of Rot/IF mice a significant elevation of excitatory amino acids, inflammatory lysophospholipids and sphingolipids occurred. Collectively, our data suggest that, when applied in combination with neurotoxin exposure, IF does not exert neuroprotective effects but rather exacerbate neuronal death by increasing the levels of excitatory amino acids and inflammatory lipids in association with altered brain membrane composition. PMID:29387000

Intermittent Fasting Applied in Combination with Rotenone Treatment Exacerbates Dopamine Neurons Degeneration in Mice.

PubMed

Tatulli, Giuseppe; Mitro, Nico; Cannata, Stefano M; Audano, Matteo; Caruso, Donatella; D'Arcangelo, Giovanna; Lettieri-Barbato, Daniele; Aquilano, Katia

2018-01-01

Intermittent fasting (IF) was suggested to be a powerful nutritional strategy to prevent the onset of age-related neurodegenerative diseases associated with compromised brain bioenergetics. Whether the application of IF in combination with a mitochondrial insult could buffer the neurodegenerative process has never been explored yet. Herein, we defined the effects of IF in C57BL/6J mice treated once per 24 h with rotenone (Rot) for 28 days. Rot is a neurotoxin that inhibits the mitochondrial complex I and causes dopamine neurons degeneration, thus reproducing the neurodegenerative process observed in Parkinson's disease (PD). IF (24 h alternate-day fasting) was applied alone or in concomitance with Rot treatment (Rot/IF). IF and Rot/IF groups showed the same degree of weight loss when compared to control and Rot groups. An accelerating rotarod test revealed that only Rot/IF mice have a decreased ability to sustain the test at the higher speeds. Rot/IF group showed a more marked decrease of dopaminergic neurons and increase in alpha-synuclein (α-syn) accumulation with respect to Rot group in the substantia nigra (SN). Through lipidomics and metabolomics analyses, we found that in the SN of Rot/IF mice a significant elevation of excitatory amino acids, inflammatory lysophospholipids and sphingolipids occurred. Collectively, our data suggest that, when applied in combination with neurotoxin exposure, IF does not exert neuroprotective effects but rather exacerbate neuronal death by increasing the levels of excitatory amino acids and inflammatory lipids in association with altered brain membrane composition.

Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

PubMed

Dudimah, Fred D; Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

2007-01-01

The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures. Copyright 2006 John Wiley & Sons, Ltd.

Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction

PubMed Central

Sanz, Alberto; Soikkeli, Mikko; Portero-Otín, Manuel; Wilson, Angela; Kemppainen, Esko; McIlroy, George; Ellilä, Simo; Kemppainen, Kia K.; Tuomela, Tea; Lakanmaa, Matti; Kiviranta, Essi; Stefanatos, Rhoda; Dufour, Eric; Hutz, Bettina; Naudí, Alba; Jové, Mariona; Zeb, Akbar; Vartiainen, Suvi; Matsuno-Yagi, Akemi; Yagi, Takao; Rustin, Pierre; Pamplona, Reinald; Jacobs, Howard T.

2010-01-01

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling. PMID:20435911

Inhibition of cell proliferation and induction of apoptosis by CDDO-Me in pancreatic cancer cells is ROS-dependent.

PubMed

Deeb, Dorrah; Gao, Xiaohua; Liu, Yong Bo; Gautam, Subhash C

2012-01-01

Oleanolic acid-derived synthetic triterpenoids are broad spectrum antiproliferative and antitumorigenic agents. In this study, we investigated the role of reactive oxygen species (ROS) in induction of apoptosis and inhibition of prosurvival Akt, NF-kappaB and mTOR signaling pro-teins by methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) in pancreatic cancer cells. Micromolar concentrations of CDDO-Me inhibited proliferation and induced apoptosis in MiaPaCa-2 and Panc-1 pancreatic cancer cells. Treatment with CDDO-Me caused the generation of hydrogen peroxide and superoxide anion and pretreatment of cells with NADPH oxidase inhibitor diphylene iodonium (DPI) or respiratory chain complex 1 inhibitor rotenone prevented ROS generation. Pretreatment with N-acetylcysteine (NAC) or overexpression of glutathione peroxidase (GPx) or superoxide dismutase-1 (SOD-1) blocked the antiproliferative effects of CDDO-Me. Likewise, NAC prevented the induction of apoptosis (annexin V-FITC binding and cleavage of PARP-1 and procaspases-3,-8 and -9) and reversed the loss of mitochondrial membrane potential and release of cytochrome c from mitochondria by CDDO-Me. CDDO-Me down-regulated p-Akt, p-mTOR and NF-kappaB (p65) but increased the activation of Erk1/2 and NAC blocked the modulation of these cell signaling proteins by CDDO-Me. Thus, the results of this study indicate that the antiproliferative and apoptosis inducing effects of CDDO-Me are mediated through a ROS-dependent mechanism and the role of ROS in modulation of signaling proteins by CDDO-Me warrants further investigation.

Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation.

PubMed

Baker, Mark A; Hetherington, Louise; Ecroyd, Heath; Roman, Shaun D; Aitken, R John

2004-01-15

The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.

Rotenone isolated from Pachyrhizus erosus displays cytotoxicity and genotoxicity in K562 cells.

PubMed

Estrella-Parra, Edgar A; Gomez-Verjan, Juan C; González-Sánchez, Ignacio; Vázquez-Martínez, Edgar Ricardo; Vergara-Castañeda, Edgar; Cerbón, Marco A; Alavez-Solano, Dagoberto; Reyes-Chilpa, Ricardo

2014-01-01

Pachyrhizus erosus (Fabaceae) is a herb commonly known as 'yam bean', which has been cultivated in México since pre-Columbian times for its edible tubers. The seeds are also known for their acaricidal and insecticidal properties due to rotenone and other isoflavonoid contents. Rotenone has exhibited cytotoxic activity against several human tumour cell lines; however, its mechanism of action is still not fully understood. In this study, we determined the cytotoxicity of rotenone isolated from P. erosus seeds on K562 human leukaemia cells. Rotenone exhibited significant cytotoxic activity (IC50 = 13.05 μM), as determined by the MTT assay. Three other isolated isoflavonoids were not cytotoxic. Rotenone genotoxicity was detected using the comet assay. Rotenone induced cell death, and caspase-3 activation as indicated by TUNEL assay, and immunocytofluorescence. Plasmid nicking assay indicated that rotenone does not interact directly with DNA.

Study Of The Specificity Of Xanthene Dye Binding To Mitochondria

NASA Astrophysics Data System (ADS)

Bunting, James R.; Kamali, Eleanor; Phan, Trung V.; Dowben, Robert M.; Matthews, J. Lester

1989-03-01

The binding of Rhodamine 123 (Rh123), Rhodamine 6G (R6G), and Rhodamine B (RhB) (from the cationic xanthene series) to isolated rat liver mitochondria maintained in State IV respiration in the presence of rotenone (NADH oxidase inhibitor) was monitored by following changes in the fluorescence signal of the dyes. Rh123 and Rh6G bind strongly with quenching, to 0.25 and 0.20, respectively, and red shift of emission maxima by 10 nm. RhB binds much less potently with slight emission enhancement of 1.2. For Rh123 added to 0.5 mg/ml mitochondria' protein, a sigmoidal relationship is obtained between percentage fluorescence quenching and log of Rh123 concentration with a 50% inflection point of 3.5x10-6M, estimating an apparent association constant of 2.9x 105M-1 for Rh123 binding. Addition of 7 uM RhB during Rh123 titration moves the sigmoidal inflection point to higher Rh123 concentrations, suggesting either RhB enhancement of binding of Rh123 fluorescence quenching by energy transfer to RhB bound. These results suggest that, to a great degree, the binding of the xanthene dyes to mitochondrial sites is specific, competitive, and probably cooperative.

Hydrogel tissue construct-based high-content compound screening.

PubMed

Lam, Vy; Wakatsuki, Tetsuro

2011-01-01

Current pharmaceutical compound screening systems rely on cell-based assays to identify therapeutic candidates and potential toxicities. However, cells grown on 2D substrata or in suspension do not exhibit the mechanical or physiological properties of cells in vivo. To address this limitation, the authors developed an in vitro, high-throughput, 3D hydrogel tissue construct (HTC)-based assay system to quantify cell and tissue mechanical properties and multiple parameters of physiology. HTC mechanics was quantified using an automated device, and physiological status was assessed using spectroscopy-based indicators that were read on microplate readers. To demonstrate the application of this system, the authors screened 4 test compounds--rotenone (ROT), cytochalasin D (CD), 2,4-dinitrophenol (DNP), and Rho kinase inhibitor (H-1152)--for their ability to modulate HTC contractility without affecting actin integrity, mitochondrial membrane potential (MMP), or viability. All 4 compounds dose-dependently reduced HTC contractility. However, ROT was toxic, DNP dissipated MMP, and CD reduced both intracellular F-actin and viability. H-1152 was found to be the best candidate compound since it reduced HTC contractility with minimal side effects. The authors propose that their HTC-based assay system can be used to screen for compounds that modulate HTC contractility and assess the underlying physiological mechanism(s) of compound activity and toxicity.

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing.

PubMed

Pistollato, Francesca; Canovas-Jorda, David; Zagoura, Dimitra; Price, Anna

2017-06-09

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Mitochondrial impacts of insecticidal formate esters in insecticide-resistant and insecticide-susceptible Drosophila melanogaster.

PubMed

Song, Cheol; Scharf, Michael E

2009-06-01

Previous research on insecticidal formate esters in flies and mosquitoes has documented toxicity profiles, metabolism characteristics and neurological impacts. The research presented here investigated mitochondrial impacts of insecticidal formate esters and their hydrolyzed metabolite formic acid in the model dipteran insect Drosophila melanogaster Meig. These studies compared two Drosophila strains: an insecticide-susceptible strain (Canton-S) and a strain resistant by cytochrome P450 overexpression (Hikone-R). In initial studies investigating inhibition of mitochondrial cytochrome c oxidase, two proven insecticidal materials (hydramethylnon and sodium cyanide) caused significant inhibition. However, for insecticidal formate esters and formic acid, no significant inhibition was identified in either fly strain. Mitochondrial impacts of formate esters were then investigated further by tracking toxicant-induced cytochrome c release from mitochondria into the cytoplasm, a biomarker of apoptosis and neurological dysfunction. Formic acid and three positive control treatments (rotenone, antimycin A and sodium cyanide) induced cytochrome c release, verifying that formic acid is capable of causing mitochondrial disruption. However, when comparing formate ester hydrolysis and cytochrome c release between Drosophila strains, formic acid liberation was only weakly correlated with cytochrome c release in the susceptible Canton-S strain (r(2) = 0.70). The resistant Hikone-R strain showed no correlation (r(2) < 0.0001) between formate ester hydrolysis and cytochrome c release. The findings of this study provide confirmation of mitochondrial impacts by insecticidal formate esters and suggest links between mitochondrial disruption, respiratory inhibition, apoptosis and formate-ester-induced neurotoxicity.

Persistence of rotenone in ponds at different temperatures

USGS Publications Warehouse

Gilderhus, P.A.; Allen, J.L.; Dawson, V.K.

1986-01-01

Two ponds were treated with liquid rotenone (5% rotenone), one with 3 mg/L at 24°C and the other with 2 mg/L at 0°C (concentrations of active rotenone were 0.15 and 0.10 mg/L, respectively). Water samples were collected and analyzed by high-performance liquid chromatography. The concentration of rotenone declined to 0.02 mg/L in 48 h in warm water and in 11 d in cold water. The half-life of rotenone was calculated at 13.9 h in warm water and 83.9 h in cold water.

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: effect of catalase overexpression.

PubMed

Barbosa, Marina R; Sampaio, Igor H; Teodoro, Bruno G; Sousa, Thais A; Zoppi, Claudio C; Queiroz, André L; Passos, Madla A; Alberici, Luciane C; Teixeira, Felipe R; Manfiolli, Adriana O; Batista, Thiago M; Cappelli, Ana Paula Gameiro; Reis, Rosana I; Frasson, Danúbia; Kettelhut, Isis C; Parreiras-e-Silva, Lucas T; Costa-Neto, Claudio M; Carneiro, Everardo M; Curi, Rui; Silveira, Leonardo R

2013-10-01

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500μM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with β-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and β-oxidation of fatty acids. Copyright © 2013 Elsevier B.V. All rights reserved.

How Parkinsonian Toxins Dysregulate the Autophagy Machinery

PubMed Central

Dagda, Ruben K.; Das Banerjee, Tania; Janda, Elzbieta

2013-01-01

Since their discovery, Parkinsonian toxins (6-hydroxydopamine, MPP+, paraquat, and rotenone) have been widely employed as in vivo and in vitro chemical models of Parkinson’s disease (PD). Alterations in mitochondrial homeostasis, protein quality control pathways, and more recently, autophagy/mitophagy have been implicated in neurotoxin models of PD. Here, we highlight the molecular mechanisms by which different PD toxins dysregulate autophagy/mitophagy and how alterations of these pathways play beneficial or detrimental roles in dopamine neurons. The convergent and divergent effects of PD toxins on mitochondrial function and autophagy/mitophagy are also discussed in this review. Furthermore, we propose new diagnostic tools and discuss how pharmacological modulators of autophagy/mitophagy can be developed as disease-modifying treatments for PD. Finally, we discuss the critical need to identify endogenous and synthetic forms of PD toxins and develop efficient health preventive programs to mitigate the risk of developing PD. PMID:24217228

Chronic inhibition of glycogen synthase kinase-3 protects against rotenone-induced cell death in human neuron-like cells by increasing BDNF secretion.

PubMed

Giménez-Cassina, Alfredo; Lim, Filip; Díaz-Nido, Javier

2012-12-07

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. Likewise, activation of glycogen synthase kinase-3 (GSK-3) has been proposed to play an important role in neurodegeneration. This multifunctional protein kinase is involved in a number of cellular functions and we previously showed that chronic inhibition of GSK-3 protects neuronal cells against mitochondrial dysfunction-elicited cell death, through a mechanism involving increased glucose metabolism and the translocation of hexokinase II (HKII) to mitochondria. Here, we sought to gain deeper insight into the molecular basis of this neuroprotection. We found that chronic inhibition of GSK-3, either genetically or pharmacologically, elicited a marked increase in brain-derived neurotrophic factor (BDNF) secretion, which in turn conferred resistance to mitochondrial dysfunction through subcellular re-distribution of HKII. These results define a molecular pathway through which chronic inhibition of GSK-3 may protect neuronal cells from death. Moreover, they highlight the potential benefits of enhanced neurotrophic factor secretion as a therapeutic approach to treat neurodegenerative diseases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in brain mitochondria from rats and dogs.

PubMed

Sugiyama, Y; Fujita, T; Matsumoto, M; Okamoto, K; Imada, I

1985-12-01

The effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in isolated brain mitochondria from rats and dogs were studied. CV-2619 was easily reduced by canine brain mitochondria in the presence of respiratory substrates. Reduced CV-2619 (2H-CV-2619) was rapidly oxidized through the cytochrome b chain, indicating that the compound functioned simply as an electron carrier of mitochondrial respiratory system. Both nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lipid peroxidations were examined in canine brain mitochondria in the presence of adenosine diphosphate (ADP) and Fe3+. NADH-cytochrome c reductase activity was sensitive to NADPH-dependent lipid peroxidation. CV-2619 (10(-5)M) strongly inhibited both types of the lipid peroxidation reactions and protected the resultant inactivation of the NADH-cytochrome c reductase activity. Activities of succinate oxidase in rat and canine brain mitochondria were virtually unaffected by CV-2619 and its metabolites (10(-5)-10(-6) M). On the other hand, CV-2619 markedly suppressed the state 3 respiration in glutamate oxidation in a dose dependent manner without any effect on the state 4 respiration and the ADP/O ratio in intact rat brain mitochondria. The inhibitory effect of CV-2619 was also observed in NADH-cytochrome c reductase, but not in NADH-2,6-dichlorophenolindophenol (DCIP) and NADH-ubiquinone reductases in canine brain mitochondria. These facts and results of inhibitor analysis suggest that the action site of CV-2619 is NADH-linked complex I in the mitochondrial respiratory chain and is different from that of inhibitors of oxidative phosphorylation such as rotenone, oligomycin and 2,4-dinitrophenol. Finally, the above findings suggest that CV-2619 acts as an electron carrier in respiratory chains and functions as an antioxidant against membrane damage caused by lipid peroxidation in brain mitochondria. It appears likely that the inhibition of oxygen consumption caused by CV-2619 is related to the effect on non-respiratory systems such as lipid peroxidation which also consumes oxygen.

Treadmill exercise alleviates nigrostriatal dopaminergic loss of neurons and fibers in rotenone-induced Parkinson rats.

PubMed

Shin, Mal-Soon; Kim, Tae-Woon; Lee, Jae-Min; Ji, Eun-Sang; Lim, Baek-Vin

2017-02-01

Parkinson disease is one of the common brain diseases caused by dopaminergic neuronal loss in the substantia nigra and dopaminergic fiber loss in the striatum. In the present study, the effects of treadmill exercise on motor performance, dopaminergic loss of neurons and fibers, and α-synuclein expression in the nigrostriatum were evaluated using rotenone-induced Parkinson rats. For the induction of Parkinson rats, 3-mg/kg rotenone was injected, once a day for 14 consecutive days. Treadmill running was conducted for 30 min once a day during 14 consecutive days. Rota-rod test for motor balance and coordination and immunohistochemistry for tyrosine hydroxylase and α-synuclein in the nigrostriatum were performed. In the present study, motor balance and coordination was disturbed by induction of rotenone-induced Parkinson disease, in contrast, treadmill exercise alleviated motor dysfunction in the rotenone-induced Parkinson rats. Nigrostriatal dopaminergic loss of neurons and fibers was occurred by induction of rotenone-induced Parkinson disease, in contrast, treadmill exercise alleviated nigrostriatal dopaminergic loss of neurons and fibers in the rotenone-induced Parkinson rats. α-Synuclein expression in the nigrostriatum was enhanced by induction of rotenone-induced Parkinson disease, in contrast, treadmill exercise suppressed α-synuclein expression in the rotenone-induced Parkinson rats. Treadmill exercise improved motor function through preservation of nigrostriatal dopaminergic neurons and fibers and suppression of nigrostriatal formation of Lewy bodies in rotenone-induced Parkinson rats.

Essential roles of caspases and their upstream regulators in rotenone-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee Jihjong; Huang, M.-S.; Yang, I-C.

2008-06-20

In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, andmore » tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity.« less

Rotenone persistence model for montane streams

USGS Publications Warehouse

Brown, Peter J.; Zale, Alexander V.

2012-01-01

The efficient and effective use of rotenone is hindered by its unknown persistence in streams. Environmental conditions degrade rotenone, but current label instructions suggest fortifying the chemical along a stream based on linear distance or travel time rather than environmental conditions. Our objective was to develop models that use measurements of environmental conditions to predict rotenone persistence in streams. Detailed measurements of ultraviolet radiation, water temperature, dissolved oxygen, total dissolved solids (TDS), conductivity, pH, oxidation–reduction potential (ORP), substrate composition, amount of organic matter, channel slope, and travel time were made along stream segments located between rotenone treatment stations and cages containing bioassay fish in six streams. The amount of fine organic matter, biofilm, sand, gravel, cobble, rubble, small boulders, slope, pH, TDS, ORP, light reaching the stream, energy dissipated, discharge, and cumulative travel time were each significantly correlated with fish death. By using logistic regression, measurements of environmental conditions were paired with the responses of bioassay fish to develop a model that predicted the persistence of rotenone toxicity in streams. This model was validated with data from two additional stream treatment reaches. Rotenone persistence was predicted by a model that used travel time, rubble, and ORP. When this model predicts a probability of less than 0.95, those who apply rotenone can expect incomplete eradication and should plan on fortifying rotenone concentrations. The significance of travel time has been previously identified and is currently used to predict rotenone persistence. However, rubble substrate, which may be associated with the degradation of rotenone by adsorption and volatilization in turbulent environments, was not previously considered.

Antioxidant mechanism of mitochondria-targeted plastoquinone SkQ1 is suppressed in aglycemic HepG2 cells dependent on oxidative phosphorylation.

PubMed

Ježek, Jan; Engstová, Hana; Ježek, Petr

2017-09-01

Previously suggested antioxidant mechanisms for mitochondria-targeted plastoquinone SkQ1 included: i) ion-pairing of cationic SkQ1 + with free fatty acid anions resulting in uncoupling; ii) SkQ1H 2 ability to interact with lipoperoxyl radical; iii) interference with electron flow at the inner ubiquinone (Q) binding site of Complex III (Q i ), involving the reduction of SkQ1 to SkQ1H 2 by ubiquinol. We elucidated SkQ1 antioxidant properties by confocal fluorescence semi-quantification of mitochondrial superoxide (J m ) and cytosolic H 2 O 2 (J c ) release rates in HepG2 cells. Only in glycolytic cells, SkQ1 prevented the rotenone-induced enhancement of J m and J c but not basal releases without rotenone. The effect ceased in glutaminolytic aglycemic cells, in which the redox parameter NAD(P)H/FAD increased after rotenone in contrast to its decrease in glycolytic cells. Autofluorescence decay indicated decreased NADPH/NADH ratios with rotenone in both metabolic modes. SkQ1 did not increase cell respiration and diminished J m established high by antimycin or myxothiazol but not by stigmatellin. The revealed SkQ1 antioxidant modes reflect its reduction to SkQ1H 2 at Complex I I Q or Complex III Q i site. Both reductions diminish electron diversions to oxygen thus attenuating superoxide formation. Resulting SkQ1H 2 oxidizes back to SkQ1at the second (flavin) Complex I site, previously indicated for MitoQ 10 . Regeneration proceeds only at lower NAD(P)H/FAD in glycolytic cells. In contrast, cyclic SkQ1 reduction/SkQ1H 2 oxidation does not substantiate antioxidant activity in intact cells in the absence of oxidative stress (neither pro-oxidant activity, representing a great advantage). A targeted delivery to oxidative-stressed tissues is suggested for the effective antioxidant therapy based on SkQ1. Copyright © 2017 Elsevier B.V. All rights reserved.

Oral supplements of inulin during gestation offsets rotenone-induced oxidative impairments and neurotoxicity in maternal and prenatal rat brain.

PubMed

Krishna, Gokul; Muralidhara

2018-05-25

Environmental insults including pesticide exposure and their entry into the immature brain are of increased concern due to their developmental neurotoxicity. Several lines of evidence suggest that maternal gut microbiota influences in utero fetal development via modulation of host's microbial composition with prebiotics. Hence we examined the hypothesis if inulin (IN) supplements during pregnancy in rats possess the potential to alleviate brain oxidative response and mitochondrial deficits employing a developmental model of rotenone (ROT) neurotoxicity. Initially, pregnant Sprague-Dawley rats were gavaged during gestational days (GDs) 6-19 with 0 (control), 10 (low), 30 (mid) or 50 (high) mg/kg bw/day of ROT to recapitulate developmental effects on general fetotoxicity (assessed by the number of fetuses, fetal body and placental weights), markers of oxidative stress and cholinergic activities in maternal brain regions and whole fetal-brain. Secondly, dams orally supplemented with inulin (2×/day, 2 g/kg/bw) on GD 0-21 were administered ROT (50 mg/kg, GD 6-19). IN supplements increased maternal cecal bacterial numbers that significantly corresponded with improved exploratory-related behavior among ROT administered rats. In addition, IN supplements improved fetal and placental weight on GD 19. IN diminished gestational ROT-induced increased reactive oxygen species levels, protein and lipid peroxidation biomarkers, and cholinesterase activity in maternal brain regions (cortex, cerebellum, and striatum) and fetal brain. Moreover, in the maternal cortex, mitochondrial assessment revealed IN protected against ROT-induced reduction in NADH cytochrome c oxidoreductase and ATPase activities. These data suggest a potential role for indigestible oligosaccharides in reducing oxidative stress-mediated developmental origins of neurodegenerative disorders. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

PubMed

Bai, Y; Hájek, P; Chomyn, A; Chan, E; Seo, B B; Matsuno-Yagi, A; Yagi, T; Attardi, G

2001-10-19

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

Automated Quantification and Integrative Analysis of 2D and 3D Mitochondrial Shape and Network Properties

PubMed Central

Nikolaisen, Julie; Nilsson, Linn I. H.; Pettersen, Ina K. N.; Willems, Peter H. G. M.; Lorens, James B.; Koopman, Werner J. H.; Tronstad, Karl J.

2014-01-01

Mitochondrial morphology and function are coupled in healthy cells, during pathological conditions and (adaptation to) endogenous and exogenous stress. In this sense mitochondrial shape can range from small globular compartments to complex filamentous networks, even within the same cell. Understanding how mitochondrial morphological changes (i.e. “mitochondrial dynamics”) are linked to cellular (patho) physiology is currently the subject of intense study and requires detailed quantitative information. During the last decade, various computational approaches have been developed for automated 2-dimensional (2D) analysis of mitochondrial morphology and number in microscopy images. Although these strategies are well suited for analysis of adhering cells with a flat morphology they are not applicable for thicker cells, which require a three-dimensional (3D) image acquisition and analysis procedure. Here we developed and validated an automated image analysis algorithm allowing simultaneous 3D quantification of mitochondrial morphology and network properties in human endothelial cells (HUVECs). Cells expressing a mitochondria-targeted green fluorescence protein (mitoGFP) were visualized by 3D confocal microscopy and mitochondrial morphology was quantified using both the established 2D method and the new 3D strategy. We demonstrate that both analyses can be used to characterize and discriminate between various mitochondrial morphologies and network properties. However, the results from 2D and 3D analysis were not equivalent when filamentous mitochondria in normal HUVECs were compared with circular/spherical mitochondria in metabolically stressed HUVECs treated with rotenone (ROT). 2D quantification suggested that metabolic stress induced mitochondrial fragmentation and loss of biomass. In contrast, 3D analysis revealed that the mitochondrial network structure was dissolved without affecting the amount and size of the organelles. Thus, our results demonstrate that 3D imaging and quantification are crucial for proper understanding of mitochondrial shape and topology in non-flat cells. In summary, we here present an integrative method for unbiased 3D quantification of mitochondrial shape and network properties in mammalian cells. PMID:24988307

An Assessment of the Effect of Rotenone on Selected Non-Target Aquatic Fauna

PubMed Central

Dalu, Tatenda; Wasserman, Ryan J.; Jordaan, Martine; Froneman, William P.; Weyl, Olaf L. F.

2015-01-01

Rotenone, a naturally occurring ketone, is widely employed for the management of invasive fish species. The use of rotenone poses serious challenges to conservation practitioners due to its impacts on non-target organisms including amphibians and macroinvertebrates. Using laboratory studies, we investigated the effects of different rotenone concentrations (0, 12.5, 25, 37.5, 50, 100 μg L-1) on selected invertebrate groups; Aeshnidae, Belostomatids, Decapods, Ephemeroptera, Pulmonata and zooplankton over a period of 18 hours. Based on field observations and body size, we hypothesized that Ephemeropterans and zooplankton would be more susceptible to rotenone than Decapods, Belostomatids and snails. Experimental results supported this hypothesis and mortality and behaviour effects varied considerably between taxa, ranging from no effect (crab Potamonuates sidneyi) to 100% mortality (Daphnia pulex and Paradiaptomus lamellatus). Planktonic invertebrates were particularly sensitive to rotenone even at very low concentrations. Future research should investigate the recovery time of invertebrate communities after the application of rotenone and conduct field assessments assessing the longer term effects of rotenone exposure on the population dynamics of those less sensitive organisms. PMID:26540301

Metabolic enhancer piracetam attenuates rotenone induced oxidative stress: a study in different rat brain regions.

PubMed

Verma, Dinesh Kumar; Joshi, Neeraj; Raju, Kunumuri Sivarama; Wahajuddin, Muhammad; Singh, Rama Kant; Singh, Sarika

2015-01-01

Piracetam is clinically being used nootropic drug but the details of its neuroprotective mechanism are not well studied. The present study was conducted to assess the effects of piracetam on rotenone induced oxidative stress by using both ex vivo and in vivo test systems. Rats were treated with piracetam (600 mg/kg b.w. oral) for seven constitutive days prior to rotenone administration (intracerebroventricular, 12 µg) in rat brain. Rotenone induced oxidative stress was assessed after 1 h and 24 h of rotenone administration. Ex vivo estimations were performed by using two experimental designs. In one experimental design the rat brain homogenate was treated with rotenone (1 mM, 2 mM and 4 mM) and rotenone+piracetam (10 mM) for 1 h. While in second experimental design the rats were pretreated with piracetam for seven consecutive days. On eighth day the rats were sacrificed, brain homogenate was prepared and treated with rotenone (1 mM, 2 mM and 4mM) for 1h. After treatment the glutathione (GSH) and malondialdehyde (MDA) levels were estimated in brain homogenate. In vivo study showed that pretreatment of piracetam offered significant protection against rotenone induced decreased GSH and increased MDA level though the protection was region specific. But the co-treatment of piracetam with rotenone did not offer significant protection against rotenone induced oxidative stress in ex vivo study. Whereas ex vivo experiments in rat brain homogenate of piracetam pretreated rats, showed the significant protection against rotenone induced oxidative stress. Findings indicated that pretreatment of piracetam significantly attenuated the rotenone induced oxidative stress though the protection was region specific. Piracetam treatment to rats led to its absorption and accumulation in different brain regions as assessed by liquid chromatography mass spectrometry/mass spectrometry. In conclusion, study indicates the piracetam is able to enhance the antioxidant capacity in brain cells in region specific manner. The study is also revealing the rationale for its clinical use in cognitive impairment and other neurological diseases.

Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.

PubMed Central

Sanders, S P; Zweier, J L; Kuppusamy, P; Harrison, S J; Bassett, D J; Gabrielson, E W; Sylvester, J T

1993-01-01

Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death. PMID:8380815

Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF-α in cerebral vascular endothelial cells

PubMed Central

Basuroy, Shyamali; Bhattacharya, Sujoy; Leffler, Charles W.; Parfenova, Helena

2009-01-01

Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The proinflammatory cytokine TNF-α causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of major cellular sources of reactive oxygen species to endothelial inflammatory response. Nitric oxide synthase and xanthine oxidase inhibitors (Nω-nitro-l-arginine and allopurinol) had no effect, while mitochondrial electron transport inhibitors (CCCP, 2-thenoyltrifluoroacetone, and rotenone) attenuated TNF-α-induced superoxide (O2•−) and apoptosis. NADPH oxidase inhibitors (diphenylene iodonium and apocynin) greatly reduced TNF-α-evoked O2•− generation and apoptosis. TNF-α rapidly increased NADPH oxidase activity in CMVEC. Nox4, the cell-specific catalytic subunit of NADPH oxidase, is highly expressed in CMVEC, contributes to basal O2•− production, and accounts for a burst of oxidative stress in response to TNF-α. Nox4 small interfering RNA, but not Nox2, knockdown prevented oxidative stress and apoptosis caused by TNF-α in CMVEC. Nox4 is colocalized with HO-2, the constitutive isoform of heme oxygenase (HO), which is critical for endothelial protection against TNF-α toxicity. The products of HO activity, bilirubin and carbon monoxide (CO, as a CO-releasing molecule, CORM-A1), inhibited Nox4-generated O2•− and apoptosis caused by TNF-α stimulation. We conclude that Nox4 is the primary source of inflammation- and TNF-α-induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO and bilirubin to combat TNF-α-induced oxidative stress by inhibiting Nox4 activity and/or by O2•− scavenging, taken together with close intracellular compartmentalization of HO-2 and Nox4 in cerebral vascular endothelium, may contribute to HO-2 cytoprotection against inflammatory cerebrovascular disease. PMID:19118162

Endothelium-specific insulin resistance leads to accelerated atherosclerosis in areas with disturbed flow patterns: a role for reactive oxygen species.

PubMed

Gage, Matthew C; Yuldasheva, Nadira Y; Viswambharan, Hema; Sukumar, Piruthivi; Cubbon, Richard M; Galloway, Stacey; Imrie, Helen; Skromna, Anna; Smith, Jessica; Jackson, Christopher L; Kearney, Mark T; Wheatcroft, Stephen B

2013-09-01

Systemic insulin resistance is associated with a portfolio of risk factors for atherosclerosis development. We sought to determine whether insulin resistance specifically at the level of the endothelium promotes atherosclerosis and to examine the potential involvement of reactive oxygen species. We cross-bred mice expressing a dominant negative mutant human insulin receptor specifically in the endothelium (ESMIRO) with ApoE(-/-) mice to examine the effect of endothelium-specific insulin resistance on atherosclerosis. ApoE(-/-)/ESMIRO mice had similar blood pressure, plasma lipids and whole-body glucose tolerance, but blunted endothelial insulin signalling, in comparison to ApoE(-/-) mice. Atherosclerosis was significantly increased in ApoE(-/-)/ESMIRO mice at the aortic sinus (226 ± 16 versus 149 ± 24 × 10(3) μm(2), P = 0.01) and lesser curvature of the aortic arch (12.4 ± 1.2% versus 9.4 ± 0.9%, P = 0.035). Relaxation to acetylcholine was blunted in aorta from ApoE(-/-)/ESMIRO mice (Emax 65 ± 41% versus 103 ± 6%, P = 0.02) and was restored by the superoxide dismutase mimetic MnTMPyP (Emax 112 ± 15% versus 65 ± 41%, P = 0.048). Basal generation of superoxide was increased 1.55 fold (P = 0.01) in endothelial cells from ApoE(-/-)/ESMIRO mice and was inhibited by the NADPH oxidase inhibitor gp91ds-tat (-12 ± 0.04%, P = 0.04), the NO synthase inhibitor L-NMMA (-8 ± 0.02%, P = 0.001) and the mitochondrial specific inhibitor rotenone (-23 ± 0.04%, P = 0.006). Insulin resistance specifically at the level of the endothelium leads to acceleration of atherosclerosis in areas with disturbed flow patterns such as the aortic sinus and the lesser curvature of the aorta. We have identified a potential role for increased generation of reactive oxygen species from multiple enzymatic sources in promoting atherosclerosis in this setting. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Rotenone persistence in freshwater ponds: Effects of temperature and sediment adsorption

USGS Publications Warehouse

Dawson, V.K.; Gingerich, W.H.; Davis, R.A.; Gilderhus, P.A.

1991-01-01

The persistence of rotenone was compared between a cement-lined pond (0.04 hectare) and an earthen-bottom pond (0.02 hectare) treated with 5 I?L Noxfish/L (250 I?g rotenone/L) during spring, summer, and fall. Water temperatures on the days of treatment in each season were 8, 22, and 15A?C, respectively. Both ponds were filled with pond water from a common source 1 week before each of the three treatments. Water samples (filtered and unfiltered) and sediment samples were analyzed by high-performance liquid chromatography to monitor the decrease of rotenone until residues were at or below the detection limit (<2.0 I?g/L for water and < 25 ng/g for sediments). The loss of rotenone from water generally followed a first-order rate ofdecay. Rotenone disappeared two to three times faster in the earthen pond than in the concrete pond. The rotenone half-life times in the spring, summer, and fall treatments were 3.7, 1.3, and 5.2 d, respectively, in the concrete pond, and 1.8, 0.7, and 1.8 d in the earthen pond. Rates of decay in both ponds were directly correlated with water temperature. Filtered water samples from both ponds contained less rotenone than unfiltered water, indicating that some rotenone was bound to suspended material. The highest concentration of rotenone in sediment samples was 102 ng/g; residues decreased to below the detection limit within 14 d in the spring treatment and within 3 d in the summer and fall treatments.

Resveratrol attenuates methylglyoxal-induced mitochondrial dysfunction and apoptosis by Sestrin2 induction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, Kyuhwa; Seo, Suho; Han, Jae Yun

2014-10-15

Methylglyoxal is found in high levels in the blood and other tissues of diabetic patients and exerts deleterious effects on cells and tissues. Previously, we reported that resveratrol, a polyphenol in grapes, induced the expression of Sestrin2 (SESN2), a novel antioxidant protein, and inhibited hepatic lipogenesis. This study investigated whether resveratrol protects cells from the methylglyoxal-induced toxicity via SESN2 induction. Methylglyoxal significantly induced cell death in HepG2 cells. However, cells pretreated with resveratrol were rescued from methylglyoxal-induced apoptosis. Resveratrol attenuated glutathione (GSH) depletion and ROS production promoted by methylglyoxal. Moreover, mitochondrial damage was observed by methylglyoxal treatment, but resveratrol restoredmore » mitochondrial function, as evidenced by the observed lack of mitochondrial permeability transition and increased ADP/ATP ratio. Resveratrol treatment inhibited SESN2 depletion elicited by methylglyoxal. SESN2 overexpression repressed methylglyoxal-induced mitochondrial dysfunction and apoptosis. Likewise, rotenone-induced cytotoxicity was not observed in SESN2 overexpressed cells. Furthermore, siRNA knockdown of SESN2 reduced the ability of resveratrol to prevent methylglyoxal-induced mitochondrial permeability transition. In addition, when mice were exposed to methylglyoxal after infection of Ad-SESN2, the plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and GSH depletion by methylglyoxal in liver was reduced in Ad-SESN2 infected mice. Our results demonstrated that resveratrol is capable of protecting cells from methylglyoxal-induced mitochondrial dysfunction and oxidative stress via SESN2 induction. - Highlights: • Resveratrol decreased methylglyoxal-induced apoptosis. • Resveratrol attenuated GSH depletion and ROS production promoted by methylglyoxal. • Resveratrol restored the mitochondrial function by Sestrin2 induction. • Induction of Sestrin2 prevented methylglyoxal-induced GSH depletion in liver.« less

Differential sensitivity of immature and mature ventral mesencephalic neurons to rotenone induced neurotoxicity in vitro.

PubMed

Satish Bollimpelli, V; Kondapi, Anand K

2015-12-25

Rotenone induced neuronal toxicity in ventral mesencephalic (VM) dopaminergic (DA) neurons in culture is widely accepted as an important model for the investigation of Parkinson's disease (PD). However, little is known about developmental stage dependent toxic effects of rotenone on VM neurons in vitro. The objective of present study is to investigate the effect of rotenone on developing VM neurons at immature versus mature stages. Primary VM neurons were cultured in the absence of glial cells. Exposure of VM neurons to rotenone for 2 days induced cell death in both immature and mature neurons in a concentration-dependent manner, but to a greater extent in mature neurons. While rotenone-treated mature VM neurons showed α-synuclein aggregation and sensitivity to DA neurons, immature VM neurons exhibited only DA neuronal sensitivity but not α-synuclein aggregation. In addition, on rotenone treatment, enhancement of caspase-3 activity and reactive oxygen species (ROS) production were higher in mature VM neurons than in immature neurons. These results suggest that even though both mature and immature VM neurons are sensitive to rotenone, their manifestations differ from each other, with only mature VM neurons exhibiting Parkinsonian conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Using gamma distribution to determine half-life of rotenone, applied in freshwater.

PubMed

Rohan, Maheswaran; Fairweather, Alastair; Grainger, Natasha

2015-09-15

Following the use of rotenone to eradicate invasive pest fish, a dynamic first-order kinetic model is usually used to determine the half-life and rate at which rotenone dissipated from the treated waterbody. In this study, we investigate the use of a stochastic gamma model for determining the half-life and rate at which rotenone dissipates from waterbodies. The first-order kinetic and gamma models produced similar values for the half-life (4.45 days and 5.33 days respectively) and days to complete dissipation (51.2 days and 52.48 days respectively). However, the gamma model fitted the data better and was more flexible than the first-order kinetic model, allowing us to use covariates and to predict a possible range for the half-life of rotenone. These benefits are particularly important when examining the influence that different environmental factors have on rotenone dissipation and when trying to predict the rate at which rotenone will dissipate during future operations. We therefore recommend that in future the gamma distribution model is used when calculating the half-life of rotenone in preference to the dynamic first-order kinetics model. Copyright © 2015 Elsevier B.V. All rights reserved.

Inhibitor-induced oxidation of the nucleus and cytosol in Arabidopsis thaliana: implications for organelle to nucleus retrograde signalling

PubMed Central

Karpinska, Barbara; Alomrani, Sarah Owdah

2017-01-01

Concepts of organelle-to-nucleus signalling pathways are largely based on genetic screens involving inhibitors of chloroplast and mitochondrial functions such as norflurazon, lincomycin (LINC), antimycin A (ANT) and salicylhydroxamic acid. These inhibitors favour enhanced cellular oxidation, but their precise effects on the cellular redox state are unknown. Using the in vivo reduction–oxidation (redox) reporter, roGFP2, inhibitor-induced changes in the glutathione redox potentials of the nuclei and cytosol were measured in Arabidopsis thaliana root, epidermal and stomatal guard cells, together with the expression of nuclear-encoded chloroplast and mitochondrial marker genes. All the chloroplast and mitochondrial inhibitors increased the degree of oxidation in the nuclei and cytosol. However, inhibitor-induced oxidation was less marked in stomatal guard cells than in epidermal or root cells. Moreover, LINC and ANT caused a greater oxidation of guard cell nuclei than the cytosol. Chloroplast and mitochondrial inhibitors significantly decreased the abundance of LHCA1 and LHCB1 transcripts. The levels of WHY1, WHY3 and LEA5 transcripts were increased in the presence of inhibitors. Chloroplast inhibitors decreased AOXA1 mRNA levels, while mitochondrial inhibitors had the opposite effect. Inhibitors that are used to characterize retrograde signalling pathways therefore have similar general effects on cellular redox state and gene expression. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’. PMID:28808105

Oxidative impairment of hippocampal long-term potentiation involves activation of protein phosphatase 2A and is prevented by ketone bodies.

PubMed

Maalouf, Marwan; Rho, Jong M

2008-11-15

Previous studies have shown that ketone bodies (KB) exert antioxidant effects in experimental models of neurological disease. In the present study, we explored the effects of the KB acetoacetate (ACA) and beta-hydroxybutyrate (BHB) on impairment of hippocampal long-term potentiation (LTP) in rats by hydrogen peroxide (H(2)O(2)) using electrophysiological, fluorescence imaging, and enzyme assay techniques. We found that: 1) a combination of ACA and BHB (1 mM each) prevented impairment of LTP by H(2)O(2) (200 microM); 2) KB significantly lowered intracellular levels of reactive oxygen species (ROS)--measured with the fluorescent indicator carboxy-H(2)DCFDA (carboxy-2',7'-dichlorodihydrofluorescein diacetate)--in CA1 pyramidal neurons exposed to H(2)O(2); 3) the effect of KB on LTP was replicated by the protein phosphatase 2A (PP2A) inhibitor fostriecin; 4) KB prevented impairment of LTP by the PP2A activator C(6) ceramide; 5) fostriecin did not prevent the increase in ROS levels in CA1 pyramidal neurons exposed to H(2)O(2), and C(6) ceramide did not increase ROS levels; 6) PP2A activity was enhanced by both H(2)O(2) and rotenone (a mitochondrial complex I inhibitor that increases endogenous superoxide production); and 7) KB inhibited PP2A activity in protein extracts from brain tissue treated with either H(2)O(2) or ceramide. We propose that oxidative impairment of hippocampal LTP is associated with PP2A activation, and that KB prevent this impairment in part by inducing PP2A inhibition through an antioxidant mechanism.

Effects of chronic low dose rotenone treatment on human microglial cells

PubMed Central

2009-01-01

Background Exposure to toxins/chemicals is considered to be a significant risk factor in the pathogenesis of Parkinson's disease (PD); one putative chemical is the naturally occurring herbicide rotenone that is now used widely in establishing PD models. We, and others, have shown that chronic low dose rotenone treatment induces excessive accumulation of Reactive Oxygen Species (ROS), inclusion body formation and apoptosis in dopaminergic neurons of animal and human origin. Some studies have also suggested that microglia enhance the rotenone induced neurotoxicity. While the effects of rotenone on neurons are well established, there is little or no information available on the effect of rotenone on microglial cells, and especially cells of human origin. The aim of the present study was to investigate the effects of chronic low dose rotenone treatment on human microglial CHME-5 cells. Methods We have shown previously that rotenone induced inclusion body formation in human dopaminergic SH-SY5Y cells and therefore used these cells as a control for inclusion body formation in this study. SH-SY5Y and CHME-5 cells were treated with 5 nM rotenone for four weeks. At the end of week 4, both cell types were analysed for the presence of inclusion bodies, superoxide dismutases and cell activation (only in CHME-5 cells) using Haematoxylin and Eosin staining, immunocytochemical and western blotting methods. Levels of active caspases and ROS (both extra and intra cellular) were measured using biochemical methods. Conclusion The results suggest that chronic low dose rotenone treatment activates human microglia (cell line) in a manner similar to microglia of animal origin as shown by others. However human microglia release excessive amounts of ROS extracellularly, do not show excessive amounts of intracellular ROS and active caspases and most importantly do not show any protein aggregation or inclusion body formation. Human microglia appear to be resistant to rotenone (chronic, low dose) induced damage. PMID:20042120

Effects of glutamate and {alpha}2-noradrenergic receptor antagonists on the development of neurotoxicity produced by chronic rotenone in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alam, Mesbah; Danysz, Wojciech; Schmidt, Werner Juergen

2009-10-15

Systemic inhibition of complex I by rotenone in rats represents a model of Parkinson's disease (PD). The aim of this study was to elucidate whether neramexane (NMDA, nicotinic {alpha}9/{alpha}10 and 5-HT{sub 3} receptor antagonist), idazoxan ({alpha}{sub 2}-adrenoceptor antagonist) or 2-methyl-6-(phenyl-ethyl)-pyrimidine (MPEP, metabotropic glutamate receptor 5 antagonist) prevents rotenone-induced parkinsonian-like behaviours and neurochemical changes in rats. Rotenone (2.5 mg/kg i.p. daily) was administered over 60 days together with saline, neramexane (5 mg/kg i.p., b.i.d.), idazoxan (2.5 mg/kg i.p., b.i.d.) or MPEP (2.5 mg/kg i.p., b.i.d.). The same doses of neramexane, idazoxan and MPEP were administered to rats treated with vehicle insteadmore » of rotenone. Treatment-related effects on parkinsonian-like behaviours, such as hypokinesia/rigidity and locomotor activity, were evaluated. Moreover, concentrations of dopamine, serotonin and their metabolites were measured in rats from each experimental group. Over the 60-day treatment period, the rotenone + saline treated animals developed hypokinesia, expressed as an increase in the bar and grid descent latencies in the catalepsy test, and a decrease in locomotor activity. Neramexane and idazoxan partially prevented the development of catalepsy in rotenone-treated rats. Co-administration of MPEP with rotenone resulted only in a decrease in descent latency in the grid test on day 60. Chronic rotenone treatment reduced concentrations of dopamine and serotonin in the anterior striatum, which was blocked by co-treatment with neramexane or idazoxan but not with MPEP. Only neramexane treatment blocked the rotenone-induced decrease in dopamine levels in the substantia nigra pars compacta. In conclusion, neramexane and idazoxan counteracted to some extent the development of parkinsonian symptoms and neurochemical alterations in the rotenone model of Parkinson's disease.« less

Molecular responses differ between sensitive silver carp and tolerant bighead carp and bigmouth buffalo exposed to rotenone

USGS Publications Warehouse

Amberg, Jon J.; Schreier, Theresa M.; Gaikowski, Mark P.

2012-01-01

Some species of fish are more tolerant of rotenone, a commonly used non-specific piscicide, than others. This species-specific tolerance to rotenone has been thought to be associated with the uptake and the efficiency at which the chemical is detoxified. However, rotenone stimulates oxidative stress and superoxides, which are also toxic. Understanding the modes in which fish physiologically respond to rotenone is important in developing improved protocols for its application in controlling aquatic nuisance species. Using a molecular approach, we investigated the physiological and molecular mechanisms of rotenone resistance. Species-specific responses were observed when rotenone-sensitive silver, Hypophthalmichthys molitrix, and both rotenone-resistant bighead carp, Hypophthalmichthys nobilis, and bigmouth buffalo, Ictiobus cyprinellus, were exposed to rotenone. Rotenone levels in plasma were highest 90 min after exposure in both silver carp and bigmouth buffalo, but bigmouth buffalo tolerated over twice the burden (ng mL-1 g-1) than silver carp. Expression of genes related with detoxification (cyp1a and gst) increased in silver carp, but either decreased or remained the same in bighead carp. Genes linked with oxidative stress in the cytosol (gpx, cat and sod1) and hsp70 increased only in silver carp after a 6-h exposure. Expression of genes associated with oxidative stress in the mitochondria (sod2 and ucp2) differed between silver carp and bighead carp. Expression of sod2 changed minimally in bighead carp, but expression of ucp2 linearly increased to nearly 85-fold of the level prior to exposure. Expression of sod2 and ucp2 did not change until 6 h in silver carp. Use of sod1 and sod2 to combat oxidative stress results in hydrogen peroxide production, while use of ucp2 produces nitric oxide, a chemical known to inhibit apoptosis. We conclude that the mechanism at which a fish handles oxidative stress plays an important role in the tolerance to rotenone.

Valeriana officinalis attenuates the rotenone-induced toxicity in Drosophila melanogaster.

PubMed

Sudati, Jéssie Haigert; Vieira, Francielli Araújo; Pavin, Sandra Sartoretto; Dias, Glaecir Roseni Mundstock; Seeger, Rodrigo Lopes; Golombieski, Ronaldo; Athayde, Margareth Linde; Soares, Félix Antunes; Rocha, João Batista Teixeira; Barbosa, Nilda Vargas

2013-07-01

In this study, we investigated the potential protective effects of Valeriana officinalis (V. officinalis) against the toxicity induced by rotenone in Drosophila melanogaster (D. melanogaster). Adult wild-type flies were concomitantly exposed to rotenone (500 μM) and V. officinalis aqueous extract (10mg/mL) in the food during 7 days. Rotenone-fed flies had a worse performance in the negative geotaxis assay (i.e. climbing capability) and open-field test (i.e. mobility time) as well as a higher incidence of mortality when compared to control group. V. officinalis treatment offered protection against these detrimental effects of rotenone. In contrast, the decreased number of crossings observed in the flies exposed to rotenone was not modified by V. officinalis. Rotenone toxicity was also associated with a marked decrease on the total-thiol content in the homogenates and cell viability of flies, which were reduced by V. officinalis treatment. Indeed, rotenone exposure caused a significant increase in the mRNA expression of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and also in the tyrosine hydroxylase (TH) gene. The expression of SOD and CAT mRNAs was normalized by V. officinalis treatment. Our results suggest that V. officinalis extract was effective in reducing the toxicity induced by rotenone in D. melanogaster as well as confirm the utility of this model to investigate potential therapeutic strategies on movement disorders, including Parkinson disease (PD). Copyright © 2013 Elsevier Inc. All rights reserved.

Inhibition of MPP+-induced mitochondrial damage and cell death by trifluoperazine and W-7 in PC12 cells.

PubMed

Lee, Chung Soo; Park, Se Young; Ko, Hyun Hee; Song, Jin Ho; Shin, Yong Kyoo; Han, Eun Sook

2005-01-01

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of trifluoperazine and W-7 on the MPP+-induced mitochondrial damage and cell death in undifferentiated PC12 cells. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) at 0.5-1 microM significantly reduced the loss of cell viability in PC12 cells treated with 500 microM MPP+. Trifluoperazine and W-7 (0.5-1 microM) inhibited the nuclear damage, the loss of the mitochondrial transmembrane potential followed by cytochrome c release, and the elevation of intracellular Ca2+ levels due to MPP+ in PC12 cells and attenuated the formation of reactive oxygen species and the depletion of GSH. Calmodulin antagonists at 5-10 microM exhibited a cytotoxic effect on PC12 cells, and compounds at 10 microM did not attenuate cytotoxicity of MPP+. Calmodulin antagonists (0.5-1 microM) significantly reduced rotenone-induced mitochondrial damage and cell death, whereas they did not attenuate cell death and elevation of intracellular Ca2+ levels due to H2O2 or ionomycin. The results show that trifluoperazine and W-7 exhibit a differential inhibitory effect against cytotoxicity of MPP+ depending on concentration. Both compounds at the concentrations less than 5 microM may attenuate the MPP+-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering the intracellular Ca2+ levels.

The pleiotropic effects of decanoic acid treatment on mitochondrial function in fibroblasts from patients with complex I deficient Leigh syndrome.

PubMed

Kanabus, Marta; Fassone, Elisa; Hughes, Sean David; Bilooei, Sara Farahi; Rutherford, Tricia; Donnell, Maura O'; Heales, Simon J R; Rahman, Shamima

2016-05-01

There is growing interest in the use of the ketogenic diet (KD) to treat inherited metabolic diseases including mitochondrial disorders. However, neither the mechanism whereby the diet may be working, nor if it could benefit all patients with mitochondrial disease, is known. This study focusses on decanoic acid (C10), a component of the medium chain triglyceride KD, and a ligand for the nuclear receptor PPAR-γ known to be involved in mitochondrial biogenesis. The effects of C10 were investigated in primary fibroblasts from a cohort of patients with Leigh syndrome (LS) caused by nuclear-encoded defects of respiratory chain complex I, using mitochondrial respiratory chain enzyme assays, gene expression microarray, qPCR and flow cytometry. Treatment with C10 increased citrate synthase activity, a marker of cellular mitochondrial content, in 50 % of fibroblasts obtained from individuals diagnosed with LS in a PPAR-γ-mediated manner. Gene expression analysis and qPCR studies suggested that treating cells with C10 supports fatty acid metabolism, through increasing ACADVL and CPT1 expression, whilst downregulating genes involved in glucose metabolism (PDK3, PDK4). PCK2, involved in blocking glucose metabolism, was upregulated, as was CAT, encoding catalase. Moreover, treatment with C10 also decreased oxidative stress in complex I deficient (rotenone treated) cells. However, since not all cells from subjects with LS appeared to respond to C10, prior cellular testing in vitro could be employed as a means for selecting individuals for subsequent clinical studies involving C10 preparations.

Voluntary aerobic exercise increases arterial resilience and mitochondrial health with aging in mice.

PubMed

Gioscia-Ryan, Rachel A; Battson, Micah L; Cuevas, Lauren M; Zigler, Melanie C; Sindler, Amy L; Seals, Douglas R

2016-11-22

Mitochondrial dysregulation and associated excessive reactive oxygen species (mtROS) production is a key source of oxidative stress in aging arteries that reduces baseline function and may influence resilience (ability to withstand stress). We hypothesized that voluntary aerobic exercise would increase arterial resilience in old mice. An acute mitochondrial stressor (rotenone) caused greater (further) impairment in peak carotid EDD in old (~27 mo., OC, n=12; -32.5±-10.5%) versus young (~7 mo., YC n=11; -5.4±- 3.7%) control male mice, whereas arteries from young and old exercising (YVR n=10 and OVR n=11, 10-wk voluntary running; -0.8±-2.1% and -8.0±4.9%, respectively) mice were protected. Ex-vivo simulated Western diet (WD, high glucose and palmitate) caused greater impairment in EDD in OC (-28.5±8.6%) versus YC (-16.9±5.2%) and YVR (-15.3±2.3%), whereas OVR (-8.9±3.9%) were more resilient (not different versus YC). Simultaneous ex-vivo treatment with mitochondria-specific antioxidant MitoQ attenuated WD-induced impairments in YC and OC, but not YVR or OVR, suggesting that exercise improved resilience to mtROS-mediated stress. Exercise normalized age-related alterations in aortic mitochondrial protein markers PGC-1α, SIRT-3 and Fis1 and augmented cellular antioxidant and stress response proteins. Our results indicate that arterial aging is accompanied by reduced resilience and mitochondrial health, which are restored by voluntary aerobic exercise.

Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity.

PubMed

Mena, Natalia P; Bulteau, Anne Laure; Salazar, Julio; Hirsch, Etienne C; Núñez, Marco T

2011-06-03

Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex I and iron accumulation are hallmarks of idiopathic Parkinson's disease, the findings reported here may have relevance for understanding the pathophysiology of this disease. Copyright © 2011 Elsevier Inc. All rights reserved.

Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yinxi; Liu, Dan; Zhang, Huifeng

Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPHmore » oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm{sup 2} induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm{sup 2}) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91{sup phox}, p47{sup phox} and p40{sup phox}); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47{sup phox} and p67{sup phox} translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to oxidative damage to DA neurons. Our findings delineated the potential role of ultrafine particles alone and in combination with pesticide rotenone in the pathogenesis of PD. - Graphical abstract: Ultrafine particles and rotenone synergistically induce the assembly of active form NADPH oxidase complex in microglia inducing oxidative damage to dopamine neurons. - Highlights: • Ultrafine carbon black promotes dopaminergic neuronal loss induced by rotenone. • The role and underlying mechanism of ultrafine particles in the pathogenesis of PD • NADPH oxidase is a potential therapeutic target of Parkinson's disease.« less

Edaravone Guards Dopamine Neurons in a Rotenone Model for Parkinson's Disease

PubMed Central

Chen, Chunnuan; Huang, Jinsha; Zhao, Ying; Zhang, Zhentao; Qiao, Xian; Feng, Yuan; Reesaul, Harrish; Zhang, Yongxue; Sun, Shenggang; Lin, Zhicheng; Wang, Tao

2011-01-01

3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), an effective free radical scavenger, provides neuroprotection in stroke models and patients. In this study, we investigated its neuroprotective effects in a chronic rotenone rat model for Parkinson's disease. Here we showed that a five-week treatment with edaravone abolished rotenone's activity to induce catalepsy, damage mitochondria and degenerate dopamine neurons in the midbrain of rotenone-treated rats. This abolishment was attributable at least partly to edaravone's inhibition of rotenone-induced reactive oxygen species production or apoptotic promoter Bax expression and its up-regulation of the vesicular monoamine transporter 2 (VMAT2) expression. Collectively, edaravone may provide novel clinical therapeutics for PD. PMID:21677777

Resveratrol Confers Protection against Rotenone-Induced Neurotoxicity by Modulating Myeloperoxidase Levels in Glial Cells

PubMed Central

Chang, Chi Young; Choi, Dong-Kug; Lee, Dae Kee; Hong, Young Jun; Park, Eun Jung

2013-01-01

Myeloperoxidase (MPO) functions as a key molecular component of the host defense system against diverse pathogens. We have previously reported that increased MPO levels and activity is a distinguishing feature of rotenone-exposed glial cells, and that either overactivation or deficiency of MPO leads to pathological conditions in the brain. Here, we provide that modulation of MPO levels in glia by resveratrol confers protective effects on rotenone-induced neurotoxicity. We show that resveratrol significantly reduced MPO levels but did not trigger abnormal nitric oxide (NO) production in microglia and astrocytes. Resveratrol-induced down-regulation of MPO, in the absence of an associated overproduction of NO, markedly attenuated rotenone-triggered inflammatory responses including phagocytic activity and reactive oxygen species production in primary microglia and astrocytes. In addition, impaired responses of primary mixed glia from Mpo −/− mice to rotenone were relieved by treatment with resveratrol. We further show that rotenone-induced neuronal injury, particularly dopaminergic cell death, was attenuated by resveratrol in neuron-glia co-cultures, but not in neurons cultured alone. Similar regulatory effects of resveratrol on MPO levels were observed in microglia treated with MPP+, another Parkinson’s disease-linked neurotoxin, supporting the beneficial effects of resveratrol on the brain. Collectively, our findings provide that resveratrol influences glial responses to rotenone by regulating both MPO and NO, and thus protects against rotenone-induced neuronal injury. PMID:23593274

Amphiphilic chitosan derivatives as carrier agents for rotenone

NASA Astrophysics Data System (ADS)

Kamari, Azlan; Aljafree, Nurul Farhana Ahmad

2017-08-01

In the present study, the feasibility of amphiphilic chitosan derivatives, namely oleoyl carboxymethyl chitosan (OCMCs), N,N-dimethylhexadecyl carboxymethyl chitosan (DCMCs) and deoxycholic acid carboxymethyl chitosan (DACMCs) as carrier agents for rotenone in water-insoluble pesticide formulations was investigated. Fourier Transform Infrared (FTIR) Spectrometer, CHN-O Elemental Analyser (CHN-O) and Transmission Electron Microscope (TEM) were used to characterise amphiphilic chitosan derivatives. The critical micelle concentration (CMC) of amphiphilic chitosan derivatives was determined using a Fluorescence Spectrometer. A High Performance Liquid Chromatography (HPLC) was used to determine the ability of OCMCs, DCMCs and DACMCs to load and release rotenone in an in vitro system. Based on TEM analysis, results have shown that amphiphilic chitosan derivatives formed self-assembly and exhibited spherical shape. The CMC values determined for OCMCs, DCMCs and DACMCs were 0.093, 0.098 and 0.468 mg/mL, respectively. The encapsulation efficiency (EE) values for the materials were more than 97.0%, meanwhile the loading capacity (LC) values were greater than 0.90%. OCMCs, DCMCs and DACMCs micelles exhibited an excellent ability to control the release of rotenone, of which 90.0% of rotenone was released within 40 to 52 h. In conclusion, OCMCs, DCMCs and DACMCs possess several key features to act as effective carrier agents for rotenone. Overall, amphiphilic chitosan derivatives produced in this study were successfully increased the solubility of rotenone by 49.0 times higher than free rotenone.

Sestrin2 Protects Dopaminergic Cells against Rotenone Toxicity through AMPK-Dependent Autophagy Activation

PubMed Central

Hou, Yi-Sheng; Guan, Jun-Jie; Xu, Hai-Dong; Wu, Feng; Sheng, Rui

2015-01-01

Dysfunction of the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) was thought to be an important pathogenic mechanism in synuclein pathology and Parkinson's disease (PD). In the present study, we investigated the role of sestrin2 in autophagic degradation of α-synuclein and preservation of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were increased in a TP53-dependent manner in Mes 23.5 cells after treatment with rotenone. Genetic knockdown of sestrin2 compromised the autophagy induction in response to rotenone, while overexpression of sestrin2 increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent fashion, as sestrin2 overexpression activated AMPK, and genetic knockdown of AMPK abrogated autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown recovered the autophagy activity. Sestrin2 overexpression ameliorated α-synuclein accumulation, inhibited caspase 3 activation, and reduced the cytotoxicity of rotenone. These results suggest that sestrin2 upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation of AMPK, and that sestrin2 exerts a protective effect on dopaminergic cells. PMID:26031332

Novel arylalkenylpropargylamines as neuroprotective, potent, and selective monoamine oxidase B inhibitors for the treatment of Parkinson's disease.

PubMed

Huleatt, Paul B; Khoo, Mui Ling; Chua, Yi Yuan; Tan, Tiong Wei; Liew, Rou Shen; Balogh, Balázs; Deme, Ruth; Gölöncsér, Flóra; Magyar, Kalman; Sheela, David P; Ho, Han Kiat; Sperlágh, Beáta; Mátyus, Péter; Chai, Christina L L

2015-02-12

To develop novel neuroprotective agents, a library of novel arylalkenylpropargylamines was synthesized and tested for inhibitory activities against monoamine oxidases. From this, a number of highly potent and selective monoamine oxidase B inhibitors were identified. Selected compounds were also tested for neuroprotection in in vitro studies with PC-12 cells treated with 6-OHDA and rotenone, respectively. It was observed that some of the compounds tested yielded a marked increase in survival in PC-12 cells treated with the neurotoxins. This indicates that these propargylamines are able to confer protection against the effects of the toxins and may also be considered as novel disease-modifying anti-Parkinsonian agents, which are much needed for the therapy of Parkinson's disease.

Assessment of ToxCast Phase II for Mitochondrial Liabilities Using a High-Throughput Respirometric Assay

PubMed Central

Wills, Lauren P.; Beeson, Gyda C.; Hoover, Douglas B.; Schnellmann, Rick G.; Beeson, Craig C.

2015-01-01

Previous high-throughput screens to identify mitochondrial toxicants used immortalized cell lines and focused on changes in mitochondrial membrane potential, which may not be sufficient and do not identify different types of mitochondrial dysfunction. Primary cultures of renal proximal tubule cells (RPTC) were examined with the Seahorse Extracellular Flux Analyzer to screen 676 compounds (5 μM; 1 h) from the ToxCast Phase II library for mitochondrial toxicants. Of the 676 compounds, 19 were classified as cytotoxicants, 376 were electron transport chain (ETC) inhibitors, and 5 were uncouplers. The remaining 276 compounds were examined after a 5-h exposure to identify slower acting mitochondrial toxicants. This experiment identified 3 cytotoxicants, 110 ETC inhibitors, and 163 compounds with no effect. A subset of the ToxCast Phase II library was also examined in immortalized human renal cells (HK2) to determine differences in susceptibility to mitochondrial toxicity. Of the 131 RPTC ETC inhibitors tested, only 14 were ETC inhibitors in HK2 cells. Of the 5 RPTC uncouplers, 1 compound was an uncoupler in HK2 cells. These results demonstrate that 73% (491/676) of the compounds in the ToxCast Phase II library compounds exhibit RPTC mitochondrial toxicity, overwhelmingly ETC inhibition. In contrast, renal HK2 cells are markedly less sensitive and only identified 6% of the compounds as mitochondrial toxicants. We suggest caution is needed when studying mitochondrial toxicity in immortalized cell lines. This information will provide mechanisms and chemical-based criteria for assessing and predicting mitochondrial liabilities of new drugs, consumer products, and environmental agents. PMID:25926417

Intravitreal delivery of AAV-NDI1 provides functional benefit in a murine model of Leber hereditary optic neuropathy.

PubMed

Chadderton, Naomi; Palfi, Arpad; Millington-Ward, Sophia; Gobbo, Oliverio; Overlack, Nora; Carrigan, Matthew; O'Reilly, Mary; Campbell, Matthew; Ehrhardt, Carsten; Wolfrum, Uwe; Humphries, Peter; Kenna, Paul F; Farrar, G Jane

2013-01-01

Leber hereditary optic neuropathy (LHON) is a mitochondrially inherited form of visual dysfunction caused by mutations in several genes encoding subunits of the mitochondrial respiratory NADH-ubiquinone oxidoreductase complex (complex I). Development of gene therapies for LHON has been impeded by genetic heterogeneity and the need to deliver therapies to the mitochondria of retinal ganglion cells (RGCs), the cells primarily affected in LHON. The therapy under development entails intraocular injection of a nuclear yeast gene NADH-quinone oxidoreductase (NDI1) that encodes a single subunit complex I equivalent and as such is mutation independent. NDI1 is imported into mitochondria due to an endogenous mitochondrial localisation signal. Intravitreal injection represents a clinically relevant route of delivery to RGCs not previously used for NDI1. In this study, recombinant adenoassociated virus (AAV) serotype 2 expressing NDI1 (AAV-NDI1) was shown to protect RGCs in a rotenone-induced murine model of LHON. AAV-NDI1 significantly reduced RGC death by 1.5-fold and optic nerve atrophy by 1.4-fold. This led to a significant preservation of retinal function as assessed by manganese enhanced magnetic resonance imaging and optokinetic responses. Intraocular injection of AAV-NDI1 overcomes many barriers previously associated with developing therapies for LHON and holds great therapeutic promise for a mitochondrial disorder for which there are no effective therapies.

Targeting deficiencies in mitochondrial respiratory complex I and functional uncoupling exerts anti-seizure effects in a genetic model of temporal lobe epilepsy and in a model of acute temporal lobe seizures.

PubMed

Simeone, Kristina A; Matthews, Stephanie A; Samson, Kaeli K; Simeone, Timothy A

2014-01-01

Mitochondria actively participate in neurotransmission by providing energy (ATP) and maintaining normative concentrations of reactive oxygen species (ROS) in both presynaptic and postsynaptic elements. In human and animal epilepsies, ATP-producing respiratory rates driven by mitochondrial respiratory complex (MRC) I are reduced, antioxidant systems are attenuated and oxidative damage is increased. We report that MRCI-driven respiration and functional uncoupling (an inducible antioxidant mechanism) are reduced and levels of H2O2 are elevated in mitochondria isolated from KO mice. Experimental impairment of MRCI in WT hippocampal slices via rotenone reduces paired-pulse ratios (PPRs) at mossy fiber-CA3 synapses (resembling KO PPRs), and exacerbates seizure-like events in vitro. Daily treatment with AATP [a combination therapy composed of ascorbic acid (AA), alpha-tocopherol (T), sodium pyruvate (P) designed to synergistically target mitochondrial impairments] improved mitochondrial functions, mossy fiber PPRs, and reduced seizure burden index (SBI) scores and seizure incidence in KO mice. AATP pretreatment reduced severity of KA-induced seizures resulting in 100% protection from the severe tonic-clonic seizures in WT mice. These data suggest that restoration of bioenergetic homeostasis in the brain may represent a viable anti-seizure target for temporal lobe epilepsy. Copyright © 2013 Elsevier Inc. All rights reserved.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans.

PubMed

Lagido, Cristina; McLaggan, Debbie; Glover, L Anne

2015-10-16

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode's ATP levels.(1-3) Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.(4,5) In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

Pharmacological Inhibition of Poly(ADP-Ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle

PubMed Central

Pirinen, Eija; Canto, Carles; Jo, Young-Suk; Morato, Laia; Zhang, Hongbo; Menzies, Keir; Williams, Evan G.; Mouchiroud, Laurent; Moullan, Norman; Hagberg, Carolina; Li, Wei; Timmers, Silvie; Imhof, Ralph; Verbeek, Jef; Pujol, Aurora; van Loon, Barbara; Viscomi, Carlo; Zeviani, Massimo; Schrauwen, Patrick; Sauve, Anthony; Schoonjans, Kristina; Auwerx, Johan

2014-01-01

SUMMARY We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function. PMID:24814482

Inhibitory Effects of Respiration Inhibitors on Aflatoxin Production

PubMed Central

Sakuda, Shohei; Prabowo, Diyan Febri; Takagi, Keiko; Shiomi, Kazuro; Mori, Mihoko; Ōmura, Satoshi; Nagasawa, Hiromichi

2014-01-01

Aflatoxin production inhibitors, which do not inhibit the growth of aflatoxigenic fungi, may be used to control aflatoxin without incurring a rapid spread of resistant strains. A respiration inhibitor that inhibits aflatoxin production was identified during a screening process for natural, aflatoxin-production inhibitors. This prompted us to evaluate respiration inhibitors as potential aflatoxin control agents. The inhibitory activities of four natural inhibitors, seven synthetic miticides, and nine synthetic fungicides were evaluated on aflatoxin production in Aspergillus parasiticus. All of the natural inhibitors (rotenone, siccanin, aptenin A5, and antimycin A) inhibited fungal aflatoxin production with IC50 values around 10 µM. Among the synthetic miticides, pyridaben, fluacrypyrim, and tolfenpyrad exhibited strong inhibitory activities with IC50 values less than 0.2 µM, whereas cyflumetofen did not show significant inhibitory activity. Of the synthetic fungicides, boscalid, pyribencarb, azoxystrobin, pyraclostrobin, and kresoxim-methyl demonstrated strong inhibitory activities, with IC50 values less than 0.5 µM. Fungal growth was not significantly affected by any of the inhibitors tested at concentrations used. There was no correlation observed between the targets of respiration inhibitors (complexes I, II, and III) and their IC50 values for aflatoxin-production inhibitory activity. This study suggests that respiration inhibitors, including commonly used pesticides, are useful for aflatoxin control. PMID:24674936

Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

PubMed

Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

2015-06-01

Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic derangement. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Azoxystrobin, a mitochondrial complex III Qo site inhibitor, exerts beneficial metabolic effects in vivo and in vitro.

PubMed

Gao, An-Hui; Fu, Yan-Yun; Zhang, Kun-Zhi; Zhang, Mei; Jiang, Hao-Wen; Fan, Li-Xia; Nan, Fa-Jun; Yuan, Chong-Gang; Li, Jia; Zhou, Yu-Bo; Li, Jing-Ya

2014-07-01

Several anti-diabetes drugs exert beneficial effects against metabolic syndrome by inhibiting mitochondrial function. Although much progress has been made toward understanding the role of mitochondrial function inhibitors in treating metabolic diseases, the potential effects of these inhibitors on mitochondrial respiratory chain complex III remain unclear. We investigated the metabolic effects of azoxystrobin (AZOX), a Qo inhibitor of complex III, in a high-fat diet-fed mouse model with insulin resistance in order to elucidate the mechanism by which AZOX improves glucose and lipid metabolism at the metabolic cellular level. Acute administration of AZOX in mice increased the respiratory exchange ratio. Chronic treatment with AZOX reduced body weight and significantly improved glucose tolerance and insulin sensitivity in high-fat diet-fed mice. AZOX treatment resulted in decreased triacylglycerol accumulation and down-regulated the expression of genes involved in liver lipogenesis. AZOX increased glucose uptake in L6 myotubes and 3T3-L1 adipocytes and inhibited de novo lipogenesis in HepG2 cells. The findings indicate that AZOX-mediated alterations to lipid and glucose metabolism may depend on AMP-activated protein kinase (AMPK) signaling. AZOX, a Qo inhibitor of mitochondrial respiratory complex III, exerts whole-body beneficial effects on the regulation of glucose and lipid homeostasis in high-fat diet-fed mice. These findings provide evidence that a Qo inhibitor of mitochondrial respiratory complex III could represent a novel approach for the treatment of obesity. Copyright © 2014 Elsevier B.V. All rights reserved.

Piracetam and vinpocetine ameliorate rotenone-induced Parkinsonism in rats.

PubMed

Zaitone, Sawsan A; Abo-Elmatty, Dina M; Elshazly, Shimaa M

2012-01-01

To evaluate the neuroprotective effect of the nootropic drugs, piracetam (PIR) and vinpocetine (VIN), in rotenone-induced Parkinsonism in rats. Sixty male rats were divided into 6 groups of 10 rats each. The groups were administered vehicle, control (rotenone, 1.5 mg/kg/48 h/6 doses, s.c.), PIR (100 and 200 mg/kg/day, p.o.) and VIN (3 and 6 mg/kg/day, p.o.). The motor performance of the rats was evaluated by the open field and pole test. Striatal dopamine level, malondialdehyde (MDA), reduced glutathione (GSH) and tumor necrosis factor-α (TNF-α) were assayed. Histopathological study of the substantia nigra was also done. Results showed that rotenone-treated rats exhibited bradykinesia and motor impairment in the open-field test. In addition, GSH level was decreased whereas MDA and TNF-α increased in striata of rotenone-treated rats as compared to vehicle-treated rats. Marked degeneration of the substantia nigra pars compacta (SNpc) neurons and depletion of striatal dopamine was also observed in the rotenone-treated rats. Treatment with PIR or VIN significantly reversed the locomotor deficits and increased striatal dopamine level. Treatment with VIN significantly (P<0.05) reduced the striatal level of MDA and GSH in comparison to rotenone group whereas TNF-α production was found to be significantly decreased in PIR group (P<0.05). VIN and PIR exhibit neuroprotective activity in rotenone-induced Parkinsonism. Hence, these nootropic agents may be considered as possible candidates in the treatment of Parkinson's disease.

Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina*

PubMed Central

Du, Jianhai; Cleghorn, Whitney M.; Contreras, Laura; Lindsay, Ken; Rountree, Austin M.; Chertov, Andrei O.; Turner, Sally J.; Sahaboglu, Ayse; Linton, Jonathan; Sadilek, Martin; Satrústegui, Jorgina; Sweet, Ian R.; Paquet-Durand, François; Hurley, James B.

2013-01-01

Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing 13C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia. PMID:24187136

MCT1 inhibitor AZD3965 increases mitochondrial metabolism, facilitating combination therapy and non-invasive magnetic resonance spectroscopy

PubMed Central

Beloueche-Babari, Mounia; Wantuch, Slawomir; Casals Galobart, Teresa; Koniordou, Markella; Parkes, Harold G; Arunan, Vaitha; Chung, Yuen-Li; Eykyn, Thomas R; Smith, Paul D; Leach, Martin O

2017-01-01

Monocarboxylate transporters (MCT) modulate tumor cell metabolism and offer promising therapeutic targets for cancer treatment. Understanding the impact of MCT blockade on tumor cell metabolism may help develop combination strategies or identify pharmacodynamic biomarkers to support the clinical development of MCT inhibitors now in clinical trials. In this study, we assessed the impact of the MCT1 inhibitor AZD3965 on cancer cell metabolism in vitro and in vivo. Exposing human lymphoma and colon carcinoma cells to AZD3965 increased MCT4-dependent accumulation of intracellular lactate, inhibiting monocarboxylate influx and efflux. AZD3965 also increased the levels of TCA cycle-related metabolites and 13C-glucose mitochondrial metabolism, enhancing oxidative pyruvate dehydrogenase and anaplerotic pyruvate carboxylase fluxes. Increased mitochondrial metabolism was necessary to maintain cell survival under drug stress. These effects were counteracted by co-administration of the mitochondrial complex I inhibitor metformin and the mitochondrial pyruvate carrier inhibitor UK5099. Improved bioenergetics were confirmed in vivo after dosing with AZD3965 in mouse xenograft models of human lymphoma. Our results reveal new metabolic consequences of MCT1 inhibition that might be exploited for therapeutic and pharmacodynamic purposes. PMID:28923861

Treadmill exercise alleviates depressive symptoms in rotenone-induced Parkinson disease rats

PubMed Central

Shin, Mal-Soon; Kim, Tae-Woon; Lee, Jae-Min; Sung, Yun-Hee; Lim, Baek-Vin

2017-01-01

Parkinson disease (PD) is characterized by selective loss of the dopaminergic neurons. The symptoms of depression following PD are closely associated with reduced activity of the serotonergic system in the dorsal raphe. We explored the antidepressive effect of exercise and its possible mechanism using the rotenone-induced PD rats. PD rats were induced by subcutaneously injection with rotenone for 14 days. The rats in the exercise groups were made to run on a treadmill for 30 min once a day during 14 consecutive days. Forced swimming test, immunohistochemistry for serotonin (5-hydroxytryptamine, 5-HT), tryptophan hydroxylase (TPH), and western blot for serotonin 1A (5-HT1A) receptor were conducted. Injection of rotenone induced PD rats. PD rats showed depressive state and treadmill exercise ameliorated this depressive state. 5-HT, TPH, and 5-HT1A receptor expressions in the dorsal raphe were suppressed by rotenone injection and treadmill exercise increased the expressions of 5-HT, TPH, and 5-HT1A receptor in the rotenone-injected rats. The present results show that treadmill exercise ameliorated depressive symptoms in the rotenone-induced PD rats. The antidepressive effect of treadmill exercise might be ascribed to the enhancement of serotonergic function through upregulation of 5-HT1A expression in the dorsal raphe. PMID:28503522

Treadmill exercise alleviates depressive symptoms in rotenone-induced Parkinson disease rats.

PubMed

Shin, Mal-Soon; Kim, Tae-Woon; Lee, Jae-Min; Sung, Yun-Hee; Lim, Baek-Vin

2017-04-01

Parkinson disease (PD) is characterized by selective loss of the dopaminergic neurons. The symptoms of depression following PD are closely associated with reduced activity of the serotonergic system in the dorsal raphe. We explored the antidepressive effect of exercise and its possible mechanism using the rotenone-induced PD rats. PD rats were induced by subcutaneously injection with rotenone for 14 days. The rats in the exercise groups were made to run on a treadmill for 30 min once a day during 14 consecutive days. Forced swimming test, immunohistochemistry for serotonin (5-hydroxytryptamine, 5-HT), tryptophan hydroxylase (TPH), and western blot for serotonin 1A (5-HT1A) receptor were conducted. Injection of rotenone induced PD rats. PD rats showed depressive state and treadmill exercise ameliorated this depressive state. 5-HT, TPH, and 5-HT1A receptor expressions in the dorsal raphe were suppressed by rotenone injection and treadmill exercise increased the expressions of 5-HT, TPH, and 5-HT1A receptor in the rotenone-injected rats. The present results show that treadmill exercise ameliorated depressive symptoms in the rotenone-induced PD rats. The antidepressive effect of treadmill exercise might be ascribed to the enhancement of serotonergic function through upregulation of 5-HT1A expression in the dorsal raphe.

Age-dependent reductions in mitochondrial respiration are exacerbated by calcium in the female rat heart.

PubMed

Hunter, J Craig; Machikas, Alexandra M; Korzick, Donna H

2012-06-01

Cardiovascular disease mortality increases rapidly after menopause by poorly defined mechanisms. Because mitochondrial function and Ca(2+) sensitivity are important regulators of cell death after myocardial ischemia, we sought to determine whether aging and/or estrogen deficiency (ovariectomy) increased mitochondrial Ca(2+) sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6 months; n = 26) and aged (24 months; n = 25), intact or ovariectomized female rats using the substrates α-ketoglutarate/malate (complex I); succinate/rotenone (complex II); ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/antimycin (complex IV). State 2 and 3 respiration was initiated by sequential addition of mitochondria and adenosine diphosphate. Ca(2+) sensitivity was assessed by Ca(2+)-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered intraperitoneally 45 minutes before euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for complexes I and II by 12% and 8%, respectively, independent of ovary status (P < 0.05). Of interest, Ca(2+) induced a greater decrease (18%-30%; P < 0.05) in complex I state 3 respiration in aged and ovariectomized animals, and mitochondrial swelling occurred twice as quickly in aged (vs adult) female rats (P < 0.05). Pretreatment with PPT increased RCI by 8% and 7% at complexes I and II, respectively (P < 0.05) but surprisingly increased Ca(2+) sensitivity. Age-dependent decreases in RCI and sensitization to Ca(2+) may explain in part the age-associated reductions in female ischemic tolerance; however, protection afforded by ER agonism involves more complex mechanisms. Copyright © 2012 Elsevier HS Journals, Inc. All rights reserved.

Age-Dependent Reductions in Mitochondrial Respiration are Exacerbated by Calcium in the Female Rat Heart

PubMed Central

Hunter, J. Craig; Machikas, Alexandra M.; Korzick, Donna H.

2012-01-01

Cardiovascular disease mortality increases rapidly following menopause by poorly defined mechanisms. Since mitochondrial function and Ca2+ sensitivity are important regulators of cell death following myocardial ischemia, we sought to determine if aging and/or estrogen deficiency (ovx) increased mitochondrial Ca2+ sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6mo; n=26) and aged (24mo; n=25), intact or ovariectomized female rats using the substrates: α-ketoglutarate/malate (Complex I); succinate/rotenone (Complex II); ascorbate/TMPD/Antimycin (Complex IV). State 2 and State 3 respiration was initiated by sequential addition of mitochondria and ADP. Ca2+ sensitivity was assessed by Ca2+-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered i.p. 45 min prior to euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for Complexes I and II by 12% and 8%, respectively, independent of ovary status (p<0.05). Of interest, Ca2+ induced a greater decrease (18–30%; p<0.05) in Complex I state 3 respiration in aged and ovx animals, and mitochondrial swelling occurred twice as quickly in aged (vs. adult) female rats (p<0.05). Pretreatment with PPT increased RCI by 8% and 7% at Complexes I and II, respectively (p<0.05) but surprisingly increased Ca2+ sensitivity. Age-dependent decreases in RCI and sensitization to Ca2+ may explain in part the age-associated reductions in female ischemic tolerance; however protection afforded by ER agonism involves more complex mechanisms. PMID:22555015

Oxygen sensitivity of mitochondrial function in rat arterial chemoreceptor cells

PubMed Central

Buckler, Keith J; Turner, Philip J

2013-01-01

The mechanism of oxygen sensing in arterial chemoreceptors is unknown but has often been linked to mitochondrial function. A common criticism of this hypothesis is that mitochondrial function is insensitive to physiological levels of hypoxia. Here we investigate the effects of hypoxia (down to 0.5% O2) on mitochondrial function in neonatal rat type-1 cells. The oxygen sensitivity of mitochondrial [NADH] was assessed by monitoring autofluorescence and increased in hypoxia with a P50 of 15 mm Hg (1 mm Hg = 133.3 Pa) in normal Tyrode or 46 mm Hg in Ca2+-free Tyrode. Hypoxia also depolarised mitochondrial membrane potential (ψm, measured using rhodamine 123) with a P50 of 3.1, 3.3 and 2.8 mm Hg in normal Tyrode, Ca2+-free Tyrode and Tyrode containing the Ca2+ channel antagonist Ni2+, respectively. In the presence of oligomycin and low carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 75 nm) ψm is maintained by electron transport working against an artificial proton leak. Under these conditions hypoxia depolarised ψm/inhibited electron transport with a P50 of 5.4 mm Hg. The effects of hypoxia upon cytochrome oxidase activity were investigated using rotenone, myxothiazol, antimycin A, oligomycin, ascorbate and the electron donor tetramethyl-p-phenylenediamine. Under these conditions ψm is maintained by complex IV activity alone. Hypoxia inhibited cytochrome oxidase activity (depolarised ψm) with a P50 of 2.6 mm Hg. In contrast hypoxia had little or no effect upon NADH (P50= 0.3 mm Hg), electron transport or cytochrome oxidase activity in sympathetic neurons. In summary, type-1 cell mitochondria display extraordinary oxygen sensitivity commensurate with a role in oxygen sensing. The reasons for this highly unusual behaviour are as yet unexplained. PMID:23671162

Mitochondrial Respiration after One Session of Calf Raise Exercise in Patients with Peripheral Vascular Disease and Healthy Older Adults.

PubMed

van Schaardenburgh, Michel; Wohlwend, Martin; Rognmo, Øivind; Mattsson, Erney J R

2016-01-01

Mitochondria are essential for energy production in the muscle cell and for this they are dependent upon a sufficient supply of oxygen by the circulation. Exercise training has shown to be a potent stimulus for physiological adaptations and mitochondria play a central role. Whether changes in mitochondrial respiration are seen after exercise in patients with a reduced circulation is unknown. The aim of the study was to evaluate the time course and whether one session of calf raise exercise stimulates mitochondrial respiration in the calf muscle of patients with peripheral vascular disease. One group of patients with peripheral vascular disease (n = 11) and one group of healthy older adults (n = 11) were included. Patients performed one session of continuous calf raises followed by 5 extra repetitions after initiation of pain. Healthy older adults performed 100 continuous calf raises. Gastrocnemius muscle biopsies were collected at baseline and 15 minutes, one hour, three hours and 24 hours after one session of calf raise exercise. A multi substrate (octanoylcarnitine, malate, adp, glutamate, succinate, FCCP, rotenone) approach was used to analyze mitochondrial respiration in permeabilized fibers. Mixed-linear model for repeated measures was used for statistical analyses. Patients with peripheral vascular disease have a lower baseline respiration supported by complex I and they increase respiration supported by complex II at one hour post-exercise. Healthy older adults increase respiration supported by electron transfer flavoprotein and complex I at one hour and 24 hours post-exercise. Our results indicate a shift towards mitochondrial respiration supported by complex II as being a pathophysiological component of peripheral vascular disease. Furthermore exercise stimulates mitochondrial respiration already after one session of calf raise exercise in patients with peripheral vascular disease and healthy older adults. ClinicalTrials.gov NCT01842412.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hossain, Quazi Sohel; Department of Biochemistry, School of Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215; Ulziikhishig, Enkhbaatar

We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated withmore » GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca{sup 2+}-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.« less

Is Reliance on Mitochondrial Respiration a “Chink in the Armor” of Therapy-Resistant Cancer?

PubMed Central

Wolf, Dieter A.

2016-01-01

Summary A series of recent reports has suggested PGC1α-driven upregulation of mitochondrial oxidative phosphorylation as a selective vulnerability of drug-resistant cancers. Accordingly, chemical inhibitors of respiration led to selective eradication of such cancer cells due to their preferential sensitivity to mitochondrial production of reactive oxygen species. These novel insights create a timely opportunity for a biomarker guided application of already existing and newly emerging mitochondrial inhibitors in recurrent drug resistant cancer, including lymphomas, melanomas, and other malignant diseases marked by increased mitochondrial respiration. PMID:25490445

Piracetam and vinpocetine ameliorate rotenone-induced Parkinsonism in rats

PubMed Central

Zaitone, Sawsan A.; Abo-Elmatty, Dina M.; Elshazly, Shimaa M.

2012-01-01

Objective: To evaluate the neuroprotective effect of the nootropic drugs, piracetam (PIR) and vinpocetine (VIN), in rotenone-induced Parkinsonism in rats. Materials and Methods: Sixty male rats were divided into 6 groups of 10 rats each. The groups were administered vehicle, control (rotenone, 1.5 mg/kg/48 h/6 doses, s.c.), PIR (100 and 200 mg/kg/day, p.o.) and VIN (3 and 6 mg/kg/day, p.o.). The motor performance of the rats was evaluated by the open field and pole test. Striatal dopamine level, malondialdehyde (MDA), reduced glutathione (GSH) and tumor necrosis factor-α (TNF-α) were assayed. Histopathological study of the substantia nigra was also done. Results: Results showed that rotenone-treated rats exhibited bradykinesia and motor impairment in the open-field test. In addition, GSH level was decreased whereas MDA and TNF-α increased in striata of rotenone-treated rats as compared to vehicle-treated rats. Marked degeneration of the substantia nigra pars compacta (SNpc) neurons and depletion of striatal dopamine was also observed in the rotenone-treated rats. Treatment with PIR or VIN significantly reversed the locomotor deficits and increased striatal dopamine level. Treatment with VIN significantly (P < 0.05) reduced the striatal level of MDA and GSH in comparison to rotenone group whereas TNF-α production was found to be significantly decreased in PIR group (P < 0.05). Conclusion: VIN and PIR exhibit neuroprotective activity in rotenone-induced Parkinsonism. Hence, these nootropic agents may be considered as possible candidates in the treatment of Parkinson's disease. PMID:23248410

Voluntary aerobic exercise increases arterial resilience and mitochondrial health with aging in mice

PubMed Central

Gioscia-Ryan, Rachel A.; Battson, Micah L.; Cuevas, Lauren M.; Zigler, Melanie C.; Sindler, Amy L.; Seals, Douglas R.

2016-01-01

Mitochondrial dysregulation and associated excessive reactive oxygen species (mtROS) production is a key source of oxidative stress in aging arteries that reduces baseline function and may influence resilience (ability to withstand stress). We hypothesized that voluntary aerobic exercise would increase arterial resilience in old mice. An acute mitochondrial stressor (rotenone) caused greater (further) impairment in peak carotid EDD in old (~27 mo., OC, n=12;−32.5±-10.5%) versus young (~7 mo., YC n=11;−5.4±- 3.7%) control male mice, whereas arteries from young and old exercising (YVR n=10 and OVR n=11, 10-wk voluntary running;−0.8±-2.1% and −8.0±4.9%, respectively) mice were protected. Ex-vivo simulated Western diet (WD, high glucose and palmitate) caused greater impairment in EDD in OC (-28.5±8.6%) versus YC (-16.9±5.2%) and YVR (-15.3±2.3%), whereas OVR (-8.9±3.9%) were more resilient (not different versus YC). Simultaneous ex-vivo treatment with mitochondria-specific antioxidant MitoQ attenuated WD-induced impairments in YC and OC, but not YVR or OVR, suggesting that exercise improved resilience to mtROS-mediated stress. Exercise normalized age-related alterations in aortic mitochondrial protein markers PGC-1α, SIRT-3 and Fis1 and augmented cellular antioxidant and stress response proteins. Our results indicate that arterial aging is accompanied by reduced resilience and mitochondrial health, which are restored by voluntary aerobic exercise. PMID:27875805

76 FR 16415 - Product Cancellation Order for Certain Pesticide Registrations

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-23

... Cube Resins other than rotenone. Rotenone. 000869-00186 Green Light Rotenone... Cube Resins other than..., Inc., P.O. Box 1019, Salem, VA 24153-3805. 270 Farnam Companies, Inc., 301 West Osborn Road, Phoenix, AZ 85013. 769 Value Gardens Supply, LLC, P.O. Box 585, Saint Joseph, MO 64502. 869 Green Light...

75 FR 44256 - Rotenone; Notice of Receipt of Requests to Voluntarily Cancel Pesticide Registrations

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-28

... that would merit its further review of the requests, or unless the registrants withdraw their requests. If these requests are granted, any sale, distribution, or use of products listed in this notice will... voluntarily cancel their rotenone registrations. The requests would not terminate the last rotenone products...

Astrocyte Mediated Protection of Fetal Cerebral Cortical Neurons from Rotenone and Paraquat

PubMed Central

Rathinam, Mary Latha; Watts, Lora Talley; Narasimhan, Madhusudhanan; Riar, Amanjot Kaur; Mahimainathan, Lenin; Henderson, George.I.

2012-01-01

Primary cultures of fetal rat cortical neurons and astrocytes were used to test the hypothesis that astrocyte-mediated control of neuronal glutathione (GSH) is a potent factor in neuroprotection against rotenone and paraquat. In neurons, rotenone (0.025 to 1μM) for 4 and 24 h decreased viability as did paraquat (2 to 100μM). Rotenone (30nM) decreased neuronal viability and GSH by 24% and 30%, while ROS were increased by 56%. Paraquat (30μM) decreased neuronal viability and GSH by 36% and 70%, while ROS were increased by 23%. When neurons were co-cultured with astrocytes, their GSH increased 1.5 fold and 5 fold at 12 and 24 h. Co-culturing with astrocytes blocked neuronal death and damage by rotenone and paraquat. Astrocyte-mediated neuroprotection was dependent on the activity of components of the γ-glutamyl cycle. These studies illustrate the importance of astrocyte-mediated glutathione homeostasis for protection of neurons from rotenone and paraquat and the role of the γ-glutamyl cycle in this neuroprotection PMID:22301167

Development and evaluation of granule and emulsifiable concentrate formulations containing Derris elliptica extract for crop pest control.

PubMed

Wiwattanapatapee, Ruedeekorn; Sae-Yun, Attawadee; Petcharat, Jiraporn; Ovatlarnporn, Chitchamai; Itharat, Arunporn

2009-12-09

Derris elliptica Benth. extracts containing rotenone have long been used as natural insecticides, but time-consuming preparation processes and the short shelf life of the extract limit their use in pest control. In this study, stable water-dispersible granules and emulsifiable concentrate liquids containing Derris extract (equivalent to 5% w/w of rotenone) were developed with simple techniques. Accelerated degradation kinetics of rotenone in the Derris extract, and in both formulations, indicated that its degradation followed first-order kinetics. The predicted half-life (t(1/2)) and shelf life (t(90%)) at 30 degrees C of rotenone in Derris extract were 520 and 79 days, respectively. Derris granules and emulsifiable concentrate clearly prolong the stability of rotenone 8-fold (t(90%) = 633 days) and 1.4-fold (t(90%) = 110 days), respectively. The study of rotenone degradation after application onto plants indicated that both formulations would be effective for up to 3 days after spraying. Preliminary efficacy testing indicated that the Derris emulsifiable concentrate was clearly more effective than Derris water-dispersible granules in controlling Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae).

Protective effect of histamine H2 receptor antagonist ranitidine against rotenone-induced apoptosis.

PubMed

Park, Hae Jeong; Kim, Hak Jae; Park, Hyun-Kyung; Chung, Joo-Ho

2009-11-01

Histamine H(2) receptor antagonists have been reported to improve the motor symptoms of Parkinson's disease (PD) patients and to exert neuroprotective effects. In this study, we investigated the protective effects of the H(2) receptor antagonist ranitidine on rotenone-induced apoptosis in human dopaminergic SH-SY5Y cells, focusing on mitogen-activated protein kinases (MAPKs) and caspases (CASPs)-mediated apoptotic events. Ranitidine blocked the rotenone-induced phosphorylation of c-Jun NH(2)-terminal protein kinase (JNK) and P38 MAPK (P38), and promoted the phosphorylation of extracellular signal-regulated protein kinase (ERK). Ranitidine also prevented the down-regulation of B-cell CLL/lymphoma 2 (BCL2) and the up-regulation of BCL2-associated X protein (BAX) by rotenone. Furthermore, ranitidine not only attenuated rotenone-induced cleavages of CASP9, poly(ADP-ribose) polymerase-1 (PARP) and CASP3, but also suppressed CASP3 enzyme activity. These results indicate that ranitidine protects against rotenone-induced apoptosis, inhibiting phosphorylation of JNK and P38, and activation of CASPs in human dopaminergic SH-SY5Y cells.

Relevance of cryptic fishes in biodiversity assessments: A case study at Buck Island Reef National Monument, St. Croix

USGS Publications Warehouse

Smith-Vaniz, W.F.; Jelks, H.L.; Rocha, L.A.

2006-01-01

Because cryptic fishes are difficult to accurately survey, they are undersampled components of coral reef habitats, and their ecological roles have been generally ignored. Fifty-eight enclosed stations were sampled in shoreline, nearshore reef, lagoon, backreef, forereef, and bank/shelf habitats with an ichthyocide (rotenone) at Buck Island Reef National Monument, St. Croix, U.S. Virgin Islands. Our samples included 55 families and 228 species, 60 previously unreported from St. Croix. Fish assemblages varied across habitat zones with the shoreline assemblage the most distinct. Only 8% of the species were present in all habitats. Multi-dimensional scaling plots of habitat characteristics and Bray-Curtis similarities of fish assemblages revealed similar patterns. Dominant and rare taxa are enumerated for each habitat sampled. Rotenone and visual census data are compared. While visual surveys accumulated more species per unit of effort, rotenone samples accumulated more species by area. Only 36% of the 228 species sampled with rotenone were visually detected, while 70% of the 115 species visually detected were also collected with rotenone. The use of rotenone is controversial but important for obtaining reasonably complete inventories of reef fishes. Misconceptions about rotenone and the advantages and limitations of alternative biodiversity assessment methods are discussed. ?? 2006 Rosenstiel School of Marine and Atmospheric Science of the University of Miami.

Toxicity of 5% rotenone to nonindigenous Asian swamp eels

USGS Publications Warehouse

Schofield, P.J.; Nico, L.G.

2007-01-01

Our primary goal was to determine whether rotenone would be a useful control against introduced populations of Asian swamp eels (family Synbranchidae, genus Monopterus). We report the results of a laboratory experiment comparing the efficacy of various rotenone concentrations (1, 2, 4, and 8 mg of 5% liquid rotenone/L of water) in killing nonindigenous swamp eels of various sizes (1-350 g) from the three known Florida populations. Although most small swamp eels were killed at concentrations of 2 and 4 mg/L. 100% mortality of adult swamp eels was achieved only at 8 mg/L. We conclude that the effective use of rotenone to control established Florida swamp eel populations would be difficult, based on the relatively high concentration of rotenone needed to kill swamp eels; the complexity of the swamp eel's habitat; and our observations of the species' habitat use and behavior, including its widespread distribution and life history characteristics (e.g., burrowing and overland movement) that enhance its invasion and survival in multiple environments. Nevertheless, control of swamp eels may be achieved in certain situations. A combination of rotenone and electroshocking may be an effective way to eradicate swamp eels from small water bodies and to control populations in larger habitats. However, we are cautious in this recommendation and provide details related to the technical aspects of this type of strategy and caveats related to the toxicity of the chemical.

MCT1 Inhibitor AZD3965 Increases Mitochondrial Metabolism, Facilitating Combination Therapy and Noninvasive Magnetic Resonance Spectroscopy.

PubMed

Beloueche-Babari, Mounia; Wantuch, Slawomir; Casals Galobart, Teresa; Koniordou, Markella; Parkes, Harold G; Arunan, Vaitha; Chung, Yuen-Li; Eykyn, Thomas R; Smith, Paul D; Leach, Martin O

2017-11-01

Monocarboxylate transporters (MCT) modulate tumor cell metabolism and offer promising therapeutic targets for cancer treatment. Understanding the impact of MCT blockade on tumor cell metabolism may help develop combination strategies or identify pharmacodynamic biomarkers to support the clinical development of MCT inhibitors now in clinical trials. In this study, we assessed the impact of the MCT1 inhibitor AZD3965 on cancer cell metabolism in vitro and in vivo Exposing human lymphoma and colon carcinoma cells to AZD3965 increased MCT4-dependent accumulation of intracellular lactate, inhibiting monocarboxylate influx and efflux. AZD3965 also increased the levels of TCA cycle-related metabolites and 13 C-glucose mitochondrial metabolism, enhancing oxidative pyruvate dehydrogenase and anaplerotic pyruvate carboxylase fluxes. Increased mitochondrial metabolism was necessary to maintain cell survival under drug stress. These effects were counteracted by coadministration of the mitochondrial complex I inhibitor metformin and the mitochondrial pyruvate carrier inhibitor UK5099. Improved bioenergetics were confirmed in vivo after dosing with AZD3965 in mouse xenograft models of human lymphoma. Our results reveal new metabolic consequences of MCT1 inhibition that might be exploited for therapeutic and pharmacodynamic purposes. Cancer Res; 77(21); 5913-24. ©2017 AACR . ©2017 American Association for Cancer Research.

Environmental toxicants as extrinsic epigenetic factors for parkinsonism: studies employing transgenic C. elegans model.

PubMed

Jadiya, Pooja; Nazir, Aamir

2012-12-01

Various human diseases are known to occur as a result of gene-environment interactions. Amongst such diseases, neurodegenerative Parkinson's disease (PD) is a complex disorder in which genetics and exposure to toxins constitute the main determinants in the onset of the disease. Many studies have reported on a link between pesticide exposure and increased risk of PD, however the role of different classes of pesticides vis-à-vis Parkinsonism has not been well elucidated. We carried out the present study to explore the role of six groups of pesticides viz botanicals, herbicides, fungicides, organophosphates, carbamates and pyrethroids on PD and and associated neurotoxic effects. These pesticides were studied using transgenic Caenorhabditis elegans model expressing human alpha synuclein protein tagged with yellow fluorescent protein [NL5901; (Punc-54::alphasynuclein::YFP+unc-119)] in the body wall muscle. Amongst all the classes of pesticides examined, botanical rotenone showed severe effects on PD pathogenesis. It significantly increased alpha synuclein aggregation and oxidative stress. Furthermore, it reduced mitochondrial and lipid content in the worms. Pesticides from other classes were observed to exert marginal effects as compared to rotenone thus suggesting that there is a class or structure specific effect of environmental chemicals vis-à-vis Parkinsonism. Hence it may be deduced that all classes of toxicants do not induce similar effects on neurodegeneration and associated events.

Functional screening of selective mitochondrial inhibitors of Plasmodium.

PubMed

Gomez-Lorenzo, Maria G; Rodríguez-Alejandre, Ane; Moliner-Cubel, Sonia; Martínez-Hoyos, María; Bahamontes-Rosa, Noemí; Gonzalez Del Rio, Rubén; Ródenas, Carolina; Fuente, Jesús de la; Lavandera, Jose Luis; García-Bustos, Jose F; Mendoza-Losana, Alfonso

2018-05-09

Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors. Copyright © 2018. Published by Elsevier Ltd.

Detailed Analysis of Apoptosis and Delayed Luminescence of Human Leukemia Jurkat T Cells after Proton Irradiation and Treatments with Oxidant Agents and Flavonoids

PubMed Central

Baran, Irina; Ganea, Constanta; Privitera, Simona; Scordino, Agata; Barresi, Vincenza; Musumeci, Francesco; Mocanu, Maria Magdalena; Condorelli, Daniele F.; Ursu, Ioan; Grasso, Rosaria; Gulino, Marisa; Garaiman, Alexandru; Musso, Nicolò; Cirrone, Giuseppe A. Pablo; Cuttone, Giacomo

2012-01-01

Following previous work, we investigated in more detail the relationship between apoptosis and delayed luminescence (DL) in human leukemia Jurkat T cells under a wide variety of treatments. We used menadione and hydrogen peroxide to induce oxidative stress and two flavonoids, quercetin, and epigallocatechin gallate, applied alone or in combination with menadione or H2O2. 62 MeV proton beams were used to irradiate cells under a uniform dose of 2 or 10 Gy, respectively. We assessed apoptosis, cell cycle distributions, and DL. Menadione, H2O2 and quercetin were potent inducers of apoptosis and DL inhibitors. Quercetin decreased clonogenic survival and the NAD(P)H level in a dose-dependent manner. Proton irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G2/M arrest. Quercetin reduced apoptosis and prolonged the G2/M arrest induced by radiation. DL spectroscopy indicated that proton irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus explaining the massive necrosis induced by 10 Gy of protons and also suggested an equivalent action of menadione and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within Complex I, probably the rotenone-binding site. PMID:22829956

Acute Mitochondrial Inhibition by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) 1/2 Inhibitors Regulates Proliferation*

PubMed Central

Ripple, Maureen O.; Kim, Namjoon; Springett, Roger

2013-01-01

The Ras-MEK1/2-ERK1/2 kinase signaling pathway regulates proliferation, survival, and differentiation and, because it is often aberrant in tumors, is a popular target for small molecule inhibition. A novel metabolic analysis that measures the real-time oxidation state of NAD(H) and the hemes of the electron transport chain and oxygen consumption within intact, living cells found that structurally distinct MEK1/2 inhibitors had an immediate, dose-dependent effect on mitochondrial metabolism. The inhibitors U0126, MIIC and PD98059 caused NAD(H) reduction, heme oxidation, and decreased oxygen consumption, characteristic of complex I inhibition. PD198306, an orally active MEK1/2 inhibitor, acted as an uncoupler. Each MEK1/2 inhibitor depleted phosphorylated ERK1/2 and inhibited proliferation, but the most robust antiproliferative effects always correlated with the metabolic failure which followed mitochondrial inhibition rather than inhibition of MEK1/2. This warrants rethinking the role of ERK1/2 in proliferation and emphasizes the importance of mitochondrial function in this process. PMID:23235157

Neuroprotective and Therapeutic Effect of Caffeine on the Rat Model of Parkinson's Disease Induced by Rotenone.

PubMed

Khadrawy, Yasser A; Salem, Ahmed M; El-Shamy, Karima A; Ahmed, Emad K; Fadl, Nevein N; Hosny, Eman N

2017-09-03

The present study aimed to investigate the protective and therapeutic effects of caffeine on rotenone-induced rat model of Parkinson's disease (PD). Rats were divided into control, PD model induced by rotenone (1.5 mg/kg intraperitoneally (i.p.) for 45 days), protected group injected with caffeine (30 mg/kg, i.p.) and rotenone for 45 days (during the development of PD model), and treated group injected with caffeine (30 mg/kg, i.p.) for 45 days after induction of PD model. The data revealed a state of oxidative and nitrosative stress in the midbrain and the striatum of animal model of PD as indicated from the increased lipid peroxidation and nitric oxide levels and the decreased reduced glutathione level and activities of glutathione-S-transferase and superoxide dismutase. Rotenone induced a decrease in acetylcholinesterase and Na + /K + -ATPase activities and an increase in tumor necrosis factor-α level in the midbrain and the striatum. Protection and treatment with caffeine ameliorated the oxidative stress and the changes in acetylcholinesterase and Na + /K + -ATPase activities induced by rotenone in the midbrain and the striatum. This was associated with improvement in the histopathological changes induced in the two areas of PD model. Caffeine protection and treatment restored the depletion of midbrain and striatal dopamine induced by rotenone and prevented decline in motor activities (assessed by open field test) and muscular strength (assessed by traction and hanging tests) and improved norepinephrine level in the two areas. The present study showed that caffeine offered a significant neuroprotection and treatment against neurochemical, histopathological, and behavioral changes in a rotenone-induced rat model of PD.

Zinc and linoleic acid pre-treatment attenuates biochemical and histological changes in the midbrain of rats with rotenone-induced Parkinsonism.

PubMed

Mbiydzenyuy, Ngala Elvis; Ninsiima, Herbert Izo; Valladares, Miriela Betancourt; Pieme, Constant Anatole

2018-05-09

Studies have suggested the supplementation of Zinc and Linoleic acid in the management of neurodegenerative disorders but none has investigated the combined effects. Little is known about the neuroprotective effects of either Zinc or Linoleic acid or their combination against development of Parkinsonism. This study was designed to investigate the neuroprotective effects of Zinc and Linoleic acid in rotenone-induced Parkinsonism in rats. Thirty-six young adult female rats weighing 100-150 g divided into six groups were used. Rats were induced with Parkinsonism by subcutaneous administration of rotenone (2.5 mg/kg) once a day for seven consecutive days. The rats received dimethyl sulfoxide (DMSO)/Olive oil or rotenone dissolved in DMSO/Olive oil. Groups III and IV received Zinc (30 mg/kg) or Linoleic acid (150 µl/kg) while group V received a combination of both, 2 weeks prior to rotenone injection. Groups II and VI served as negative (rotenone group) and positive (Levodopa groups) controls respectively. Oxidative stress levels were assessed by estimating Lipid peroxidation (MDA), total antioxidant capacity, Superoxide dismutase, reduced Glutathione (GSH), glutathione peroxidase and catalase in the midbrain. Histological examination was done to assess structural changes in the midbrain. There was a significant prevention in lipid peroxidation and decrease in the antioxidant status in intervention-treated groups as compared to the rotenone treated group. In addition, histological examination revealed that Parkinsonian rat brains exhibited neuronal damage. Cell death and reduction in neuron size induced by rotenone was prevented by treatment with zinc, linoleic acid and their combination. These results suggest that zinc and linoleic acid and their combination showed significant neuroprotective activity most likely due to the antioxidant effect.

Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production

PubMed Central

Sensi, Stefano L.; Yin, Hong Z.; Carriedo, Sean G.; Rao, Shyam S.; Weiss, John H.

1999-01-01

Synaptically released Zn2+ can enter and cause injury to postsynaptic neurons. Microfluorimetric studies using the Zn2+-sensitive probe, Newport green, examined levels of [Zn2+]i attained in cultured cortical neurons on exposure to N-methyl-d-asparte, kainate, or high K+ (to activate voltage-sensitive Ca2+ channels) in the presence of 300 μM Zn2+. Indicating particularly high permeability through Ca2+-permeable α-amino3-hydroxy-5-methyl-4-isoxazolepropionic-acid/kainate (Ca-A/K) channels, micromolar [Zn2+]i rises were observed only after kainate exposures and only in neurons expressing these channels [Ca-A/K(+) neurons]. Further studies using the oxidation-sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species (ROS) generation that paralleled the [Zn2+]i rises, with rapid oxidation observed only in the case of Zn2+ entry through Ca-A/K channels. Indicating a mitochondrial source of this ROS generation, hydroethidine oxidation was inhibited by the mitochondrial electron transport blocker, rotenone. Additional evidence for a direct interaction between Zn2+ and mitochondria was provided by the observation that the Zn2+ entry through Ca-A/K channels triggered rapid mitochondrial depolarization, as assessed by using the potential-sensitive dye tetramethylrhodamine ethylester. Whereas Ca2+ influx through Ca-A/K channels also triggers ROS production, the [Zn2+]i rises and subsequent ROS production are of more prolonged duration. PMID:10051656

EPR Detection of Cellular and Mitochondrial Superoxide Using Cyclic Hydroxylamines

PubMed Central

Dikalov, Sergey I.; Kirilyuk, Igor A.; Voinov, Maxim; Grigor’ev, Igor A.

2014-01-01

Superoxide (O2•) has been implicated in the pathogenesis of many human diseases but detection of the O2• radicals in biological systems is limited due to inefficiency of O2• spin trapping and lack of site-specific information. In this work we studied production of extracellular, intracellular and mitochondrial O2• in neutrophils, cultured endothelial cells and isolated mitochondria using new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O2• producing stable nitroxides and allowed site-specific O2• detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O2•. Inhibition of EPR signal by SOD2 overexpression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O2• both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected increase in cytoplasm O2• stimulated by PMA but only CM-H and mitoTEMPO-H showed increase in rotenone-induced mitochondrial O2•. These data show that new set of hydroxylamine spin probes provide unique information about site-specific production of O2• radical in extracellular or intracellular compartments, cytoplasm or mitochondria. PMID:21128732

Synchronization of calcium waves by mitochondrial substrates in Xenopus laevis oocytes

NASA Astrophysics Data System (ADS)

Jouaville, Laurence S.; Ichas, François; Holmuhamedov, Ekhson L.; Camacho, Patricia; Lechleiter, James D.

1995-10-01

INXenopus oocytes, as well as other cells, inositol-l,4,5-tris-phosphate (Ins(l,4,5)P3)-induced Ca2+ release1-4 is an excitable process that generates propagating Ca2+ waves5-7 that annihilate upon collision8-12. The fundamental property responsible for excitability13 appears to be the Ca2+ dependency of the Ins(l,4,5)P3 receptor9. Here we report that Ins(l,4,5)P3-induced Ca2+ wave activity is strengthened by oxidizable substrates that energize mitochondria, increasing Ca2+ wave amplitude, velocity and interwave period. The effects of pyruvate/malate are blocked by ruthenium red at the Ca2+ uniporter, by rotenone at complex I, and by antimycin A at complex III, and are subsequently rescued at complex IV by ascorbate tetramethylphenylenediamine (TMPD)14. Our data reveal that potential-driven mitochondrial Ca2+ uptake is a major factor in the regulation of Ins(l,4,5)P3-induced Ca2+ release and clearly demonstrate a physiological role of mitochondria in intracellular Ca2+ signalling.

Novel sila-amide derivatives of N-acetylcysteine protects platelets from oxidative stress-induced apoptosis.

PubMed

Paul, Manoj; Thushara, Ram M; Jagadish, Swamy; Zakai, Uzma I; West, Robert; Kemparaju, Kempaiah; Girish, Kesturu S

2017-02-01

Oxidative stress-induced platelet apoptosis is one among the many causes for the development and progression of many disorders like cardiovascular diseases, arthritis, Alzheimer's disease and many chronic inflammatory responses. Many studies have demonstrated the less optimal effect of N-acetyl cysteine (NAC) in oxidative stress-induced cellular damage. This could be due to its less lipophilicity which makes it difficult to enter the cellular membrane. Therefore in the present study, lipophilic sila-amide derivatives (6a and 6b) synthesized through the reaction of NAC with 3-Aminopropyltrimethylsilane and aminomethyltrimethylsilane were used to determine their protective property against oxidative stress-induced platelet apoptosis. At a concentration of 10 µM, compound 6a and 6b were able to significantly inhibit Rotenone/H 2 O 2 induced platelet apoptotic markers like reactive oxygen species, intracellular calcium level, mitochondrial membrane potential, cytochrome c release from mitochondrial to the cytosol, caspase-9 and -3 activity and phosphatidylserine externalization. Therefore, the compounds can be extrapolated as therapeutic agents to protect platelets from oxidative stress-induced platelet apoptosis and its associated complications.

Short-term exposure to low doses of rotenone induces developmental, biochemical, behavioral, and histological changes in fish.

PubMed

Melo, Karina Motta; Oliveira, Rhaul; Grisolia, Cesar Koppe; Domingues, Inês; Pieczarka, Julio Cesar; de Souza Filho, José; Nagamachi, Cleusa Yoshiko

2015-09-01

Rotenone, a natural compound derived from plants of the genera Derris and Lonchocarpus, is used worldwide as a pesticide and piscicide. This study aims to assess short-term toxicity of rotenone to early-life stages of the fish Danio rerio and Poecilia reticulata using a wide and integrative range of biomarkers (developmental, biochemical, behavioral, and histopathological). Moreover, the species sensitivity distribution (SSD) approach was used to compare rotenone acute toxicity to fish species. Toxicity tests were based on the OECD protocols, fish embryo toxicity test (for D. rerio embryos), and fish acute toxicity test (for P. reticulata juveniles). D. rerio embryos were used to estimate lethal concentrations and analyze embryonic and enzymatic alterations (activity of catalase, glutathione-S-transferase, and cholinesterase), while P. reticulata juveniles were used for the assessment of histological damage in the gills and liver. Rotenone induced significant mortality in zebrafish embryos with a 96-h lethal concentration 50% (LC50) = 12.2 μg/L. Rotenone was embryotoxic, affecting the development of D. rerio embryos, which showed cardiac edema; tail deformities; loss of equilibrium; and a general delay characterized by lack of tail detachment, delayed somite formation, yolk sac absorption, and lack of pigmentation. Biochemical biomarker inhibition was observed for concentrations ≥1 μg/L for CAT and glutathione-S-transferase (GST) and for cholinesterase (ChE) in concentration from 10 μg/L. Behavioral changes were observed for P. reticulata juveniles exposed to concentrations equal to or above 25 μg/L of rotenone; moreover, histological damage in the liver and gills of fish exposed to concentrations equal to or above 2.5 μg/L could be observed. A hazard concentration 5% (HC5) of 3.2 μg/L was estimated considering the acute toxicity data for different fish species (n = 49). Lethal and sublethal effects of rotenone raise a concern about its effects on nontarget fish species, especially because rotenone and its metabolite rotenolone are frequently reported in the microgram range in natural environments for several days after field applications. Rotenone should be used with caution. Given the high toxicity and wide range of sublethal effects here reported, further studies in a chronic exposure scenario are recommended.

BCL2 and BCL(X)L selective inhibitors decrease mitochondrial ATP production in breast cancer cells and are synthetically lethal when combined with 2-deoxy-D-glucose.

PubMed

Lucantoni, Federico; Düssmann, Heiko; Llorente-Folch, Irene; Prehn, Jochen H M

2018-05-25

Cancer cells display differences regarding their engagement of glycolytic vs. mitochondrial oxidative phosphorylation (OXPHOS) pathway. Triple negative breast cancer, an aggressive form of breast cancer, is characterized by elevated glycolysis, while estrogen receptor positive breast cancer cells rely predominantly on OXPHOS. BCL2 proteins control the process of mitochondrial outer membrane permeabilization during apoptosis, but also regulate cellular bioenergetics. Because BCL2 proteins are overexpressed in breast cancer and targetable by selective antagonists, we here analysed the effect of BCL2 and BCL(X)L selective inhibitors, Venetoclax and WEHI-539, on mitochondrial bioenergetics and cell death. Employing single cell imaging using a FRET-based mitochondrial ATP sensor, we found that MCF7 breast cancer cells supplied with mitochondrial substrates reduced their mitochondrial ATP production when treated with Venetoclax or WEHI-539 at concentrations that per se did not induce cell death. Treatments with lower concentrations of both inhibitors also reduced the length of the mitochondrial network and the dynamics, as evaluated by quantitative confocal microscopy. We next tested the hypothesis that mitochondrial ATP production inhibition with BCL2 or BCL(X)L antagonists was synthetically lethal when combined with glycolysis inhibition. Treatment with 2-deoxy-D-glucose in combination with Venetoclax or WEHI-539 synergistically reduced the cellular bioenergetics of ER+ and TNBC breast cancer cells and abolished their clonogenic potential. Synthetic lethality was also observed when cultures were grown in 3D spheres. Our findings demonstrate that BCL2 antagonists exert potent effects on cancer metabolism independent of cell death-inducing effects, and demonstrate a synthetic lethality when these are applied in combination with glycolysis inhibitors.

BCL2 and BCL(X)L selective inhibitors decrease mitochondrial ATP production in breast cancer cells and are synthetically lethal when combined with 2-deoxy-D-glucose

PubMed Central

Lucantoni, Federico; Düssmann, Heiko; Llorente-Folch, Irene; Prehn, Jochen H.M.

2018-01-01

Cancer cells display differences regarding their engagement of glycolytic vs. mitochondrial oxidative phosphorylation (OXPHOS) pathway. Triple negative breast cancer, an aggressive form of breast cancer, is characterized by elevated glycolysis, while estrogen receptor positive breast cancer cells rely predominantly on OXPHOS. BCL2 proteins control the process of mitochondrial outer membrane permeabilization during apoptosis, but also regulate cellular bioenergetics. Because BCL2 proteins are overexpressed in breast cancer and targetable by selective antagonists, we here analysed the effect of BCL2 and BCL(X)L selective inhibitors, Venetoclax and WEHI-539, on mitochondrial bioenergetics and cell death. Employing single cell imaging using a FRET-based mitochondrial ATP sensor, we found that MCF7 breast cancer cells supplied with mitochondrial substrates reduced their mitochondrial ATP production when treated with Venetoclax or WEHI-539 at concentrations that per se did not induce cell death. Treatments with lower concentrations of both inhibitors also reduced the length of the mitochondrial network and the dynamics, as evaluated by quantitative confocal microscopy. We next tested the hypothesis that mitochondrial ATP production inhibition with BCL2 or BCL(X)L antagonists was synthetically lethal when combined with glycolysis inhibition. Treatment with 2-deoxy-D-glucose in combination with Venetoclax or WEHI-539 synergistically reduced the cellular bioenergetics of ER+ and TNBC breast cancer cells and abolished their clonogenic potential. Synthetic lethality was also observed when cultures were grown in 3D spheres. Our findings demonstrate that BCL2 antagonists exert potent effects on cancer metabolism independent of cell death-inducing effects, and demonstrate a synthetic lethality when these are applied in combination with glycolysis inhibitors. PMID:29899841

Dynamic monitoring of p53 translocation to mitochondria for the analysis of specific inhibitors using luciferase-fragment complementation.

PubMed

Noda, Natsumi; Awais, Raheela; Sutton, Robert; Awais, Muhammad; Ozawa, Takeaki

2017-12-01

Intracellular protein translocation plays a pivotal role in regulating complex biological processes, including cell death. The tumor suppressor p53 is a transcription factor activated by DNA damage and oxidative stress that also translocates from the cytosol into the mitochondrial matrix to facilitate necrotic cell death. However, specific inhibitors of p53 mitochondrial translocation are largely unknown. To explore the inhibitors of p53, we developed a bioluminescent probe to monitor p53 translocation from cytosol to mitochondria using luciferase fragment complementation assays. The probe is composed of a novel pair of luciferase fragments, the N-terminus of green click beetle luciferase CBG68 (CBGN) and multiple-complement luciferase fragment (McLuc1). The combination of luciferase fragments showed significant luminescence intensity and high signal-to-background ratio. When the p53 connected with McLuc1 translocates from cytosol into mitochondrial matrix, CBGN in mitochondrial matrix enables to complement with McLuc1, resulting in the restoration of the luminescence. The luminescence intensity was significantly increased under hydrogen peroxide-induced oxidative stress following the complementation of CBGN and McLuc1. Pifithrin-μ, a selective inhibitor of p53 mitochondrial translocation, prevented the mitochondrial translocation of the p53 probe in a concentration-dependent manner. Furthermore, the high luminescence intensity made it easier to visualize the p53 translocation at a single cell level under a bioluminescence microscope. This p53 mitochondrial translocation assay is a new tool for high-throughput screening to identify novel p53 inhibitors, which could be developed as drugs to treat diseases in which necrotic cell death is a major contributor. © 2017 Wiley Periodicals, Inc.

Inhibiting Mitochondrial DNA Ligase IIIα Activates Caspase 1-Dependent Apoptosis in Cancer Cells.

PubMed

Sallmyr, Annahita; Matsumoto, Yoshihiro; Roginskaya, Vera; Van Houten, Bennett; Tomkinson, Alan E

2016-09-15

Elevated levels of DNA ligase IIIα (LigIIIα) have been identified as a biomarker of an alteration in DNA repair in cancer cells that confers hypersensitivity to a LigIIIα inhibitor, L67, in combination with a poly (ADP-ribose) polymerase inhibitor. Because LigIIIα functions in the nucleus and mitochondria, we examined the effect of L67 on these organelles. Here, we show that, although the DNA ligase inhibitor selectively targets mitochondria, cancer and nonmalignant cells respond differently to disruption of mitochondrial DNA metabolism. Inhibition of mitochondrial LigIIIα in cancer cells resulted in abnormal mitochondrial morphology, reduced levels of mitochondrial DNA, and increased levels of mitochondrially generated reactive oxygen species that caused nuclear DNA damage. In contrast, these effects did not occur in nonmalignant cells. Furthermore, inhibition of mitochondrial LigIIIα activated a caspase 1-dependent apoptotic pathway, which is known to be part of inflammatory responses induced by pathogenic microorganisms in cancer, but not nonmalignant cells. These results demonstrate that the disruption of mitochondrial DNA metabolism elicits different responses in nonmalignant and cancer cells and suggests that the abnormal response in cancer cells may be exploited in the development of novel therapeutic strategies that selectively target cancer cells. Cancer Res; 76(18); 5431-41. ©2016 AACR. ©2016 American Association for Cancer Research.

Succinate-driven reverse electron transport in the respiratory chain of plant mitochondria. The effects of rotenone and adenylates in relation to malate and oxaloacetate metabolism.

PubMed Central

Rustin, P; Lance, C

1991-01-01

The effects of rotenone on the succinate-driven reduction of matrix nicotinamide nucleotides were investigated in Percoll-purified mitochondria from potato (Solanum tuberosum) tubers. Depending on the presence of ADP or ATP, rotenone caused an increase or a decrease in the level of reduction of the matrix nicotinamide nucleotides. The increase in the reduction induced by rotenone in the presence of ADP was linked to the oxidation of the malate resulting from the oxidation of succinate. Depending on the experimental conditions, malic enzyme (at pH 6.6 or in the presence of added CoA) or malate dehydrogenase (at pH 7.9) were involved in this oxidation. At pH 7.9, the oxaloacetate produced progressively inhibited the succinate dehydrogenase. In the presence of ATP the production of oxaloacetate was stopped, and succinate dehydrogenase was protected from inhibition by oxaloacetate. However, previously accumulated oxaloacetate transitorily decreased the level of the reduction of the NAD+ driven by succinate, by causing the reversal of the malate dehydrogenase reaction. Under these conditions (i.e. presence of ATP), rotenone strongly inhibited the reduction of NAD+ by succinate-driven reverse electron flow. No evidence for an active reverse electron transport through a rotenone-insensitive path could be obtained. The inhibitory effect of rotenone was masked if malate had previously accumulated, owing to the malate-oxidizing enzymes which reduced part or all of the matrix NAD+. PMID:2001241

Re-evaluation of the H+/site ratio of mitochondrial electron transport with the oxygen pulse technique.

PubMed

Brand, M D; Reynafarje, B; Lehninger, A L

1976-09-25

The number of protons ejected per pair of electrons passing each energy-conserving site in the electron transport chain (the H+/site ratio) has been investigated in rat liver mitochondria by means of the oxygen pulse technique introduced by Mitchell and Moyle (1967) (Biochem. J. 105, 1147-1162). The usual H+/site values of 2.0 observed by this method were found to be substantially underestimated as a result of the influx of phosphate into the mitochondria. This was shown by three different kinds of experiments. 1. Addition of N-ethylmaleimide or mersalyl, inhibitors of mitochondrial phosphate transport, increased the H+/site ratio from 2.0 to 3.0. The dependence of this effect on the concentration of either inhibitor was identical with that for inhibition of phosphate transport. Added phosphate diminished the H+/site ratio to values below 2.0 in the absence of N-ethylmaleimide. N-Ethylmaleimide protected the elevated H+/site ratio of 3.0 against the deleterious effect of added phosphate, but did not prevent a lowering effect of weak acid anions such as 3-hydroxybutyrate. 2. Prior washing of mitochondria to remove the endogenous phosphate that leaks out during the anaerobic preincubation led to H+/site ratios near 3.0, which were not increased by N-ethylmaleimide. Addition of low concentrations of phosphate to such phosphate-depleted mitochondria decreased the H+/site ratio to 2.0; addition of N-ethylmaleimide returned the ratio to 3.0. 3. Lowering the temperature to 5 degrees, which slows down phosphate transport, led to H+/site values of 3.0 even in the absence of N-ethylmaleimide. The H+/site ratio of 3.0 observed in the absence of phosphate movements was not dependent on any narrowly limited set of experimental conditions. It occurred with either Ca2+ or K+ (in the presence of valinomycin) as mobile permeant cation. It was independent of the concentration of succinate, oxygen, mitochondria, or rotenone, additions of Ca2+, Li+, or Na+ and was independent of medium pH between 6.5 and 7.5. Inhibitors of the transport of ions or acids other than phosphate did not affect the H+/site ratio. These results indicate that re-uptake of endogenous phosphate, lost from mitochondria during anaerobic preincubation, reduces the observed H+ ejection and leads to underestimated H+/site ratios of 2.0 in the oxygen pulse method. When phosphate movements are eliminated by the procedures described above, the observed H+/site ratio is about 3.0. This value appears to be closer to the true H+/site ratio for the primary H+ ejection process during electron transport.

Recombinant Buckwheat Trypsin Inhibitor Induces Mitophagy by Directly Targeting Mitochondria and Causes Mitochondrial Dysfunction in Hep G2 Cells.

PubMed

Wang, Zhuanhua; Li, Shanshan; Ren, Rong; Li, Jiao; Cui, Xiaodong

2015-09-09

Mitochondria are essential targets for cancer chemotherapy and other disease treatments. Recombinant buckwheat trypsin inhibitor (rBTI), a member of the potato type I proteinase inhibitor family, was derived from tartary buckwheat extracts. Our results showed that rBTI directly targeted mitochondria and induced mitochondrial fragmentation and mitophagy. This occurs through enhanced depolarization of the mitochondrial membrane potential, increasing reactive oxygen species (ROS) generation associated with the rise of the superoxide dismutase and catalase activity and glutathione peroxidase (GSH) content, and changes in the GSH/oxidized glutathione ratio. Mild and transient ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 mitophagy to remove dysfunctional mitochondria. Furthermore, rBTI could directly induce mitochondrial fragmentation. It was also noted that rBTI highly increased colocalization of mitochondria in treated cells compared to nontreated cells. Tom 20, a subunit of the translocase of the mitochondrial outer membrane complex responsible for recognizing mitochondrial presequences, may be the direct target of rBTI.

Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mena, Natalia P.; Millennium Institute of Cell Dynamics and Biotechnology, Santiago; Bulteau, Anne Laure

2011-06-03

Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters aremore » involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex I and iron accumulation are hallmarks of idiopathic Parkinson's disease, the findings reported here may have relevance for understanding the pathophysiology of this disease.« less

High-throughput screening identifies artesunate as selective inhibitor of cancer stemness: Involvement of mitochondrial metabolism.

PubMed

Subedi, Amit; Futamura, Yushi; Nishi, Mayuko; Ryo, Akihide; Watanabe, Nobumoto; Osada, Hiroyuki

2016-09-02

Cancer stem cells (CSCs) have robust systems to maintain cancer stemness and drug resistance. Thus, targeting such robust systems instead of focusing on individual signaling pathways should be the approach allowing the identification of selective CSC inhibitors. Here, we used the alkaline phosphatase (ALP) assay to identify inhibitors for cancer stemness in induced cancer stem-like (iCSCL) cells. We screened several compounds from natural product chemical library and evaluated hit compounds for their efficacy on cancer stemness in iCSCL tumorspheres. We identified artesunate, an antimalarial drug, as a selective inhibitor of cancer stemness. Artesunate induced mitochondrial dysfunction that selectively inhibited cancer stemness of iCSCL cells, indicating an essential role of mitochondrial metabolism in cancer stemness. Copyright © 2016 Elsevier Inc. All rights reserved.

Synergism of antifungal activity between mitochondrial respiration inhibitors and kojic acid

USDA-ARS?s Scientific Manuscript database

Co-application of certain types of compounds with conventional antimicrobial drugs results in the enhancement of efficacy of drugs through a mechanism termed chemosensitization. We show that kojic acid (KA), a natural product, is a potent chemosensitizer to complex III inhibitors of mitochondrial re...

Demethoxycurcumin, a Natural Derivative of Curcumin Abrogates Rotenone-induced Dopamine Depletion and Motor Deficits by Its Antioxidative and Anti-inflammatory Properties in Parkinsonian Rats

PubMed Central

Ramkumar, Muthu; Rajasankar, Srinivasagam; Gobi, Veerapan Venkatesh; Janakiraman, Udaiyappan; Manivasagam, Thamilarasan; Thenmozhi, Arokiasamy Justin; Essa, Musthafa Mohamed; Chidambaram, Ranganathan; Chidambaram, Saravana Babu; Guillemin, Giles J.

2018-01-01

Background: Parkinson's disease (PD) is a progressive neurodegenerative disorder (NDD) associated with the loss of dopaminergic neurons in the substantia nigra and subsequently has an effect on motor function and coordination. The pathology of PD is multifactorial, in which neuroinflammation and oxidative damage are the two of the main protagonists. Objectives: The present study aims to assess the potential antioxidant and anti-inflammatory effects of demethoxycurcumin (DMC), a natural derivative of curcumin, against rotenone-induced PD in rats. Materials and Methods: Rats were randomized and divided into six groups: control, rotenone (0.5 mg/kg/day, intraperitoneal in sunflower oil) treated for 7 days, rotenone and DMC (5, 10, and 20 mg/kg b.w) cotreated, and DMC (20 mg/kg b.w) alone treated groups. Results: Based on the dopamine concentration and biochemical estimations, the effective dose of DMC was selected and the chronic study was performed. At the end of the experimental period, behavioral studies and protein expression patterns of inflammatory markers were analyzed. Rotenone treatment led to motor dysfunctions, neurochemical deficits, and oxidative stress and enhanced expressions of inflammatory markers, whereas oral administration of DMC attenuated all the above. Conclusion: Even though further research is needed to prove its efficacy in clinical trial, the results of our study showed that DMC may offer a promising and new therapeutic lead for the treatment of NDDs including PD. SUMMARY Curcumin and their derivatives have been shown to be potent neuroprotective effectDemethoxycurcumin (DMC) amolerated the rotenone induced behavioural alterationsDMC abrogated the rotenone induced dopamine deficitsDMC attenuated the rotenone induced oxidative stressDMC diminished the rotenone mediated inflammation. Abbreviations used: COX-2: Cyclooxygenase-2; DA: Dopamine; DMC: Demethoxycurcumin; DMRT: Duncan's multiple range test; GSH: Reduced glutathione; GPx: Glutathione peroxidase; IL-1 β: Interleukin-1 β; IL-6: Interleukin-6; iNOS: Inducible nitric oxide synthase; PD: Parkinson's disease; SN: Substantia nigra; SOD: Superoxide dismutase; TBARS: Thiobarbituric acid reactive substances; TNF-α: Tumor necrosis factor-α. PMID:29576695

The metabolomic signature of Leber's hereditary optic neuropathy reveals endoplasmic reticulum stress.

PubMed

Chao de la Barca, Juan Manuel; Simard, Gilles; Amati-Bonneau, Patrizia; Safiedeen, Zainab; Prunier-Mirebeau, Delphine; Chupin, Stéphanie; Gadras, Cédric; Tessier, Lydie; Gueguen, Naïg; Chevrollier, Arnaud; Desquiret-Dumas, Valérie; Ferré, Marc; Bris, Céline; Kouassi Nzoughet, Judith; Bocca, Cinzia; Leruez, Stéphanie; Verny, Christophe; Miléa, Dan; Bonneau, Dominique; Lenaers, Guy; Martinez, M Carmen; Procaccio, Vincent; Reynier, Pascal

2016-11-01

Leber's hereditary optic neuropathy (MIM#535000), the commonest mitochondrial DNA-related disease, is caused by mutations affecting mitochondrial complex I. The clinical expression of the disorder, usually occurring in young adults, is typically characterized by subacute, usually sequential, bilateral visual loss, resulting from the degeneration of retinal ganglion cells. As the precise action of mitochondrial DNA mutations on the overall cell metabolism in Leber's hereditary optic neuropathy is unknown, we investigated the metabolomic profile of the disease. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites in fibroblasts from 16 patients with Leber's hereditary optic neuropathy and eight healthy control subjects. Latent variable-based statistical methods were used to identify discriminating metabolites. One hundred and twenty-four of the metabolites were considered to be accurately quantified. A supervised orthogonal partial least squares discriminant analysis model separating patients with Leber's hereditary optic neuropathy from control subjects showed good predictive capability (Q 2cumulated = 0.57). Thirty-eight metabolites appeared to be the most significant variables, defining a Leber's hereditary optic neuropathy metabolic signature that revealed decreased concentrations of all proteinogenic amino acids, spermidine, putrescine, isovaleryl-carnitine, propionyl-carnitine and five sphingomyelin species, together with increased concentrations of 10 phosphatidylcholine species. This signature was not reproduced by the inhibition of complex I with rotenone or piericidin A in control fibroblasts. The importance of sphingomyelins and phosphatidylcholines in the Leber's hereditary optic neuropathy signature, together with the decreased amino acid pool, suggested an involvement of the endoplasmic reticulum. This was confirmed by the significantly increased phosphorylation of PERK and eIF2α, as well as the greater expression of C/EBP homologous protein and the increased XBP1 splicing, in fibroblasts from affected patients, all these changes being reversed by the endoplasmic reticulum stress inhibitor, TUDCA (tauroursodeoxycholic acid). Thus, our metabolomic analysis reveals a pharmacologically-reversible endoplasmic reticulum stress in complex I-related Leber's hereditary optic neuropathy fibroblasts, a finding that may open up new therapeutic perspectives for the treatment of Leber's hereditary optic neuropathy with endoplasmic reticulum-targeting drugs. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Inhibition of stress-inducible HSP70 impairs mitochondrial proteostasis and function.

PubMed

Leu, Julia I-Ju; Barnoud, Thibaut; Zhang, Gao; Tian, Tian; Wei, Zhi; Herlyn, Meenhard; Murphy, Maureen E; George, Donna L

2017-07-11

Protein quality control is an important component of survival for all cells. The use of proteasome inhibitors for cancer therapy derives from the fact that tumor cells generally exhibit greater levels of proteotoxic stress than do normal cells, and thus cancer cells tend to be more sensitive to proteasome inhibition. However, this approach has been limited in some cases by toxicity to normal cells. Recently, the concept of inhibiting proteostasis in organelles for cancer therapy has been advanced, in part because it is predicted to have reduced toxicity for normal cells. Here we demonstrate that a fraction of the major stress-induced chaperone HSP70 (also called HSPA1A or HSP72, but hereafter HSP70) is abundantly present in mitochondria of tumor cells, but is expressed at quite low or undetectable levels in mitochondria of most normal tissues and non-tumor cell lines. We show that treatment of tumor cells with HSP70 inhibitors causes a marked change in mitochondrial protein quality control, loss of mitochondrial membrane potential, reduced oxygen consumption rate, and loss of ATP production. We identify several nuclear-encoded mitochondrial proteins, including polyadenylate binding protein-1 (PABPC1), which exhibit decreased abundance in mitochondria following treatment with HSP70 inhibitors. We also show that targeting HSP70 function leads to reduced levels of several mitochondrial-encoded RNA species that encode components of the electron transport chain. Our data indicate that small molecule inhibitors of HSP70 represent a new class of organelle proteostasis inhibitors that impair mitochondrial function in cancer cells, and therefore constitute novel therapeutics.

Seahorse Xfe24 Extracellular Flux Analyzer-based analysis of cellular respiration in Caenorhabditis elegans

PubMed Central

Luz, Anthony L.; Smith, Latasha L.; Rooney, John P.

2015-01-01

Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and inter- as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler) and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters (basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity and proton leak) of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans. PMID:26523474

PDE 5 inhibitor improves insulin sensitivity by enhancing mitochondrial function in adipocytes.

PubMed

Yu, Hea Min; Chung, Hyo Kyun; Kim, Koon Soon; Lee, Jae Min; Hong, Jun Hwa; Park, Kang Seo

2017-11-04

Adipocytes are involved in many metabolic disorders. It was recently reported that phosphodiesterase type 5 (PDE5) is expressed in human adipose tissue. In addition, PDE5 inhibitors have been shown to improve insulin sensitivity in humans. However, the mechanism underlying the role of PDE5 inhibitors as an insulin sensitizer remains largely unknown. The present study was undertaken to investigate the role of the PDE5 inhibitor udenafil in insulin signaling in adipocytes and whether this is mediated through the regulation of mitochondrial function. To study the mechanism underlying the insulin sensitizing action of PDE5 inhibitors, we evaluated quantitative changes in protein or mRNA levels of mitochondrial oxidative phosphorylation (OxPhos) complex, oxygen consumption rate (OCR), and fatty acid oxidation with varying udenafil concentrations in 3T3-L1 cells. Our cell study suggested that udenafil enhanced the insulin signaling pathway in 3T3-L1 cells. Following udenafil treatment, basal mitochondrial OCR, maximal OxPhos capacity, and OxPhos gene expression significantly increased. Finally, we examined whether udenafil can affect the fatty acid oxidation process. Treatment of 3T3-L1 cells with udenafil (10 and 20 μM) significantly increased fatty acid oxidation rate in a dose-dependent manner. In addition, the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) significantly increased. We demonstrated that the PDE5 inhibitor udenafil enhances insulin sensitivity by improving mitochondrial function in 3T3-L1 cells. This might be the mechanism underlying the PDE5 inhibitor-enhanced insulin signaling in adipocytes. This also suggests that udenafil may provide benefit in the treatment of type 2 diabetes and other related cardiovascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure.

PubMed

Rurek, Michal; Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

2015-01-01

The biogenesis of the cauliflower curd mitochondrial proteome was investigated under cold, heat and the recovery. For the first time, two dimensional fluorescence difference gel electrophoresis was used to study the plant mitochondrial complexome in heat and heat recovery. Particularly, changes in the complex I and complex III subunits and import proteins, and the partial disintegration of matrix complexes were observed. The presence of unassembled subunits of ATP synthase was accompanied by impairment in mitochondrial translation of its subunit. In cold and heat, the transcription profiles of mitochondrial genes were uncorrelated. The in-gel activities of respiratory complexes were particularly affected after stress recovery. Despite a general stability of respiratory chain complexes in heat, functional studies showed that their activity and the ATP synthesis yield were affected. Contrary to cold stress, heat stress resulted in a reduced efficiency of oxidative phosphorylation likely due to changes in alternative oxidase (AOX) activity. Stress and stress recovery differently modulated the protein level and activity of AOX. Heat stress induced an increase in AOX activity and protein level, and AOX1a and AOX1d transcript level, while heat recovery reversed the AOX protein and activity changes. Conversely, cold stress led to a decrease in AOX activity (and protein level), which was reversed after cold recovery. Thus, cauliflower AOX is only induced by heat stress. In heat, contrary to the AOX activity, the activity of rotenone-insensitive internal NADH dehydrogenase was diminished. The relevance of various steps of plant mitochondrial biogenesis to temperature stress response and recovery is discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells.

PubMed

Sun, Wen; Wu, Xiaxia; Gao, Hongwei; Yu, Jie; Zhao, Wenwen; Lu, Jin-Jian; Wang, Jinhua; Du, Guanhua; Chen, Xiuping

2017-07-01

Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca 2+ ) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium accumulation is a critical mediator in MAM-induced necroptosis through sustained JNK activation and mitochondrial ROS production. Our study also provided new insights into the molecular regulation of necroptosis in human colon cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

RIP1-mediated mitochondrial dysfunction and ROS production contributed to tumor necrosis factor alpha-induced L929 cell necroptosis and autophagy.

PubMed

Ye, Yuan-Chao; Wang, Hong-Ju; Yu, Lu; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2012-12-01

Tumor necrosis factor alpha (TNFα) induces necroptosis and autophagy; however, the detailed molecular mechanism is not fully understood. In this study, we found that TNFα administration caused mitochondrial dysfunction and reactive oxygen species (ROS) production, which led to necroptosis and autophagy in murine fibrosarcoma L929 cells. Notably, the RIP1 (serine-threonine kinase receptor-interacting protein 1, a main adaptor protein of necroptosis) specific inhibitor necrostatin-1 (Nec-1) recovered mitochondrial dysfunction and ROS production due to TNFα administration. Moreover, pan-caspase inhibitor z-VAD-fmk (zVAD) increased RIP1 expression and exacerbated TNFα-induced mitochondrial dysfunction and ROS production, indicating that RIP1 led to mitochondrial dysfunction and ROS production. In addition, cytochrome c release from mitochondria was accompanied with TNFα administration, and Nec-1 blocked the release of cytochrome c upon TNFα administration, while zVAD enhanced the release. These further suggested that RIP1 induced mitochondrial dysfunction accompanied with cytochrome c release. Furthermore, autophagy inhibitor 3-methyladenine (3MA) did not affect RIP1 expression as well as mitochondrial dysfunction and ROS production. Together with our previous publication that autophagy was a downstream consequence of necroptosis, we concluded that TNFα induced mitochondrial dysfunction accompanied with ROS production and cytochrome c release via RIP1, leading to necroptosis and resulting autophagic cell death. Copyright © 2012 Elsevier B.V. All rights reserved.

Relative toxicities of similar formulations of pyrethrum and rotenone to fish and immature stoneflies

USGS Publications Warehouse

Bridges, W.R.; Cope, O.B.

1965-01-01

The insecticidal properties of rotenone and pyrethrum have been known for many years. Both toxicants have long histories of extensive use for control of a variety of insect pests. Rotenone has been also employed as a management tool by fishery biologists to rehabilitate fishing waters, and its toxicity to fish in a number of ecological situations is well established. Pyrethrum's toxicity to fish is less well known.

Probing neurodegeneration and aging: A PET approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

VanBrocklin, H.F.

1995-12-31

Positron Emission Tomography (PET) imaging has received wide application to the study of the aging brain and its diseases, most notably Parkinson`s Disease (PD) and Alzheimer`s Disease (AD). Basic neurological processes such as blood flow and glucose metabolism have been most often measured. Radioligands developed for specific neurochemical systems have amplified the flow and metabolism studies by more precisely defining the changes associated with degenerative processes. Our present research focuses on two additional applications of radiopharmaceutical development and PET imaging - (1) investigating the fundamental mechanisms of neurodegeneration and aging, and (2) assessing novel therapeutic intervention for PD with PETmore » imaging. We have synthesized fluorine-18 labeled analogs of rotenone, a natural product that possesses high affinity to Complex I of the mitochondrial electron transport chain, and evaluated their potential to study changes in neuronal mitochondrial density and function. A large body evidence points to mitochondrial dysfunction as a key factor in aging and neurodegeneration. We are also currently evaluating the use of genetically transfected cells to treat PD. Primates are being imaged with [{sup 18}F]flouro-m-L-tyrosine before and after MPTP Parkinsonian type lesioning and following implantation of genetically altered cells capable of secreting tyrosine hydroxylase into the lesioned area. The ability to develop and apply PET probes has significantly enhanced the understanding of normal, aging, and degenerative processes of the brain.« less

Characterization of the NADH:ubiquinone oxidoreductase (complex I) in the trypanosomatid Phytomonas serpens (Kinetoplastida).

PubMed

Cermáková, Petra; Verner, Zdenek; Man, Petr; Lukes, Julius; Horváth, Anton

2007-06-01

NADH dehydrogenase activity was characterized in the mitochondrial lysates of Phytomonas serpens, a trypanosomatid flagellate parasitizing plants. Two different high molecular weight NADH dehydrogenases were characterized by native PAGE and detected by direct in-gel activity staining. The association of NADH dehydrogenase activities with two distinct multisubunit complexes was revealed in the second dimension performed under denaturing conditions. One subunit present in both complexes cross-reacted with the antibody against the 39 kDa subunit of bovine complex I. Out of several subunits analyzed by MS, one contained a domain characteristic for the LYR family subunit of the NADH:ubiquinone oxidoreductases. Spectrophotometric measurement of the NADH:ubiquinone 10 and NADH:ferricyanide dehydrogenase activities revealed their different sensitivities to rotenone, piericidin, and diphenyl iodonium.

Mitochondrial dysfunction enhances cisplatin resistance in human gastric cancer cells via the ROS-activated GCN2-eIF2α-ATF4-xCT pathway

PubMed Central

Wang, Sheng-Fan; Chen, Meng-Shian; Chou, Yueh-Ching; Ueng, Yune-Fang; Yin, Pen-Hui; Yeh, Tien-Shun; Lee, Hsin-Chen

2016-01-01

Mitochondrial DNA mutations and defects in mitochondrial enzymes have been identified in gastric cancers, and they might contribute to cancer progression. In previous studies, mitochondrial dysfunction was induced by oligomycin-enhanced chemoresistance to cisplatin. Herein, we dissected the regulatory mechanism for mitochondrial dysfunction-enhanced cisplatin resistance in human gastric cancer cells. Repeated cisplatin treatment-induced cisplatin-resistant cells exhibited high SLC7A11 (xCT) expression, and xCT inhibitors (sulfasalazine or erastin), xCT siRNA, or a GSH synthesis inhibitor (buthionine sulphoximine, BSO) could sensitize these cells to cisplatin. Clinically, the high expression of xCT was associated with a poorer prognosis for gastric cancer patients under adjuvant chemotherapy. Moreover, we found that mitochondrial dysfunction enhanced cisplatin resistance and up-regulated xCT expression, as well as intracellular glutathione (GSH). The xCT inhibitors, siRNA against xCT or BSO decreased mitochondrial dysfunction-enhanced cisplatin resistance. We further demonstrated that the upregulation of the eIF2α-ATF4 pathway contributed to mitochondrial dysfunction-induced xCT expression, and activated eIF2α kinase GCN2, but not PERK, stimulated the eIF2α-ATF4-xCT pathway in response to mitochondrial dysfunction-increased reactive oxygen species (ROS) levels. In conclusion, our results suggested that the ROS-activated GCN2-eIF2α-ATF4-xCT pathway might contribute to mitochondrial dysfunction-enhanced cisplatin resistance and could be a potential target for gastric cancer therapy. PMID:27708226

Mitochondrial dysfunction enhances cisplatin resistance in human gastric cancer cells via the ROS-activated GCN2-eIF2α-ATF4-xCT pathway.

PubMed

Wang, Sheng-Fan; Chen, Meng-Shian; Chou, Yueh-Ching; Ueng, Yune-Fang; Yin, Pen-Hui; Yeh, Tien-Shun; Lee, Hsin-Chen

2016-11-08

Mitochondrial DNA mutations and defects in mitochondrial enzymes have been identified in gastric cancers, and they might contribute to cancer progression. In previous studies, mitochondrial dysfunction was induced by oligomycin-enhanced chemoresistance to cisplatin. Herein, we dissected the regulatory mechanism for mitochondrial dysfunction-enhanced cisplatin resistance in human gastric cancer cells. Repeated cisplatin treatment-induced cisplatin-resistant cells exhibited high SLC7A11 (xCT) expression, and xCT inhibitors (sulfasalazine or erastin), xCT siRNA, or a GSH synthesis inhibitor (buthionine sulphoximine, BSO) could sensitize these cells to cisplatin. Clinically, the high expression of xCT was associated with a poorer prognosis for gastric cancer patients under adjuvant chemotherapy. Moreover, we found that mitochondrial dysfunction enhanced cisplatin resistance and up-regulated xCT expression, as well as intracellular glutathione (GSH). The xCT inhibitors, siRNA against xCT or BSO decreased mitochondrial dysfunction-enhanced cisplatin resistance. We further demonstrated that the upregulation of the eIF2α-ATF4 pathway contributed to mitochondrial dysfunction-induced xCT expression, and activated eIF2α kinase GCN2, but not PERK, stimulated the eIF2α-ATF4-xCT pathway in response to mitochondrial dysfunction-increased reactive oxygen species (ROS) levels. In conclusion, our results suggested that the ROS-activated GCN2-eIF2α-ATF4-xCT pathway might contribute to mitochondrial dysfunction-enhanced cisplatin resistance and could be a potential target for gastric cancer therapy.

Bruton's tyrosine kinase inhibition increases BCL-2 dependence and enhances sensitivity to venetoclax in chronic lymphocytic leukemia.

PubMed

Deng, J; Isik, E; Fernandes, S M; Brown, J R; Letai, A; Davids, M S

2017-10-01

Although the BTK inhibitor ibrutinib has transformed the management of patients with chronic lymphocytic leukemia (CLL), it does not induce substantial apoptosis in vitro, and as such the mechanisms underlying its ability to kill CLL cells are not well understood. Acalabrutinib, a more specific BTK inhibitor now in development, also appears to be highly effective in CLL, but the connection of its mechanism with CLL cell death is also unclear. Using dynamic BH3 profiling, we analyzed alterations in the function of the mitochondrial apoptotic pathway induced by ibrutinib and acalabrutinib. We studied CLL patient samples treated ex vivo with both drugs, as well as primary samples from CLL patients on clinical trials of both drugs. We found that BTK inhibition enhances mitochondrial BCL-2 dependence without significantly altering overall mitochondrial priming. Enhancement of BCL-2 dependence was accompanied by an increase in the pro-apoptotic protein BIM. In contrast, treatment with the selective BCL-2 inhibitor venetoclax enhanced overall mitochondrial priming without increasing BCL-2 dependence. Pre-treatment of CLL cells with either BTK inhibitor, whether ex vivo or in vivo in patients, enhanced killing by venetoclax. Our data suggest that BTK inhibition enhances mitochondrial BCL-2 dependence, supporting the ongoing development of clinical trials combining BTK and BCL-2 inhibition.

Bruton’s tyrosine kinase inhibition increases BCL-2 dependence and enhances sensitivity to venetoclax in chronic lymphocytic leukemia

PubMed Central

Deng, Jing; Isik, Elif; Fernandes, Stacey M.; Brown, Jennifer R.; Letai, Anthony; Davids, Matthew S.

2017-01-01

Although the BTK inhibitor ibrutinib has transformed the management of patients with CLL, it does not induce substantial apoptosis in vitro, and as such the mechanisms underlying its ability to kill CLL cells are not well understood. Acalabrutinib, a more specific BTK inhibitor now in development, also appears to be highly effective in CLL, but the connection of its mechanism with CLL cell death is also unclear. Using dynamic BH3 profiling, we analyzed alterations in the function of the mitochondrial apoptotic pathway induced by ibrutinib and acalabrutinib. We studied CLL patient samples treated ex vivo with both drugs, as well as primary samples from CLL patients on clinical trials of both drugs. We found that BTK inhibition enhances mitochondrial BCL-2 dependence without significantly altering overall mitochondrial priming. Enhancement of BCL-2 dependence was accompanied by an increase in the pro-apoptotic protein BIM. In contrast, treatment with the selective BCL-2 inhibitor venetoclax enhanced overall mitochondrial priming without increasing BCL-2 dependence. Pre-treatment of CLL cells with either BTK inhibitor, whether ex vivo or in vivo in patients, enhanced killing by venetoclax. Our data suggest that BTK inhibition enhances mitochondrial BCL2 dependence, supporting the ongoing development of clinical trials combining BTK and BCL-2 inhibition. PMID:28111464

EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines.

PubMed

Dikalov, Sergey I; Kirilyuk, Igor A; Voinov, Maxim; Grigor'ev, Igor A

2011-04-01

Superoxide (O₂ⁱ⁻) has been implicated in the pathogenesis of many human diseases, but detection of the O(2)(•-) radicals in biological systems is limited due to inefficiency of O₂ⁱ⁻ spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O₂ⁱ⁻ in neutrophils, cultured endothelial cells and isolated mitochondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O₂ⁱ⁻, producing stable nitroxides and allowing site-specific cO₂ⁱ⁻ detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O₂ⁱ⁻. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O₂ⁱ⁻ both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected an increase in cytoplasm O₂ⁱ⁻ stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O₂ⁱ⁻. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O₂ⁱ⁻ radical in extracellular or intracellular compartments, cytoplasm or mitochondria.

Critical contribution of RIPK1 mediated mitochondrial dysfunction and oxidative stress to compression-induced rat nucleus pulposus cells necroptosis and apoptosis.

PubMed

Chen, Songfeng; Lv, Xiao; Hu, Binwu; Zhao, Lei; Li, Shuai; Li, Zhiliang; Qing, Xiangcheng; Liu, Hongjian; Xu, Jianzhong; Shao, Zengwu

2018-04-28

The aim of this study was to investigate whether RIPK1 mediated mitochondrial dysfunction and oxidative stress contributed to compression-induced nucleus pulposus (NP) cells necroptosis and apoptosis, together with the interplay relationship between necroptosis and apoptosis in vitro. Rat NP cells underwent various periods of 1.0 MPa compression. To determine whether compression affected mitochondrial function, we evaluated the mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP), mitochondrial ultrastructure and ATP content. Oxidative stress-related indicators reactive oxygen species, superoxide dismutase and malondialdehyde were also assessed. To verify the relevance between oxidative stress and necroptosis together with apoptosis, RIPK1 inhibitor necrostatin-1(Nec-1), mPTP inhibitor cyclosporine A (CsA), antioxidants and small interfering RNA technology were utilized. The results established that compression elicited a time-dependent mitochondrial dysfunction and elevated oxidative stress. Nec-1 and CsA restored mitochondrial function and reduced oxidative stress, which corresponded to decreased necroptosis and apoptosis. CsA down-regulated mitochondrial cyclophilin D expression, but had little effects on RIPK1 expression and pRIPK1 activation. Additionally, we found that Nec-1 largely blocked apoptosis; whereas, the apoptosis inhibitor Z-VAD-FMK increased RIPK1 expression and pRIPK1 activation, and coordinated regulation of necroptosis and apoptosis enabled NP cells survival more efficiently. In contrast to Nec-1, SiRIPK1 exacerbated mitochondrial dysfunction and oxidative stress. In summary, RIPK1-mediated mitochondrial dysfunction and oxidative stress play a crucial role in NP cells necroptosis and apoptosis during compression injury. The synergistic regulation of necroptosis and apoptosis may exert more beneficial effects on NP cells survival, and ultimately delaying or even retarding intervertebral disc degeneration.

Ameliorative effect of Sida cordifolia in rotenone induced oxidative stress model of Parkinson's disease.

PubMed

Khurana, Navneet; Gajbhiye, Asmita

2013-12-01

Present study focused on the evaluation of aqueous extract of Sida cordifolia (AESC), and its different fractions; hexane (HFSC), chloroform (CFSC) and aqueous (AFSC), against rotenone induced biochemical, neurochemical, histopathological and behavioral alterations in a rat model of Parkinson's disease (PD). An estimation of the level of thiobarbituric acid reactive substances (TBARS), glutathione (GSH) and catalase (CAT) along with superoxide anion generation (SAG) in different brain regions (cortex, midbrain and cerebellum) was carried out to assess biochemical changes. Behavioral evaluation tests (catalepsy, rearing behavior and posture instability) and neurochemical estimations (norepinephrine, dopamine and serotonin level) along with histopathological evaluations of different brain regions were also performed. The varying doses (50, 100, 250mg/kg; p.o.) of different test treatments (AESC, HFSC, CFSC and AFSC) were co-administered along with rotenone (2mg/kg; s.c.), for a period of 35 days to rats of various groups and compared with rotenone per se (negative control) and l-deprenyl (positive control; 10mg/kg; p.o.) treated groups for the above mentioned parameters. The increase in catalepsy and posture instability along with decrease in rearing behavior observed due to rotenone treatment was significantly attenuated by co-treatment with varying doses of AESC and AFSC. Results of the histopathological studies of different brain regions of rats showed eosinophilic lesions in the mid brain region due to rotenone treatment. The eosinophilic lesions were significantly attenuated in co-treated groups of AESC-100mg/kg and AFSC-100mg/kg. Rotenone induced oxidative damage, revealed by increased level of TBARS, SAG and decreased level of GSH and CAT in mid brain region of rats, was attenuated by the co-treatment of AESC and AFSC. The rotenone induced decrease of dopamine level in the midbrain region of rats was also attenuated by co-treatment of AESC-100mg/kg and AFSC-100mg/kg. The maximum effect in all the above activities was observed in AFSC (100mg/kg) treated group, which was comparable to l-deprenyl treated group. The HFSC and CFSC co-treatment failed to show significant attenuation of rotenone induced damage. These results indicate the possible therapeutic potential of most polar fraction of AESC i.e. AFSC in PD by virtue of its antioxidative actions. Copyright © 2013 Elsevier Inc. All rights reserved.

Succinate modulation of H2O2 release at NADH:ubiquinone oxidoreductase (Complex I) in brain mitochondria

PubMed Central

Zoccarato, Franco; Cavallini, Lucia; Bortolami, Silvia; Alexandre, Adolfo

2007-01-01

Complex I (NADH:ubiquinone oxidoreductase) is responsible for most of the mitochondrial H2O2 release, both during the oxidation of NAD-linked substrates and during succinate oxidation. The much faster succinate-dependent H2O2 production is ascribed to Complex I, being rotenone-sensitive. In the present paper, we report high-affinity succinate-supported H2O2 generation in the absence as well as in the presence of GM (glutamate/malate) (1 or 2 mM of each). In brain mitochondria, their only effect was to increase from 0.35 to 0.5 or to 0.65 mM the succinate concentration evoking the semi-maximal H2O2 release. GM are still oxidized in the presence of succinate, as indicated by the oxygen-consumption rates, which are intermediate between those of GM and of succinate alone when all substrates are present together. This effect is removed by rotenone, showing that it is not due to inhibition of succinate influx. Moreover, α-oxoglutarate production from GM, a measure of the activity of Complex I, is decreased, but not stopped, by succinate. It is concluded that succinate-induced H2O2 production occurs under conditions of regular downward electron flow in Complex I. Succinate concentration appears to modulate the rate of H2O2 release, probably by controlling the hydroquinone/quinone ratio. PMID:17477844

A novel method for determining human ex vivo submaximal skeletal muscle mitochondrial function

PubMed Central

Hey-Mogensen, Martin; Gram, Martin; Jensen, Martin Borch; Lund, Michael Taulo; Hansen, Christina Neigaard; Scheibye-Knudsen, Morten; Bohr, Vilhelm A; Dela, Flemming

2015-01-01

Abstract Despite numerous studies, there is no consensus about whether mitochondrial function is altered with increased age. The novelty of the present study is the determination of mitochondrial function at submaximal activity rates, which is more physiologically relevant than the ex vivo functionality protocols used previously. Muscle biopsies were taken from 64 old or young male subjects (aged 60–70 or 20–30 years). Aged subjects were recruited as trained or untrained. Muscle biopsies were used for the isolation of mitochondria and subsequent measurements of DNA repair, anti-oxidant capacity and mitochondrial protein levels (complexes I–V). Mitochondrial function was determined by simultaneous measurement of oxygen consumption, membrane potential and hydrogen peroxide emission using pyruvate + malate (PM) or succinate + rotenone (SR) as substrates. Proton leak was lower in aged subjects when determined at the same membrane potential and was unaffected by training status. State 3 respiration was lower in aged untrained subjects. This effect, however, was alleviated in aged trained subjects. H2O2 emission with PM was higher in aged subjects, and was exacerbated by training, although it was not changed when using SR. However, with a higher manganese superoxide dismuthase content, the trained aged subjects may actually have lower or similar mitochondrial superoxide emission compared to the untrained subjects. We conclude that ageing and the physical activity level in aged subjects are both related to changes in the intrinsic functionality of the mitochondrion in skeletal muscle. Both of these changes could be important factors in determining the metabolic health of the aged skeletal muscle cell. Key points The present study utilized a novel method aiming to investigate mitochondrial function in human skeletal muscle at submaximal levels and at a predefined membrane potential. The effect of age and training status was investigated using a cross-sectional design. Ageing was found to be related to decreased leak regardless of training status. Increased training status was associated with increased mitochondrial hydrogen peroxide emission. PMID:26096709

Effect of injected rotenone on the production and composition of urine from the rainbow trout (Salmo gairdneri)

USGS Publications Warehouse

Erickson, D.A.; Gingerich, W.H.

1986-01-01

Renal function was evaluated in adult rainbow trout (Salmo gairdneri) dosed i.a. with rotenone at 225 and 275 μg/kg. The chemical composition of urine samples and urine flow rates collected over a 5-h pretreatment period were compared with hourly urine samples collected over a 5-h posttreatment period. Significant increases in osmolality and in concentrations of sodium, potassium, chloride, glucose, and total protein were observed in the urine of treated fish. Urine solute concentrations reached maximum values within 1 to 3 h after treatment and decreased thereafter, indicating that the effects were reversible. Concentrations of sodium and chloride were highly correlated in 2-h posttreatment urine samples at the low (r = 0.922) and high (r = 0.981) rotenone treatments. Urine flow rates were reduced in trout at each dose of rotenone but the decrease in volume of urine voided was not dose-dependent. In a separate study, [14C]polyethylene glycol was used as a filtration marker to determine the effect of rotenone treatment (225 &mu:g/kg) on urine flow rate, glomerular filtration rate, and renal water reabsorption. We showed that posttreatment urine flow rates were reduced partly by reduced glomerular filtration and partly by increased water reabsorption. Transient increases in plasma osmolality and hematocrit also were observed 0.5 h after rotenone treatment.

Mitochondrial Respiration after One Session of Calf Raise Exercise in Patients with Peripheral Vascular Disease and Healthy Older Adults

PubMed Central

Wohlwend, Martin; Rognmo, Øivind; Mattsson, Erney J. R.

2016-01-01

Purpose Mitochondria are essential for energy production in the muscle cell and for this they are dependent upon a sufficient supply of oxygen by the circulation. Exercise training has shown to be a potent stimulus for physiological adaptations and mitochondria play a central role. Whether changes in mitochondrial respiration are seen after exercise in patients with a reduced circulation is unknown. The aim of the study was to evaluate the time course and whether one session of calf raise exercise stimulates mitochondrial respiration in the calf muscle of patients with peripheral vascular disease. Methods One group of patients with peripheral vascular disease (n = 11) and one group of healthy older adults (n = 11) were included. Patients performed one session of continuous calf raises followed by 5 extra repetitions after initiation of pain. Healthy older adults performed 100 continuous calf raises. Gastrocnemius muscle biopsies were collected at baseline and 15 minutes, one hour, three hours and 24 hours after one session of calf raise exercise. A multi substrate (octanoylcarnitine, malate, adp, glutamate, succinate, FCCP, rotenone) approach was used to analyze mitochondrial respiration in permeabilized fibers. Mixed-linear model for repeated measures was used for statistical analyses. Results Patients with peripheral vascular disease have a lower baseline respiration supported by complex I and they increase respiration supported by complex II at one hour post-exercise. Healthy older adults increase respiration supported by electron transfer flavoprotein and complex I at one hour and 24 hours post-exercise. Conclusion Our results indicate a shift towards mitochondrial respiration supported by complex II as being a pathophysiological component of peripheral vascular disease. Furthermore exercise stimulates mitochondrial respiration already after one session of calf raise exercise in patients with peripheral vascular disease and healthy older adults. Trial Registration ClinicalTrials.gov NCT01842412 PMID:27760222

Rotenone and Paraquat do not Directly Activate Microglia or Induce Inflammatory Cytokine Release

PubMed Central

Klintworth, Heather; Garden, Gwenn; Xia, Zhengui

2009-01-01

Both epidemiological and pathological data suggest an inflammatory response including microglia activation and neuro-inflammation in the Parkinsonian brain. Treatments with lipopolysacchride (LPS), rotenone and paraquat have been used as models for Parkinson’s disease, as they cause dopaminergic neuron degeneration in culture and in animals. Recent studies have suggested that rotenone and paraquat induce neuro-inflammation, however, it is not known if they can directly activate microglia. Here, we use primary cultured microglia to address this question. Microglia activation was analyzed by morphological changes and release of nitric oxide and inflammatory cytokines. Treatment with LPS was used as a positive control. While LPS induced morphological changes characteristic of microglial activation and release of nitric oxide and inflammatory cytokines, rotenone and paraquat did not. Our results suggest that paraquat and rotenone do not act directly on microglia and that neuro-inflammation and microglial activation in animals treated with these agents is likely non-cell autonomous, and may occur as a result of dopaminergic neuron damage or factors released by neurons and other cells. PMID:19559752

Rapid method for measuring rotenone in water at piscicidal concentrations

USGS Publications Warehouse

Dawson, V.K.; Harman, P.D.; Schultz, D.P.; Allen, J.L.

1983-01-01

A high-performance liquid chromatography (HPLC) procedure that is rapid, specific, and sensitive (limit of detection <0.005 mg/liter) was developed for monitoring application and degradation rates of rotenone. For analysis, a water sample is buffered to pH 5 and injected through a Sep Pak(R) C18 disposable cartridge. The cartridge adsorbs and retains the rotenone which then can be eluted quantitatively from the cartridge with a small volume of methanol. This step effectively concentrates the sample and provides sample cleanup. The methanol extract is analyzed directly by HPLC on an MCH 10 reverse-phase column; methanol: water (75:25, volume : volume) is the mobile phase and flow rate is 1.5 ml/minute. The rotenone is detected by ultraviolet spectrophotometry at a wavelength of 295 nm.

Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

PubMed

Löw, Hans; Crane, Frederick L; Morré, D James

2012-11-01

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

Role of cellular bioenergetics in smooth muscle cell proliferation induced by platelet-derived growth factor.

PubMed

Perez, Jessica; Hill, Bradford G; Benavides, Gloria A; Dranka, Brian P; Darley-Usmar, Victor M

2010-05-13

Abnormal smooth muscle cell proliferation is a hallmark of vascular disease. Although growth factors are known to contribute to cell hyperplasia, the changes in metabolism associated with this response, particularly mitochondrial respiration, remain unclear. Given the increased energy requirements for proliferation, we hypothesized that PDGF (platelet-derived growth factor) would stimulate glycolysis and mitochondrial respiration and that this elevated bioenergetic capacity is required for smooth muscle cell hyperplasia. To test this hypothesis, cell proliferation, glycolytic flux and mitochondrial oxygen consumption were measured after treatment of primary rat aortic VSMCs (vascular smooth muscle cells) with PDGF. PDGF increased basal and maximal rates of glycolytic flux and mitochondrial oxygen consumption; enhancement of these bioenergetic pathways led to a substantial increase in the mitochondrial reserve capacity. Interventions with the PI3K (phosphoinositide 3-kinase) inhibitor LY-294002 or the glycolysis inhibitor 2-deoxy-D-glucose abrogated PDGF-stimulated proliferation and prevented augmentation of glycolysis and mitochondrial reserve capacity. Similarly, when L-glucose was substituted for D-glucose, PDGF-dependent proliferation was abolished, as were changes in glycolysis and mitochondrial respiration. Interestingly, LDH (lactate dehydrogenase) protein levels and activity were significantly increased after PDGF treatment. Moreover, substitution of L-lactate for D-glucose was sufficient to increase mitochondrial reserve capacity and cell proliferation after treatment with PDGF; these effects were inhibited by the LDH inhibitor oxamate. These results suggest that glycolysis, by providing substrates that enhance the mitochondrial reserve capacity, plays an essential role in PDGF-induced cell proliferation, underscoring the integrated metabolic response required for proliferation of VSMCs in the diseased vasculature.

Ginsenoside Rg3 attenuates sepsis-induced injury and mitochondrial dysfunction in liver via AMPK-mediated autophagy flux.

PubMed

Xing, Wei; Yang, Lei; Peng, Yue; Wang, Qianlu; Gao, Min; Yang, Mingshi; Xiao, Xianzhong

2017-08-31

Sepsis-led mitochondrial dysfunction has become a critical pathophysiological procedure in sepsis. Since ginsenosides have been applied in the treatment of mitochondrial dysfunction, ginsenoside Rg3 was employed to study its effects on the mitochondrial dysfunction induced by sepsis. The apoptosis rate, oxygen consumption rate (OCR), reactive oxygen species (ROS), antioxidant glutathione (GSH) pools, and mitochondrial transmembrane potential (MTP) were determined in LPS-induced sepsis hepatocytes treated with different concentrations of Rg3. Then, the protein expression levels of mitochondrial biogenesis related transcription factors, autophagy-related proteins, and AMP-activated protein kinase (AMPK) signal pathway related proteins were determined by Western blotting in both in vitro and in vivo sepsis models. Rg3 shows functions of promotion of OCR, attenuation of ROS, and maintenance of GSH pools, and its conjugating activity in the in vitro sepsis models. Rg3-treated cells were observed to have a higher MTP value compared with the LPS only induced cells. Moreover, Rg3 treatment can inhibit mitochondrial dysfunction via increasing the protein expression levels of mitochondrial biogenesis related transcription factors. Rg3 treatment has the function of inhibitor of apoptosis of human primary hepatocytes, and Rg3 can up-regulate the autophagy-related proteins and activate AMPK signal pathway in sepsis models. Meanwhile, the mitochondrial protective function exerted by Rg3 decreased after the autophagy inhibitors or AMPK inhibitor treatment in LPS-induced human primary hepatocytes. Rg3 can improve mitochondrial dysfunction by regulating autophagy in mitochondria via activating the AMPK signal pathway, thus protecting cell and organ injuries caused by sepsis. © 2017 The Author(s).

Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors

PubMed Central

Issaq, Sameer H; Teicher, Beverly A; Monks, Anne

2014-01-01

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy. PMID:24553119

Apoptosis inducing factor (AIF) mediates lethal redox stress induced by menadione.

PubMed

Wiraswati, Hesti Lina; Hangen, Emilie; Sanz, Ana Belén; Lam, Ngoc-Vy; Reinhardt, Camille; Sauvat, Allan; Mogha, Ariane; Ortiz, Alberto; Kroemer, Guido; Modjtahedi, Nazanine

2016-11-22

Mitochondrial apoptosis inducing factor (AIF) is a redox-active enzyme that participates to the biogenesis/maintenance of complex I of the respiratory chain, yet also contributes to catabolic reactions in the context of regulated cell death when AIF translocates to the cytosol and to the nucleus. Here we explore the contribution of AIF to cell death induced by menadione (2-methyl-1,4-naphtoquinone; also called vitamin K3) in conditions in which this pro-oxidant does not cause the mitochondrial release of AIF, yet causes caspase-independent cell killing. Depletion of AIF from human cancer cells reduced the cytotoxicity of menadione. This cytoprotective effect was accompanied by the maintenance of high levels of reduced glutathione (GSH), which are normally depleted by menadione. In addition, AIF depletion reduced the arylation of cellular proteins induced by menadione. This menadione-triggered arylation, which can be measured by a fluorescence assay, is completely suppressed by addition of exogenous glutathione or N-acetyl cysteine. Complex I inhibition by Rotenone did not mimic the cytoprotective action of AIF depletion. Altogether, these results are compatible with the hypothesis that mitochondrion-sessile AIF facilitates lethal redox cycling of menadione, thereby precipitating protein arylation and glutathione depletion.

Apoptosis inducing factor (AIF) mediates lethal redox stress induced by menadione

PubMed Central

Wiraswati, Hesti Lina; Hangen, Emilie; Sanz, Ana Belén; Lam, Ngoc-Vy; Reinhardt, Camille; Sauvat, Allan; Mogha, Ariane; Ortiz, Alberto

2016-01-01

Mitochondrial apoptosis inducing factor (AIF) is a redox-active enzyme that participates to the biogenesis/maintenance of complex I of the respiratory chain, yet also contributes to catabolic reactions in the context of regulated cell death when AIF translocates to the cytosol and to the nucleus. Here we explore the contribution of AIF to cell death induced by menadione (2-methyl-1,4-naphtoquinone; also called vitamin K3) in conditions in which this pro-oxidant does not cause the mitochondrial release of AIF, yet causes caspase-independent cell killing. Depletion of AIF from human cancer cells reduced the cytotoxicity of menadione. This cytoprotective effect was accompanied by the maintenance of high levels of reduced glutathione (GSH), which are normally depleted by menadione. In addition, AIF depletion reduced the arylation of cellular proteins induced by menadione. This menadione-triggered arylation, which can be measured by a fluorescence assay, is completely suppressed by addition of exogenous glutathione or N-acetyl cysteine. Complex I inhibition by Rotenone did not mimic the cytoprotective action of AIF depletion. Altogether, these results are compatible with the hypothesis that mitochondrion-sessile AIF facilitates lethal redox cycling of menadione, thereby precipitating protein arylation and glutathione depletion. PMID:27738311

Rotenone formulation fate in Lake Davis following the 2007 treatment.

PubMed

Vasquez, Martice E; Rinderneck, Janna; Newman, Julie; McMillin, Stella; Finlayson, Brian; Mekebri, Abdou; Crane, David; Tjeerdema, Ronald S

2012-05-01

In September 2007, Lake Davis (near Portola, California) was treated by the California Department of Fish and Game with CFT Legumine, a rotenone formulation, to eradicate the invasive northern pike (Esox lucius). The objective of this report is to describe the fate of the five major formulation constituents-rotenone, rotenolone, methyl pyrrolidone (MP), diethylene glycol monethyl ether (DEGEE), and Fennedefo 99-in water, sediment, and brown bullhead catfish (Ameiurus nebulosus; a rotenone-resistant species) by determination of their half-lives (t(1/2)) and pseudo first-order dissipation rate constants (k). The respective t(1/2) values in water for rotenone, rotenolone, MP, DEGEE, and Fennedefo 99 were 5.6, 11.1, 4.6, 7.7, and 13.5 d; in sediments they were 31.1, 31.8, 10.0, not able to calculate, and 48.5 d; and in tissues were 6.1, 12.7, 3.7, 3.2, and 10.4 d, respectively. Components possessing low water solubility values (rotenone and rotenolone) persisted longer in sediments (not detectable after 157 d) and tissues (<212 d) compared with water, whereas the water-miscible components (MP and DEGEE) dissipated more quickly from all matrices, except for Fennedefo 99, which was the most persistent in water (83 d). None of the constituents was found to bioaccumulate in tissues as a result of treatment. In essence, the physicochemical properties of the chemical constituents effectively dictated their fate in the lake following treatment. Copyright © 2012 SETAC.

Phenformin-Induced Mitochondrial Dysfunction Sensitizes Hepatocellular Carcinoma for Dual Inhibition of mTOR.

PubMed

Veiga, Sonia Rosa; Ge, Xuemei; Mercer, Carol A; Hernández-Alvarez, María Isabel; Thomas, Hala Elnakat; Hernández-Losa, Javier; Ramón Y Cajal, Santiago; Zorzano, Antonio; Thomas, George; Kozma, Sara C

2018-04-24

Hepatocellular carcinoma (HCC) ranks second in cancer mortality and has limited therapeutic options. We recently described the synergistic effect of allosteric and ATP-site competitive inhibitors against the mammalian target of rapamycin (mTOR) for the treatment of HCC. However, such inhibitors induce glycemia and increase mitochondrial efficiency. Here we determined whether the mitochondrial complex I inhibitor Phenformin could reverse both side effects, impose an energetic-stress on cancer cells and suppress the growth of HCC. Human HCC cell lines were used in vitro to access the signaling and energetic impact of mTOR inhibitors and Phenformin, either alone or in combination. Next, the therapeutic utility of these drugs alone or in combination was investigated pre-clinically in human orthotopic tumors implanted in mice, by analyzing their impact on the tumor burden and overall survival. We found Phenformin caused mitochondrial dysfunction and fragmentation, inducing a compensatory shift to glycolysis. In contrast, dual inhibition of mTOR impaired cell growth and glycolysis, while increasing mitochondrial fusion and efficiency. In a mouse model of human HCC, dual inhibition of mTOR, together with Phenformin, was highly efficacious in controlling tumor burden. However, more striking, pretreatment with Phenformin sensitized tumors to dual inhibition of mTOR, leading to a dramatic improvement in survival. Treatment of HCC cells in vitro with the biguanide Phenformin causes a metabolic shift to glycolysis, mitochondrial dysfunction and fragmentation, and dramatically sensitizes orthotopic liver tumors to dual inhibition of mTOR. We therefore propose this therapeutic approach should be tested clinically in HCC. Copyright ©2018, American Association for Cancer Research.

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification

PubMed Central

Dejonghe, Wim; Kuenen, Sabine; Mylle, Evelien; Vasileva, Mina; Keech, Olivier; Viotti, Corrado; Swerts, Jef; Fendrych, Matyáš; Ortiz-Morea, Fausto Andres; Mishev, Kiril; Delang, Simon; Scholl, Stefan; Zarza, Xavier; Heilmann, Mareike; Kourelis, Jiorgos; Kasprowicz, Jaroslaw; Nguyen, Le Son Long; Drozdzecki, Andrzej; Van Houtte, Isabelle; Szatmári, Anna-Mária; Majda, Mateusz; Baisa, Gary; Bednarek, Sebastian York; Robert, Stéphanie; Audenaert, Dominique; Testerink, Christa; Munnik, Teun; Van Damme, Daniël; Heilmann, Ingo; Schumacher, Karin; Winne, Johan; Friml, Jiří; Verstreken, Patrik; Russinova, Eugenia

2016-01-01

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane. PMID:27271794

RNA-Seq Expression Analysis of Enteric Neuron Cells with Rotenone Treatment and Prediction of Regulated Pathways.

PubMed

Guan, Qiang; Wang, Xijin; Jiang, Yanyan; Zhao, Lijuan; Nie, Zhiyu; Jin, Lingjing

2017-02-01

The enteric nervous system (ENS) is involved in the initiation and development of the pathological process of Parkinson's disease (PD). The effect of rotenone on the ENS may trigger the progression of PD through the central nervous system (CNS). In this study, we used RNA-sequencing (RNA-seq) analysis to examine differential expression genes (DEGs) and pathways induced by in vitro treatment of rotenone in the enteric nervous cells isolated from rats. We identified 45 up-regulated and 30 down-regulated genes. The functional categorization revealed that the DEGs were involved in the regulation of cell differentiation and development, response to various stimuli, and regulation of neurogenesis. In addition, the pathway and network analysis showed that the Mitogen Activated Protein Kinase (MAPK), Toll-like receptor, Wnt, and Ras signaling pathways were intensively involved in the effect of rotenone on the ENS. Additionally, the quantitative real-time polymerase chain reaction result for the selected seven DEGs matched those of the RNA-seq analysis. Our results present a significant step in the identification of DEGs and provide new insight into the progression of PD in the rotenone-induced model.

Agaricus blazei extract abrogates rotenone-induced dopamine depletion and motor deficits by its anti-oxidative and anti-inflammatory properties in Parkinsonic mice.

PubMed

Venkatesh Gobi, Veerappan; Rajasankar, Srinivasagam; Ramkumar, Muthu; Dhanalakshmi, Chinnasamy; Manivasagam, Thamilarasan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed; Chidambaram, Ranganathan; Kalandar, Ameer

2017-06-19

Neuroinflammation and oxidative damage are the two main malfactors that play an important role in the pathogenesis of experimental and clinical Parkinson's disease (PD). The current study was aimed to study the possible anti-oxidant and anti-inflammatory effects of the methanolic extract of Agaricus blazei (A. blazei) against rotenone-induced PD in mice. Male Albino mice were randomized and divided into the following groups: control, treated with rotenone (1 mg/kg/day), co-treated with rotenone and A. blazei (50, 100, and 200 mg/kg b.w.), and treated with A. blazei alone (200 mg/kg b.w.). After the end of the experimental period, behavioral studies, biochemical estimations, and protein expression patterns of inflammatory markers were studied. Rotenone treatment exhibited enhanced motor impairments, neurochemical deficits, oxidative stress, and inflammation, whereas oral administration of A. blazei extract attenuated the above-said indices. Even though further research is needed to prove its efficacy in clinical studies, the results of our study concluded that A. blazei extract offers a promising and new therapeutic lead for treatment of PD.

Effect of rainbow trout size on response to rotenone and antimycin

USGS Publications Warehouse

Brown, Peter J.; Johnson, Heather; Zale, Alexander V.

2011-01-01

The piscicides rotenone and antimycin are commonly used to eradicate unwanted fish populations. However, the relationships (if present) between their toxicities and fish sizes are unknown and could be especially important when bioassay fish are used to detect piscicide presence and effectiveness. Size-mediated toxicity could lead to either excessive or inadequate piscicide applications if bioassay fish are larger or smaller than the fish being eradicated. The relationships between time to death and weight of rainbow trout Oncorhynchus mykiss (0.7–574.0 g) at an antimycin concentration of 7.5 μg/L and a rotenone concentration of 12.5 μg/L were determined. Antimycin took significantly longer than rotenone to kill rainbow trout at concentrations typically used in eradication projects. Significant positive relationships existed between fish size and time to death for rotenone and antimycin exposures and were probably caused by size-mediated differences in metabolic rate; however, these relationships accounted for less than 21% of the variation in time to death. Smaller fish appeared to be affected by the chemicals more quickly, but their deaths did not consistently occur before the deaths of larger fish.

Toxicity of copper sulfate and rotenone to Chinese mystery snail (Bellamya chinensis)

USGS Publications Warehouse

Haak, Danielle M.; Stephen, Bruce J.; Kill, Robert A.; Smeenk, Nicholas A.; Allen, Craig R.; Pope, Kevin L.

2014-01-01

The Chinese mystery snail (Bellamya chinensis) is a freshwater snail native to Southeast Asia, Japan, and Russia and is currently classified as an invasive species in at least 27 states in the USA. The species tolerates a wide range of environmental conditions, making management of established populations difficult. We tested the efficacy of two traditional chemical treatments, rotenone and copper sulfate, on the elimination of adult Chinese mystery snails in laboratory experiments. All snails (N=50) survived 72-hour exposure to rotenone-treated lake water, and 96% (N=25) survived 72-hour exposure to pre-determined rotenone concentrations of 0.25, 2.5, and 25.0 mg/L. All snails (N=10) survived exposure to 1.25 mg/L copper sulfate solution, 90% (N=10) survived exposure to 2.50 mg/L copper sulfate solution, and 80% (N=5) survived exposure to 5.0 mg/L copper sulfate solution. Neither rotenone nor copper sulfate effectively killed adult Chinese mystery snails in laboratory experiments, most likely due to their relatively large size, thick shell, and operculum. Therefore, it appears that populations will be very difficult to control once established, and management should focus on preventing additional spread or introductions of this species.

Neuroprotective Effects of Filgrastim in Rotenone-Induced Parkinson's Disease in Rats: Insights into its Anti-Inflammatory, Neurotrophic, and Antiapoptotic Effects.

PubMed

Azmy, Mariama S; Menze, Esther T; El-Naga, Reem N; Tadros, Mariane G

2018-01-11

All current treatments of Parkinson's disease (PD) focus on enhancing the dopaminergic effects and providing symptomatic relief; however, they cannot delay the disease progression. Filgrastim, a recombinant methionyl granulocyte colony-stimulating factor, demonstrated neuroprotection in many neurodegenerative and neurological diseases. This study aimed to assess the neuroprotective effects of filgrastim in rotenone-induced rat model of PD and investigate the potential underlying mechanisms of filgrastim actions. The effects of two doses of filgrastim (20 and 40 μg/kg) on spontaneous locomotion, catalepsy, body weight, histology, and striatal dopamine (DA) content, as well as tyrosine hydroxylase (TH) and α-synuclein expression, were evaluated. Then, the effective dose was further tested for its potential anti-inflammatory, neurotrophic, and antiapoptotic effects. Filgrastim (40 μg/kg) prevented rotenone-induced motor deficits, weight reduction, striatal DA depletion, and histological damage. Besides, it significantly inhibited rotenone-induced decrease in TH expression and increase in α-synuclein immunoreactivity in the midbrains and striata of the rats. These effects were associated with reduction of rotenone-induced neuroinflammation, apoptosis, and brain-derived neurotrophic factor depletion. Collectively, these results suggest that filgrastim might be a good candidate for management of PD.

Effect of a high dose of simvastatin on muscle mitochondrial metabolism and calcium signaling in healthy volunteers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Galtier, F., E-mail: f-galtier@chu-montpellier.fr; INSERM, CIC 1001, 80 Avenue Augustin Fliche, 34295 Montpellier Cedex 5; CPID, Faculté de Pharmacie, 15 Av. Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, Montpellier

Statin use may be limited by muscle side effects. Although incompletely understood to date, their pathophysiology may involve oxidative stress and impairments of mitochondrial function and of muscle Ca{sup 2+} homeostasis. In order to simultaneously assess these mechanisms, 24 male healthy volunteers were randomized to receive either simvastatin for 80 mg daily or placebo for 8 weeks. Blood and urine samples and a stress test were performed at baseline and at follow-up, and mitochondrial respiration and Ca{sup 2+} spark properties were evaluated on a muscle biopsy 4 days before the second stress test. Simvastatin-treated subjects were separated according to theirmore » median creatine kinase (CK) increase. Simvastatin treatment induced a significant elevation of aspartate amino transferase (3.38 ± 5.68 vs − 1.15 ± 4.32 UI/L, P < 0.001) and CK (− 24.3 ± 99.1 ± 189.3vs 48.3 UI/L, P = 0.01) and a trend to an elevation of isoprostanes (193 ± 408 vs12 ± 53 pmol/mmol creatinine, P = 0.09) with no global change in mitochondrial respiration, lactate/pyruvate ratio or Ca{sup 2+} sparks. However, among statin-treated subjects, those with the highest CK increase displayed a significantly lower Vmax rotenone succinate and an increase in Ca{sup 2+} spark amplitude vs both subjects with the lowest CK increase and placebo-treated subjects. Moreover, Ca{sup 2+} spark amplitude was positively correlated with treatment-induced CK increase in the whole group (r = 0.71, P = 0.0045). In conclusion, this study further supports that statin induced muscular toxicity may be related to alterations in mitochondrial respiration and muscle calcium homeostasis independently of underlying disease or concomitant medication. -- Highlights: ► Statin use may be limited by side effects, particularly myopathy. ► Statins might impair mitochondrial function and muscle Ca2+ signaling in muscle. ► This was tested among healthy volunteers receiving simvastatin 80 mg daily for 8 weeks. ► CK increase was associated with alterations in Ca2+ sparks and mitochondrial function.« less

Mitochondrial oxidative stress is the achille's heel of melanoma cells resistant to Braf-mutant inhibitor

PubMed Central

André, Fanny; Jonneaux, Aurélie; Scalbert, Camille; Garçon, Guillaume; Malet-Martino, Myriam; Balayssac, Stéphane; Rocchi, Stephane; Savina, Ariel; Formstecher, Pierre; Mortier, Laurent; Kluza, Jérome; Marchetti, Philippe

2013-01-01

Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy. Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses. Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α. However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown. We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients. Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α. Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib. Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol. Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition. PMID:24161908

Manganese ions enhance mitochondrial H2O2 emission from Krebs cycle oxidoreductases by inducing permeability transition.

PubMed

Bonke, Erik; Siebels, Ilka; Zwicker, Klaus; Dröse, Stefan

2016-10-01

Manganese-induced toxicity has been linked to mitochondrial dysfunction and an increased generation of reactive oxygen species (ROS). We could recently show in mechanistic studies that Mn 2+ ions induce hydrogen peroxide (H 2 O 2 ) production from the ubiquinone binding site of mitochondrial complex II (II Q ) and generally enhance H 2 O 2 formation by accelerating the rate of superoxide dismutation. The present study with intact mitochondria reveals that manganese additionally enhances H 2 O 2 emission by inducing mitochondrial permeability transition (mPT). In mitochondria fed by NADH-generating substrates, the combination of Mn 2+ and different respiratory chain inhibitors led to a dynamically increasing H 2 O 2 emission which was sensitive to the mPT inhibitor cyclosporine A (CsA) as well as Ru-360, an inhibitor of the mitochondrial calcium uniporter (MCU). Under these conditions, flavin-containing enzymes of the mitochondrial matrix, e.g. the mitochondrial 2-oxoglutaratedehydrogenase (OGDH), were major sources of ROS. With succinate as substrate, Mn 2+ stimulated ROS production mainly at complex II, whereby the applied succinate concentration had a marked effect on the tendency for mPT. Also Ca 2+ increased the rate of H 2 O 2 emission by mPT, while no direct effect on ROS-production of complex II was observed. The present study reveals a complex scenario through which manganese affects mitochondrial H 2 O 2 emission: stimulating its production from distinct sites (e.g. site II Q ), accelerating superoxide dismutation and enhancing the emission via mPT which also leads to the loss of soluble components of the mitochondrial antioxidant systems and favors the ROS production from flavin-containing oxidoreductases of the Krebs cycle. Copyright © 2016 Elsevier Inc. All rights reserved.

Uptake, biotransformation, and elimination of rotenone by bluegills (Lepomis macrochirus )

USGS Publications Warehouse

Gingerich, W.H.; Rach, J.J.

1985-01-01

Yearling bluegills (Lepomis macrochirus) were exposed to sublethal concentrations of [14C]rotenone (5.2 μg/l) for 30 days in a continuous flow exposure system and then transferred to clean, flowing water for an additional 21-day depuration period. Rates of uptake and elimination and profile of the rotenoid metabolites in head, viscera, and carcass components were evaluated by 14C counting and by high performance liquid chromatography. Total [14C]rotenone derived activity was relatively uniform in all body components within 3 days after initial exposure and remained constant during the ensuing 27 days of exposure. Initial uptake rate coefficients were highest in viscera (Ku = 80· h -1) and were nearly identical for head (Ku = 14 · h) and carcass (Ku = 10 · h-1). Analyses of tissue extracts by high performance liquid chromatography confirmed the presence of at least six biotransformation products of rotenone. More than 60% of the activity extracted from viscera was present as a single peak which represented a compound that was extremely soluble in water. Rotenone composed only 0.3% of the extractable activity in viscera taken from fish exposed to rotenone for 30 days; however, rotenone accounted for 15.4% of extractable activity in the head and 20.1% in the carcass components. Rotenolone and 6',7'-dihydro-6'-,7'--dihydroxyrotenolone were tentatively identified as oxidation products in all tissue extracts. Elimination of 14C activity from all body components was biphasic; both phases followed first-order kinetics. The rate of elimination was nearly equal for all body components during the initial phase but was most rapid from viscera during the second phase of elimination. Bioconcentration factors for the head, viscera, and carcass were 165, 3,550, and 125, respectively, when calculated on the basis of total 14C activity but only 25.4, 11, and 26 when calculated as the concentration of parent material.

Rotenone

Integrated Risk Information System (IRIS)

Rotenone ; CASRN 83 - 79 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effects )

75 FR 40823 - Rotenone; Cancellation Order for Amendments to Terminate Uses

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-14

... only available in hard copy, at the Office of Pesticide Programs (OPP) Regulatory Public Docket in Rm.... The commenter expressed her opinion that no living thing should be exposed to rotenone products. The...

Regulated Cell Death of Lymphoma Cells after Graded Mitochondrial Damage is Differentially Affected by Drugs Targeting Cell Stress Responses.

PubMed

Lombardo, Tomás; Folgar, Martín Gil; Salaverry, Luciana; Rey-Roldán, Estela; Alvarez, Elida M; Carreras, María C; Kornblihtt, Laura; Blanco, Guillermo A

2018-05-01

Collapse of the mitochondrial membrane potential (MMP) is often considered the initiation of regulated cell death (RCD). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is an uncoupler of the electron transport chain (ETC) that facilitates the translocation of protons into the mitochondrial matrix leading to the collapse of the MMP. Several cell stress responses such as mitophagy, mitochondrial biogenesis and the ubiquitin proteasome system may differentially contribute to restrain the initiation of RCD depending on the extent of mitochondrial damage. We induced graded mitochondrial damage after collapse of MMP with the mitochondrial uncoupler CCCP in Burkitt's lymphoma cells, and we evaluated the effect of several drugs targeting cell stress responses over RCD at 72 hr, using a multiparametric flow cytometry approach. CCCP caused collapse of MMP after 30 min., massive mitochondrial fission, oxidative stress and increased mitophagy within the 5-15 μM low-dose range (LDR) of CCCP. Within the 20-50 μM high-dose range (HDR), CCCP caused lysosomal destabilization and rupture, thus precluding mitophagy and autophagy. Cell death after 72 hr was below 20%, with increased mitochondrial mass (MM). The inhibitors of mitophagy 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (Mdivi-1) and vincristine (VCR) increased cell death from CCCP within the LDR, while valproic acid (an inducer of mitochondrial biogenesis) also increased MM and cell death within the LDR. The proteasome inhibitor, MG132, increased cell death only in the HDR. Doxycycline, an antibiotic that disrupts mitochondrial biogenesis, had no effect on cell survival, while iodoacetamide, an inhibitor of glycolysis, increased cell death at the HDR. We conclude that mitophagy influenced RCD of lymphoma cells after MMP collapse by CCCP only within the LDR, while proteasome activity and glycolysis contributed to survival in the HDR under extensive mitochondria and lysosome damage. © 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

The first fatal case of yam bean and rotenone toxicity in Thailand.

PubMed

Narongchai, Paitoon; Narongchai, Siripun; Thampituk, Suparat

2005-07-01

The first fatal case of Yam bean and Rotenone toxicity in Thailand was studied at Forensic Medicine, Chiang Mai, Thailand. A Chinese Taiwan man, 59 years old, was found dead after Yam bean ingestion. Yam bean toxicity and death have been found very rarely in the world and has not been reported in Thailand The Yam bean plant is grown widely in Northern Thailand. But many people know that mature pods, seeds and filage of the Yam bean, except the tuberous root, are very toxic. The victim ate a lot of Yam bean seeds and died within 2 hours with respiratory failure. The authors detected Rotenone substance in Yam bean seeds, gastric content and 72 ng/ml blood by HPLC. Also generalized microscopic hemorrhage in the brain, lungs, liver and adrenal glands which were of characteristic pathology were detected. The authors concluded that the cause of death was asphyxia from Yam bean or Rotenone toxicity.

Tianma Gouteng Yin, a Traditional Chinese Medicine decoction, exerts neuroprotective effects in animal and cellular models of Parkinson’s disease

PubMed Central

Liu, Liang-Feng; Song, Ju-Xian; Lu, Jia-Hong; Huang, Ying-Yu; Zeng, Yu; Chen, Lei-Lei; Durairajan, Siva Sundara Kumar; Han, Quan-Bin; Li, Min

2015-01-01

Tianma Gouteng Yin (TGY) is a traditional Chinese medicine (TCM) decoction widely used to treat symptoms associated with typical Parkinson’s disease (PD). In this study, the neuroprotective effects of water extract of TGY were tested on rotenone-intoxicated and human α-synuclein transgenic Drosophila PD models. In addition, the neuroprotective effect of TGY was also evaluated in the human dopaminergic neuroblastoma SH-SY5Y cell line treated with rotenone and the rotenone intoxicated hemi-parkinsonian rats. In rotenone-induced PD models, TGY improved survival rate, alleviated impaired locomotor function of Drosophila, mitigated the loss of dopaminergic neurons in hemi-parkinsonian rats and alleviated apoptotic cell death in SH-SY5Y cells; in α-synuclein transgenic Drosophila, TGY reduced the level of α-synuclein and prevented degeneration of dopaminergic neurons. Conclusively, TGY is neuroprotective in PD models both in vivo and in vitro. PMID:26578166

SIRT3 aggravates metformin-induced energy stress and apoptosis in ovarian cancer cells.

PubMed

Wu, Yao; Gao, Wei-Nan; Xue, Ya-Nan; Zhang, Li-Chao; Zhang, Juan-Juan; Lu, Sheng-Yao; Yan, Xiao-Yu; Yu, Hui-Mei; Su, Jing; Sun, Lian-Kun

2018-06-15

Increasing evidence suggests that mitochondrial respiratory chain complex I participates in carcinogenesis and cancer progression by providing energy and maintaining mitochondrial function. However, the role of complex I in ovarian cancer is largely unknown. In this study we showed that metformin, considered to be an inhibitor of complex I, simultaneously inhibited cell growth and induced mitochondrial-related apoptosis in human ovarian cancer cells. Metformin interrupted cellular energy metabolism mainly by causing damage to complex I that impacted mitochondrial function. Additionally, treatment with metformin increased the activation of sirtuin 3 (SIRT3), a mitochondrial deacetylase. We demonstrated that SIRT3 overexpression aggravated metformin-induced apoptosis, energy stress and mitochondrial dysfunction. Moreover, treatment with metformin or SIRT3 overexpression increased activation of AMP-activated protein kinase (AMPK), a major sensor of cellular energy status. AMPK compensated for energy loss by increasing glycolysis. The impact of this was assessed by reducing glucose levels in the media or by using inhibitors (2-deoxyglucose, Compound C) of glycolysis and AMPK. The combination of these factors with metformin intensified cytotoxicity through further downregulation of ATP. Our study outlines an important role for SIRT3 in the antitumor effect of mitochondrial complex I inhibitors in human ovarian cancer cells. This effect appears to be mediated by induction of energy stress and apoptosis. Strategies that target the mitochondria could be enhanced by modulating glycolysis to further aggravate energy stress that may increase the antitumor effect. Copyright © 2018 Elsevier Inc. All rights reserved.

Magnetic Resonance Imaging of Mitochondrial Dysfunction and Metabolic Activity, Accompanied by Overproduction of Superoxide.

PubMed

Bakalova, Rumiana; Georgieva, Ekaterina; Ivanova, Donika; Zhelev, Zhivko; Aoki, Ichio; Saga, Tsuneo

2015-12-16

This study shows that a mitochondria-penetrating nitroxide probe (mito-TEMPO) allows detection of superoxide and visualization of mitochondrial dysfunction in living cells due to the effect of T1 shortening in MRI. Mitochondrial dysfunction was induced by treatment of cells with rotenone and 2-methoxyestradiol (2-ME/Rot). The MRI measurements were performed on 7T MRI. The 2-ME/Rot-treated cells were characterized by overproduction of superoxide, which was confirmed by a conventional dihydroethidium test. In the presence of mito-TEMPO, the intensity of MRI signal in 2-ME/Rot-treated cells was ∼30-40% higher, in comparison with that in untreated cells or culture media. In model (cell-free) systems, we observed that superoxide, but not hydrogen peroxide, increased the intensity of T1-weighted MRI signal of mito-TEMPO. Moreover, the superoxide restores the T1-weighted MRI contrast of mito-TEMPOH, a noncontrast (diamagnetic) analogue of mito-TEMPO. This was also confirmed by using EPR spectroscopy. The results demonstrate that superoxide radical is involved in the enhancement of T1-weighted MRI contrast in living cells, in the absence and presence of mito-TEMPO. This report gives a direction for discovering new opportunities for functional MRI, for detection of metabolic activity, accompanied by overproduction of superoxide, as well as by disturbance of the balance between superoxide and hydrogen peroxide, a very important approach to clarify the fine molecular mechanisms in the regulation of many pathologies. The visualization of mitochondrial activity in real-time can be crucial to clarify the molecular mechanism of the functional MRI in its commonly accepted definition, as a method for detection of neurovascular coupling.

Mitochondrial DNA replication, nucleoside reverse-transcriptase inhibitors, and AIDS cardiomyopathy.

PubMed

Lewis, William

2003-01-01

Nucleoside reverse-transcriptase inhibitors (NRTIs) in combination with other antiretrovirals (HAART) are the cornerstones of current AIDS therapy, but extensive use brought mitochondrial side effects to light. Clinical experience, pharmacological, cell, and molecular biological evidence links altered mitochondrial (mt-) DNA replication to the toxicity of NRTIs in many tissues, and conversely, mtDNA replication defects and mtDNA depletion in target tissues are observed. Organ-specific pathological changes or diverse systemic effects result from and are frequently attributed to HAART in which NRTIs are included. The shared features of mtDNA depletion and energy depletion became key observations and related the clinical and in vivo experimental findings to inhibition of mtDNA replication by NRTI triphosphates in vitro. Subsequent to those findings, other observations suggested that mitochondrial energy deprivation is concomitant with or the result of mitochondrial oxidative stress in AIDS (from HIV, for example) or from NRTI therapy itself. Copyright 2003, Elsevier Science (USA)

Protective effects of Forsythia suspense extract with antioxidant and anti-inflammatory properties in a model of rotenone induced neurotoxicity.

PubMed

Zhang, Shuai; Shao, Si-Yuan; Song, Xiu-Yun; Xia, Cong-Yuan; Yang, Ya-Nan; Zhang, Pei-Cheng; Chen, Nai-Hong

2016-01-01

The present study investigated the neuroprotective effects of Forsythia suspense extract in a rotenone-induced neurotoxic model. FS8, one of the herbal extracts, markedly protected PC12 cells against rotenone toxicity and was selected for the in vivo study. Gavage administration of FS8 (50 and 200mg/kg, but not 10mg/kg) for 25 days significantly improved the behavior function, decreased the loss of dopaminergic neurons in substantia nigra (SN), and maintained the level of dopamine in striatum after unilateral infusion of rotenone in SN. Wherein, the protective effects of FS8 at the dose of 200mg/kg were better than selegiline. Further study indicated the excellent antioxidant activity of FS8 on the 5th and 21st days after intranigral injection of rotenone. Moreover, FS8 could inhibit microglia activity and accumulation in SN, and obviously decreased the expression of pro-inflammatory molecules (IL-6, TNF-α, iNOS and COX-2), which indicated the anti-inflammatory effects of FS8. In the PI3K/Akt/NF-κB and MAPK pathways, FS8 significantly down-regulated the protein expression of p-PI3K, p-Akt, p-IκB, p-P65, cleaved Caspase 8, p-p38 and p-JNK but not p-mTOR, cleaved Caspase 3 and p-ERK. Therefore, FS8 protected dopamine neurons against rotenone toxicity via antioxidant and anti-inflammatory effects, which suggested the promising application of FS8 in the prevention and treatment of Parkinson disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Distribution and accumulation of rotenone in tissues of warm water fishes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rach, J.J.; Gingerich, W.H.

1986-03-01

The tissue distribution of rotenoid residues was determined in tissues of common carp Cyprinus carpio (88.2 g), bluegills Lepomis macrochirus (47.9 g), and yellow perch Perca flavescens (67.7 g) after the fish were exposed to 50..mu..g/L of rotenone-6a/sup 14/C(15.9 x 10/sup 4/ Bq/..mu..M). Exposures were terminated 1 h after the fish were moribund and failed to respond to gentle prodding. The fish were dissected, and various tissues were oxidized to determine /sup 14/C-rotenone accumulation and distribution. Major rotenone metabolites were identified by gradient-elution high performance liquid chromatography. The exposure time required for fish to reach total incapacitation was 3 hmore » for bluegills and yellow perch, and 11.25 h for common carp. The mean rotenoid concentrations (..mu..g/100g fish) in the whole body were 22.4 in yellow perch, 39.7 in bluegills, and 107.8 in common carp. The percentage of rotenone-derived /sup 14/C activity was higher in the carcass components than in the head or viscera; the skin and bone contained about 60% of the carcass activity. The highest relative rotenoid concentrations were found in the liver, bile, gills, brain, and heart. Percentages of total rotenoid material as parent rotenone were highest in yellow perch (70.0 in the viscera and 84.4 in the fillet), followed by those for bluegills (22.7 and 27.8) and common carp (9.7 and 48.5).« less

Inhibition of mitochondrial fragmentation diminishes Huntington’s disease–associated neurodegeneration

PubMed Central

Guo, Xing; Disatnik, Marie-Helene; Monbureau, Marie; Shamloo, Mehrdad; Mochly-Rosen, Daria; Qi, Xin

2013-01-01

Huntington’s disease (HD) is the result of expression of a mutated Huntingtin protein (mtHtt), and is associated with a variety of cellular dysfunctions including excessive mitochondrial fission. Here, we tested whether inhibition of excessive mitochondrial fission prevents mtHtt-induced pathology. We developed a selective inhibitor (P110-TAT) of the mitochondrial fission protein dynamin-related protein 1 (DRP1). We found that P110-TAT inhibited mtHtt-induced excessive mitochondrial fragmentation, improved mitochondrial function, and increased cell viability in HD cell culture models. P110-TAT treatment of fibroblasts from patients with HD and patients with HD with iPS cell–derived neurons reduced mitochondrial fragmentation and corrected mitochondrial dysfunction. P110-TAT treatment also reduced the extent of neurite shortening and cell death in iPS cell–derived neurons in patients with HD. Moreover, treatment of HD transgenic mice with P110-TAT reduced mitochondrial dysfunction, motor deficits, neuropathology, and mortality. We found that p53, a stress gene involved in HD pathogenesis, binds to DRP1 and mediates DRP1-induced mitochondrial and neuronal damage. Furthermore, P110-TAT treatment suppressed mtHtt-induced association of p53 with mitochondria in multiple HD models. These data indicate that inhibition of DRP1-dependent excessive mitochondrial fission with a P110-TAT–like inhibitor may prevent or slow the progression of HD. PMID:24231356

Downregulation of mitochondrial UQCRB inhibits cancer stem cell-like properties in glioblastoma.

PubMed

Jung, Narae; Kwon, Ho Jeong; Jung, Hye Jin

2018-01-01

Glioblastoma stem cell targeted therapies have become a powerful strategy for the treatment of this deadliest brain tumor. We demonstrate for the first time that downregulation of mitochondrial ubiquinol-cytochrome c reductase binding protein (UQCRB) inhibits the cancer stem cell-like properties in human glioblastoma cells. The synthetic small molecules targeting UQCRB significantly suppressed not only the self-renewal capacity such as growth and neurosphere formation, but also the metastatic potential such as migration and invasion of glioblastoma stem‑like cells (GSCs) derived from U87MG and U373MG at subtoxic concentrations. Notably, the UQCRB inhibitors repressed c‑Met-mediated downstream signal transduction and hypoxia‑inducible factor‑1α (HIF‑1α) activation, thereby reducing the expression levels of GSC markers including CD133, Nanog, Oct4 and Sox2 in the GSCs. Furthermore, the UQCRB inhibitors decreased mitochondrial ROS generation and mitochondrial membrane potential in the GSCs, indicating that they regulate the mitochondrial function in GSCs. Indeed, the knockdown of UQCRB gene by UQCRB siRNA significantly inhibited the cancer stem cell-like phenotypes as well as the expression of stemness markers by blocking mitochondrial ROS/HIF‑1α/c‑Met pathway in U87MG GSCs. These findings suggest that UQCRB and its inhibitors could be a new therapeutic target and lead compounds for eliminating cancer stem cells in glioblastoma.

Curcumin Monoglucoside Shows Improved Bioavailability and Mitigates Rotenone Induced Neurotoxicity in Cell and Drosophila Models of Parkinson's Disease.

PubMed

Pandareesh, M D; Shrivash, M K; Naveen Kumar, H N; Misra, K; Srinivas Bharath, M M

2016-11-01

Curcumin (CUR), a dietary polyphenol has diverse pharmacologic effects, but is limited by poor bioavailability. This is probably due to decreased solubility, cellular uptake and stability. In order to enhance its solubility and bioavailability, we synthesized the CUR bioconjugate curcumin monoglucoside (CMG) and tested its bioavailability, neuroprotective and anti-apoptotic propensity against rotenone (ROT) induced toxicity in N27 dopaminergic neuronal cells and Drosophila models. Our results elucidate that CMG showed improved bioavailability than CUR in N27 cells. Pre-treatment with CMG protected against ROT neurotoxicity and exerted antioxidant effects by replenishing cellular glutathione levels and significantly decreasing reactive species. CMG pre-treatment also restored mitochondrial complex I and IV activities inhibited by ROT. ROT-induced nuclear damage was also restored by CMG as confirmed by comet assay. CMG induced anti-apoptotic effects was substantiated by decreased phosporylation of JNK3 and c-jun, which in turn decreased the cleavage of pro-caspase 3. Q-PCR analysis of redox genes showed up-regulation of NOS2 and down-regulation of NQO1 upon ROT exposure and this was attenuated by CMG pre-treatment. Studies in the Drosophila ROT model revealed that, CMG administration showed better survival rate and locomotor activity, improved antioxidant activity and dopamine content than ROT treated group and was comparable with the CUR group. Based on these data, we surmise that CMG has improved bioavailability and offered neuroprotection comparable with CUR, against ROT-induced toxicity both in dopaminergic neuronal cell line and Drosophila models, with therapeutic implications for PD.

Disrupting mitochondrial Ca2+ homeostasis causes tumor-selective TRAIL sensitization through mitochondrial network abnormalities.

PubMed

Ohshima, Yohei; Takata, Natsuhiko; Suzuki-Karasaki, Miki; Yoshida, Yukihiro; Tokuhashi, Yasuaki; Suzuki-Karasaki, Yoshihiro

2017-10-01

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent with high tumor-selective cytotoxicity. The congenital and acquired resistance of some cancer types including malignant melanoma and osteosarcoma impede the current TRAIL therapy of these cancers. Since fine tuning of the intracellular Ca2+ level is essential for cell function and survival, Ca2+ dynamics could be a promising target for cancer treatment. Recently, we demonstrated that mitochondrial Ca2+ removal increased TRAIL efficacy toward malignant melanoma and osteosarcoma cells. Here we report that mitochondrial Ca2+ overload leads to tumor-selective sensitization to TRAIL cytotoxicity. Treatment with the mitochondrial Na+/Ca2+ exchanger inhibitor CGP-37157 and oxidative phosphorylation inhibitor antimycin A and FCCP resulted in a rapid and persistent mitochondrial Ca2+ rise. These agents also increased TRAIL sensitivity in a tumor-selective manner with a switching from apoptosis to a nonapoptotic cell death. Moreover, we found that mitochondrial Ca2+ overload led to increased mitochondrial fragmentation, while mitochondrial Ca2+ removal resulted in mitochondrial hyperfusion. Regardless of their reciprocal actions on the mitochondrial dynamics, both interventions commonly exacerbated TRAIL-induced mitochondrial network abnormalities. These results expand our previous study and suggest that an appropriate level of mitochondrial Ca2+ is essential for maintaining the mitochondrial dynamics and the survival of these cells. Thus, disturbing mitochondrial Ca2+ homeostasis may serve as a promising approach to overcome the TRAIL resistance of these cancers with minimally compromising the tumor-selectivity.

Mitochondria as new therapeutic targets for eradicating cancer stem cells: Quantitative proteomics and functional validation via MCT1/2 inhibition.

PubMed

Lamb, Rebecca; Harrison, Hannah; Hulit, James; Smith, Duncan L; Lisanti, Michael P; Sotgia, Federica

2014-11-30

Here, we used quantitative proteomics analysis to identify novel therapeutic targets in cancer stem cells and/or progenitor cells. For this purpose, mammospheres from two ER-positive breast cancer cell lines (MCF7 and T47D) were grown in suspension using low-attachment plates and directly compared to attached monolayer cells grown in parallel. This allowed us to identify a subset of proteins that were selectively over-expressed in mammospheres, relative to epithelial monolayers. We focused on mitochondrial proteins, as they appeared to be highly upregulated in both MCF7 and T47D mammospheres. Key mitochondrial-related enzymes involved in beta-oxidation and ketone metabolism were significantly upregulated in mammospheres, as well as proteins involved in mitochondrial biogenesis, and specific protein inhibitors of autophagy/mitophagy. Overall, we identified >40 "metabolic targets" that were commonly upregulated in both MCF7 and T47D mammospheres. Most of these "metabolic targets" were also transcriptionally upregulated in human breast cancer cells in vivo, validating their clinical relevance. Based on this analysis, we propose that increased mitochondrial biogenesis and decreased mitochondrial degradation could provide a novel mechanism for the accumulation of mitochondrial mass in cancer stem cells. To functionally validate our observations, we utilized a specific MCT1/2 inhibitor (AR-C155858), which blocks the cellular uptake of two types of mitochondrial fuels, namely ketone bodies and L-lactate. Our results indicate that inhibition of MCT1/2 function effectively reduces mammosphere formation, with an IC-50 of ~1 µM, in both ER-positive and ER-negative breast cancer cell lines. Very similar results were obtained with oligomycin A, an inhibitor of the mitochondrial ATP synthase. Thus, the proliferative clonal expansion of cancer stem cells appears to require oxidative mitochondrial metabolism, related to the re-use of monocarboxylic acids, such as ketones or L-lactate. Our findings have important clinical implications for exploiting mitochondrial metabolism to eradicate cancer stem cells and to prevent recurrence, metastasis and drug resistance in cancer patients. Importantly, a related MCT1/2 inhibitor (AZD3965) is currently in phase I clinical trials in patients with advanced cancers: http://clinicaltrials.gov/show/NCT01791595.

Monitoring the change of mitochondrial morphology and its metabolism of the breast cancer cells with the treatment of Hsp70 inhibitor during heat shock by fluorescence imaging

NASA Astrophysics Data System (ADS)

Yu, Biying; Yang, Hongqin; Zhang, Xiaoman; Li, Hui

2016-10-01

Heat shock (HS) is one of the best-studied exogenous cellular stresses, and all cellular compartments and metabolic processes are involved in HS response. The heat shock proteins (Hsps) expression enhanced during HS mainly localized in subcellular compartments, such as cytosol, endoplasmic reticulum and mitochandria. The major inducible heat shock protein 70 (Hsp70) modulate cellular homeostasis and promote cellular survival by blocking a caspase independent cell death through its association with apoptosis inducing factor. Mitochondria as the critical elements of HS response that participate in key metabolic reactions, and the changes in mitochonrial morphology may impact on mitochondrial metabolism. In this paper, the changes of mitorchondrial morphology in breast cancer cell have been monitored in real time after heat shock (43 °) by the fluorescence imaging, and the influence of Hsp70 inhibitor on mitochandrial structures have also been investigated. Then the information of mitochondrial metabolism which can be characterized by the level of the mitochondrial membrane potential has also been obtained wihout/with the treatment of Hsp70 inhibitor. Our data indicated that the mitochandrial morphology were related with the mitochandrial membrane potential, and the mitochandrial membrane potential was influenced significantly with the treatment of Hsp70 inhibitor during HS.

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3β inhibitor, SB216763.

PubMed

Brooks, Morgan M; Neelam, Sudha; Fudala, Rafal; Gryczynski, Ignacy; Cammarata, Patrick R

2013-01-01

Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3β (GSK-3β) in regulating mMPT. Using direct inhibition of GSK-3β with the GSK-3β inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential. Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O₂) followed by exposure to atmospheric oxygen (approximately 21% O₂). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability. Treatment of HLE-B3 cells with SB216763 (12 µM), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment. Inhibition of GSK-3β activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3β activity by the GSK-3β inhibitor SB216763 provides positive protection against mitochondrial depolarization.

Novel Cancer Therapeutics with Allosteric Modulation of the Mitochondrial C-Raf-DAPK Complex by Raf Inhibitor Combination Therapy.

PubMed

Tsai, Yi-Ta; Chuang, Mei-Jen; Tang, Shou-Hung; Wu, Sheng-Tang; Chen, Yu-Chi; Sun, Guang-Huan; Hsiao, Pei-Wen; Huang, Shih-Ming; Lee, Hwei-Jen; Yu, Cheng-Ping; Ho, Jar-Yi; Lin, Hui-Kuan; Chen, Ming-Rong; Lin, Chung-Chih; Chang, Sun-Yran; Lin, Victor C; Yu, Dah-Shyong; Cha, Tai-Lung

2015-09-01

Mitochondria are the powerhouses of cells. Mitochondrial C-Raf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its N-terminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-Raf(S338)) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPK(S308)), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEF(C-Raf-/-) or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity. In the presence of pDAPK(S308), this drug-target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-Raf(S338) and pDAPK(S308) translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPK(S308) to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPK(S308) modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPK(S308) may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation. ©2015 American Association for Cancer Research.

N-Methyl, N-propynyl-2-phenylethylamine (MPPE), a Selegiline Analog, Attenuates MPTP-induced Dopaminergic Toxicity with Guaranteed Behavioral Safety: Involvement of Inhibitions of Mitochondrial Oxidative Burdens and p53 Gene-elicited Pro-apoptotic Change.

PubMed

Shin, Eun-Joo; Nam, Yunsung; Lee, Ji Won; Nguyen, Phuong-Khue Thi; Yoo, Ji Eun; Tran, The-Vinh; Jeong, Ji Hoon; Jang, Choon-Gon; Oh, Young J; Youdim, Moussa B H; Lee, Phil Ho; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2016-11-01

Selegiline is a monoamine oxidase-B (MAO-B) inhibitor with anti-Parkinsonian effects, but it is metabolized to amphetamines. Since another MAO-B inhibitor N-Methyl, N-propynyl-2-phenylethylamine (MPPE) is not metabolized to amphetamines, we examined whether MPPE induces behavioral side effects and whether MPPE affects dopaminergic toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Multiple doses of MPPE (2.5 and 5 mg/kg/day) did not show any significant locomotor activity and conditioned place preference, whereas selegiline (2.5 and 5 mg/kg/day) significantly increased these behavioral side effects. Treatment with MPPE resulted in significant attenuations against decreases in mitochondrial complex I activity, mitochondrial Mn-SOD activity, and expression induced by MPTP in the striatum of mice. Consistently, MPPE significantly attenuated MPTP-induced oxidative stress and MPPE-mediated antioxidant activity appeared to be more pronounced in mitochondrial-fraction than in cytosolic-fraction. Because MPTP promoted mitochondrial p53 translocation and p53/Bcl-xL interaction, it was also examined whether mitochondrial p53 inhibitor pifithrin-μ attenuates MPTP neurotoxicity. MPPE, selegiline, or pifithrin-μ significantly attenuated mitochondrial p53/Bcl-xL interaction, impaired mitochondrial transmembrane potential, cytosolic cytochrome c release, and cleaved caspase-3 in wild-type mice. Subsequently, these compounds significantly ameliorated MPTP-induced motor impairments. Neuroprotective effects of MPPE appeared to be more prominent than those of selegiline. MPPE or selegiline did not show any additional protective effects against the attenuation by p53 gene knockout, suggesting that p53 gene is a critical target for these compounds. Our results suggest that MPPE possesses anti-Parkinsonian potentials with guaranteed behavioral safety and that the underlying mechanism of MPPE requires inhibition of mitochondrial oxidative stress, mitochondrial translocation of p53, and pro-apoptotic process.

Activation of mitochondrial calpain and release of apoptosis-inducing factor from mitochondria in RCS rat retinal degeneration.

PubMed

Mizukoshi, Sayuri; Nakazawa, Mitsuru; Sato, Kota; Ozaki, Taku; Metoki, Tomomi; Ishiguro, Sei-ichi

2010-09-01

The present study was performed to investigate changes of cytosolic and mitochondrial calpain activities, and effects of intravitreously injected calpain inhibitor on photoreceptor apoptosis in Royal College of Surgeon's (RCS) rats. Time courses of activities for both cytosolic and mitochondrial calpains and amount of calpastatin in RCS rat retina were analyzed by subcellular fractionation, calpain assay and western blotting. Calpain assay was colorimetrically performed using Suc-LLVY-Glo as substrate. Effects of intravitreously injected calpain inhibitor (ALLN and PD150606) on RCS rat retinal degeneration were analyzed by TUNEL staining. Effects of mitochondrial calpain activity on activation and translocation of apoptosis-inducing factor (AIF) were analyzed by western blotting. Mitochondrial calpain started to be significantly activated at postnatal (p) 28 days in RCS rat retina, whereas cytosolic micro-calpain was activated at p 35 days, although specific activity of mitochondrial calpain was 13% compared to cytosolic micro-calpain. Intravitreously injected ALLN and PD150606 effectively inhibited photoreceptor apoptosis only when injected at p 25 days, but did not inhibit photoreceptor apoptosis when injected at p 32 days. Parts of AIF were truncated/activated by mitochondrial calpains and translocated to the nucleus. These results suggest that 1), calpain presents not only in the cytosolic fraction but also in the mitochondrial fraction in RCS rat retina; 2), mitochondrial calpain is activated earlier than cytosolic calpain during retinal degeneration in RCS rats; 3), photoreceptor apoptosis may be regulated by not only calpain systems but also other mechanisms; 4), mitochondrial calpain may activate AIF to induce apoptosis; and 5), calpain inhibitors may be partially effective to inhibit photoreceptor apoptosis in RCS rats. The present study provides new insights into the molecular basis for photoreceptor apoptosis in RCS rats and the future possibility of new pharmaceutical treatments for retinitis pigmentosa. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver.

PubMed

Gusdon, Aaron M; Fernandez-Bueno, Gabriel A; Wohlgemuth, Stephanie; Fernandez, Jenelle; Chen, Jing; Mathews, Clayton E

2015-09-10

Aberrant mitochondrial function, including excessive reactive oxygen species (ROS) production, has been implicated in the pathogenesis of human diseases. The use of mitochondrial inhibitors to ascertain the sites in the electron transport chain (ETC) resulting in altered ROS production can be an important tool. However, the response of mouse mitochondria to ETC inhibitors has not been thoroughly assessed. Here we set out to characterize the differences in phenotypic response to ETC inhibitors between the more energetically demanding brain mitochondria and less energetically demanding liver mitochondria in commonly utilized C57BL/6J mice. We show that in contrast to brain mitochondria, inhibiting distally within complex I or within complex III does not increase liver mitochondrial ROS production supported by complex I substrates, and liver mitochondrial ROS production supported by complex II substrates occurred primarily independent of membrane potential. Complex I, II, and III enzymatic activities and membrane potential were equivalent between liver and brain and responded to ETC. inhibitors similarly. Brain mitochondria exhibited an approximately two-fold increase in complex I and II supported respiration compared with liver mitochondria while exhibiting similar responses to inhibitors. Elevated NADH transport and heightened complex II-III coupled activity accounted for increased complex I and II supported respiration, respectively in brain mitochondria. We conclude that important mechanistic differences exist between mouse liver and brain mitochondria and that mouse mitochondria exhibit phenotypic differences compared with mitochondria from other species.

Glycation inhibitors extend yeast chronological lifespan by reducing advanced glycation end products and by back regulation of proteins involved in mitochondrial respiration.

PubMed

Kazi, Rubina S; Banarjee, Reema M; Deshmukh, Arati B; Patil, Gouri V; Jagadeeshaprasad, Mashanipalya G; Kulkarni, Mahesh J

2017-03-06

Advanced Glycation End products (AGEs) are implicated in aging process. Thus, reducing AGEs by using glycation inhibitors may help in attenuating the aging process. In this study using Saccharomyces cerevisiae yeast system, we show that Aminoguanidine (AMG), a well-known glycation inhibitor, decreases the AGE modification of proteins in non-calorie restriction (NR) (2% glucose) and extends chronological lifespan (CLS) similar to that of calorie restriction (CR) condition (0.5% glucose). Proteomic analysis revealed that AMG back regulates the expression of differentially expressed proteins especially those involved in mitochondrial respiration in NR condition, suggesting that it switches metabolism from fermentation to respiration, mimicking CR. AMG induced back regulation of differentially expressed proteins could be possibly due to its chemical effect or indirectly by glycation inhibition. To delineate this, Metformin (MET), a structural analog of AMG and a mild glycation inhibitor and Hydralazine (HYD), another potent glycation inhibitor but not structural analog of AMG were used. HYD was more effective than MET in mimicking AMG suggesting that glycation inhibition was responsible for restoration of differentially expressed proteins. Thus glycation inhibitors particularly AMG, HYD and MET extend yeast CLS by reducing AGEs, modulating the expression of proteins involved in mitochondrial respiration and possibly by scavenging glucose. This study reports the role of glycation in aging process. In the non-caloric restriction condition, carbohydrates such as glucose promote protein glycation and reduce CLS. While, the inhibitors of glycation such as AMG, HYD, MET mimic the caloric restriction condition by back regulating deregulated proteins involved in mitochondrial respiration which could facilitate shift of metabolism from fermentation to respiration and extend yeast CLS. These findings suggest that glycation inhibitors can be potential molecules that can be used in management of aging. Copyright © 2017 Elsevier B.V. All rights reserved.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

PubMed

Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

2015-08-21

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

PubMed Central

Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

2015-01-01

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

Mitochondrial ATF2 translocation contributes to apoptosis induction and BRAF inhibitor resistance in melanoma through the interaction of Bim with VDAC1.

PubMed

Gao, Zongwei; Shang, Qingjuan; Liu, Zhaoyun; Deng, Chun; Guo, Chunbao

2015-11-03

The mitochondrial accumulation of ATF2 is involved in tumor suppressor activities via cytochrome c release in melanoma cells. However, the signaling pathways that connect mitochondrial ATF2 accumulation and cytochrome c release are not well documented. Several melanoma cell lines, B16F10, K1735M2, A375 and A375-R1, were treated with paclitaxel and vemurafenib to test the function of mitochondrial ATF2 and its connection to Bim and voltage-dependent anion channel 1 (VDAC1). Immunoprecipitation analysis was performed to investigate the functional interaction between the involved proteins. VDAC1 oligomerization was evaluated using an EGS-based crosslinking assay. The expression and migration of ATF2 to the mitochondria accounted for paclitaxel stimuli and acquired resistance to BRAF inhibitors. Mitochondrial ATF2 facilitated Bim stabilization through the inhibition of its degradation by the proteasome, thereby promoting cytochrome c release and inducing apoptosis in B16F10 and A375 cells. Studies using B16F10 and A375 cells genetically modified for ATF2 indicated that mitochondrial ATF2 was able to dissociate Bim from the Mcl-1/Bim complex to trigger VDAC1 oligomerization. Immunoprecipitation analysis revealed that Bim interacts with VDAC1, and this interaction was remarkably enhanced during apoptosis. These results reveal that mitochondrial ATF2 is associated with the induction of apoptosis and BRAF inhibitor resistance through Bim activation, which might suggest potential novel therapies for the targeted induction of apoptosis in melanoma therapy.

Mitochondrial ATF2 translocation contributes to apoptosis induction and BRAF inhibitor resistance in melanoma through the interaction of Bim with VDAC1

PubMed Central

Deng, Chun; Guo, Chunbao

2015-01-01

Background The mitochondrial accumulation of ATF2 is involved in tumor suppressor activities via cytochrome c release in melanoma cells. However, the signaling pathways that connect mitochondrial ATF2 accumulation and cytochrome c release are not well documented. Methods Several melanoma cell lines, B16F10, K1735M2, A375 and A375-R1, were treated with paclitaxel and vemurafenib to test the function of mitochondrial ATF2 and its connection to Bim and voltage-dependent anion channel 1 (VDAC1). Immunoprecipitation analysis was performed to investigate the functional interaction between the involved proteins. VDAC1 oligomerization was evaluated using an EGS-based crosslinking assay. Results The expression and migration of ATF2 to the mitochondria accounted for paclitaxel stimuli and acquired resistance to BRAF inhibitors. Mitochondrial ATF2 facilitated Bim stabilization through the inhibition of its degradation by the proteasome, thereby promoting cytochrome c release and inducing apoptosis in B16F10 and A375 cells. Studies using B16F10 and A375 cells genetically modified for ATF2 indicated that mitochondrial ATF2 was able to dissociate Bim from the Mcl-1/Bim complex to trigger VDAC1 oligomerization. Immunoprecipitation analysis revealed that Bim interacts with VDAC1, and this interaction was remarkably enhanced during apoptosis. Conclusion These results reveal that mitochondrial ATF2 is associated with the induction of apoptosis and BRAF inhibitor resistance through Bim activation, which might suggest potential novel therapies for the targeted induction of apoptosis in melanoma therapy. PMID:26462148

Environmental conditions affecting the efficiency and efficacy of piscicides for use in nonnative fish eradication

USGS Publications Warehouse

Brown, Peter James

2010-01-01

Conservation of native fish is a pressing issue for fisheries managers. Conservation efforts often require eliminating threats posed by nonnative fish by eradicating them with piscicides. The piscicides rotenone and antimycin are used for eradication but their application is often inefficient or ineffective. My goal was to increase the efficiency and efficacy of nonnative fish eradication using piscicides. I identified environmental conditions affecting piscicide application, researched methods to overcome these problems, and provided tools that piscicide applicators can use to make piscicide application more efficient and effective. Rotenone and antimycin were exposed to varying levels of sunlight, turbulence, and dissolved organic matter (DOM) to determine the effect these environmental conditions have on piscicides. Bioassay fish were used to determine the toxicity of the piscicides. Sunlight and turbulence affected rotenone and antimycin but DOM did not. Increasing the concentration of chemical can increase the resistance to the effects of these environmental conditions; however, the effects of these conditions are considerable in natural settings. Observations of bioassay fish in stream applications of rotenone were used to develop a statistical model to predict the persistence of the piscicide. The model can be used to predict rotenone persistence in small montane streams and to estimate where rotenone concentrations need to be fortified. I measured the mixing rate of a chemical plume in different channel morphologies and at center or edge applications. Center application had a significantly shorter mixing distance than edge application, but mixing distance was not different among meandering, straight, and riffle/pool morphologies. Application of my findings will increase the efficiency and efficacy of native fish conservation using piscicides.

Rotenone and elevated extracellular potassium concentration induce cell-specific fibrillation of α-synuclein in axons of cholinergic enteric neurons in the guinea-pig ileum.

PubMed

Sharrad, D F; Chen, B N; Gai, W P; Vaikath, N; El-Agnaf, O M; Brookes, S J H

2017-04-01

Parkinson's disease is a progressive neurodegenerative disorder that results in the widespread loss of select classes of neurons throughout the nervous system. The pathological hallmarks of Parkinson's disease are Lewy bodies and neurites, of which α-synuclein fibrils are the major component. α-Synuclein aggregation has been reported in the gut of Parkinson's disease patients, even up to a decade before motor symptoms, and similar observations have been made in animal models of disease. However, unlike the central nervous system, the nature of α-synuclein species that form these aggregates and the classes of neurons affected in the gut are unclear. We have previously reported selective expression of α-synuclein in cholinergic neurons in the gut (J Comp Neurol. 2013; 521:657), suggesting they may be particularly vulnerable to degeneration in Parkinson's disease. In this study, we used immunohistochemistry to detect α-synuclein oligomers and fibrils via conformation-specific antibodies after rotenone treatment or prolonged exposure to high [K + ] in ex vivo segments of guinea-pig ileum maintained in organotypic culture. Rotenone and prolonged raising of [K + ] caused accumulation of α-synuclein fibrils in the axons of cholinergic enteric neurons. This took place in a time- and, in the case of rotenone, concentration-dependent manner. Rotenone also caused selective necrosis, indicated by increased cellular autofluorescence, of cholinergic enteric neurons, labeled by ChAT-immunoreactivity, also in a concentration-dependent manner. To our knowledge, this is the first report of rotenone causing selective loss of a neurochemical class in the enteric nervous system. Cholinergic enteric neurons may be particularly susceptible to Lewy pathology and degeneration in Parkinson's disease. © 2016 John Wiley & Sons Ltd.

Mitochondria-Division Inhibitor 1 Protects Against Amyloid-β induced Mitochondrial Fragmentation and Synaptic Damage in Alzheimer's Disease.

PubMed

Reddy, P Hemachandra; Manczak, Maria; Yin, XiangLing

2017-01-01

The purpose our study was to determine the protective effects of mitochondria division inhibitor 1 (Mdivi1) in Alzheimer's disease (AD). Mdivi1 is hypothesized to reduce excessive fragmentation of mitochondria and mitochondrial dysfunction in AD neurons. Very little is known about whether Mdivi1 can confer protective effects in AD. In the present study, we sought to determine the protective effects of Mdivi1 against amyloid-β (Aβ)- and mitochondrial fission protein, dynamin-related protein 1 (Drp1)-induced excessive fragmentation of mitochondria in AD progression. We also studied preventive (Mdivi1+Aβ42) and intervention (Aβ42+Mdivi1) effects against Aβ42 in N2a cells. Using real-time RT-PCR and immunoblotting analysis, we measured mRNA and protein levels of mitochondrial dynamics, mitochondrial biogenesis, and synaptic genes. We also assessed mitochondrial function by measuring H2O2, lipid peroxidation, cytochrome oxidase activity, and mitochondrial ATP. MTT assays were used to assess the cell viability. Aβ42 was found to impair mitochondrial dynamics, lower mitochondrial biogenesis, lower synaptic activity, and lower mitochondrial function. On the contrary, Mdivi1 enhanced mitochondrial fusion activity, lowered fission machinery, and increased biogenesis and synaptic proteins. Mitochondrial function and cell viability were elevated in Mdivi1-treated cells. Interestingly, Mdivi1 pre- and post-treated cells treated with Aβ showed reduced mitochondrial dysfunction, and maintained cell viability, mitochondrial dynamics, mitochondrial biogenesis, and synaptic activity. The protective effects of Mdivi1 were stronger in N2a+Aβ42 pre-treated with Mdivi1, than in N2a+Aβ42 cells than Mdivi1 post-treated cells, indicating that Mdivi1 works better in prevention than treatment in AD like neurons.

NADH-ubiquinone oxidoreductase activity in the kinetoplasts of the plant trypanosomatid Phytomonas serpens.

PubMed

González-Halphen, Diego; Maslov, Dmitri A

2004-03-01

NADH-ubiquinone oxidoreductase activity is present in mitochondrial lysates of Phytomonas serpens. Rotenone at 2-10 microM inhibited the activity 50-75%, indicating that it belongs to respiratory complex I. The activity was also inhibited 50-60% in the presence of 10-30 nM atovaquone suggesting that inhibition of complex I represents a likely mechanism of the known antileishmanial activity of this drug. The complex was partially purified by chromatography on DEAE-Sepharose CL-6B and gel-filtration on Sepharose CL-2B. The NADH:ubiquinone oxidoreductase activity in this preparation was completely inactivated by 20 nM atovaquone. The partially purified complex was present in a low amount and its subunits could not be discerned by staining with Coomassie. However, one of its components, a homologue of the 39 kDa subunit of the bovine complex I, was identified immunochemically in the original lysate and in the partially purified material.

Interaction between subclinical doses of the Parkinson's disease associated gene, α-synuclein, and the pesticide, rotenone, precipitates motor dysfunction and nigrostriatal neurodegeneration in rats.

PubMed

Naughton, Carol; O'Toole, Daniel; Kirik, Deniz; Dowd, Eilís

2017-01-01

In most patients, Parkinson's disease is thought to emerge after a lifetime of exposure to, and interaction between, various genetic and environmental risk factors. One of the key genetic factors linked to this condition is α-synuclein, and the α-synuclein protein is pathologically associated with idiopathic cases. However, α-synuclein pathology is also present in presymptomatic, clinically "normal" individuals suggesting that environmental factors, such as Parkinson's disease-linked agricultural pesticides, may be required to precipitate Parkinson's disease in these individuals. In this context, the aim of this study was to assess the behavioural and neuropathological impact of exposing rats with a subclinical load of α-synuclein to subclinical doses of the organic pesticide, rotenone. Rats were randomly assigned to two groups for intra-nigral infusion of AAV 2/5- GFP or AAV 2/5 -α-synuclein. Post viral motor function was assessed at 8, 10 and 12 weeks in the Corridor, Stepping and Whisker tests of lateralised motor function. At week 12, animals were performance-matched to receive a subsequent intra-striatal challenge of the organic pesticide rotenone (or its vehicle) to yield four final groups (Control, Rotenone, AAV 2/5 -α-synuclein and Combined). Behavioural testing resumed one week after rotenone surgery and continued for 5 weeks. We found that, when administered alone, neither intra-nigral AAV-α-synuclein nor intra-striatal rotenone caused sufficient nigrostriatal neurodegeneration to induce a significant motor impairment in their own right. However, when these were administered sequentially to the same rats, the interaction between the two Parkinsonian challenges significantly exacerbated nigrostriatal neurodegeneration which precipitated a pronounced impairment in motor function. These results indicate that exposing rats with a subclinical α-synuclein-induced pathology to the pesticide, rotenone, profoundly exacerbates their Parkinsonian neuropathology and dysfunction, and highlights the potential importance of this interaction in the etiology of, and in driving the pathogenesis of Parkinson's disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluating the piscicide rotenone as an option for eradication of invasive Mozambique tilapia in a Hawaiian brackish-water wetland complex

USGS Publications Warehouse

Nico, Leo; Englund, Ronald A.; Jelks, Howard L.

2015-01-01

Mozambique tilapia Oreochromis mossambicus were recently discovered in ‘Aimakapā Fishpond, a 12-hectare brackish-water wetland complex in Kaloko-Honokōhau National Historical Park, on the Island of Hawai’i. As a possible eradication method, we evaluated rotenone, a natural piscicide used in fish management and the active ingredient in plants traditionally used by indigenous Hawaiians for capturing fish. To assess rotenone’s efficacy in killing tilapia and effects on non-target species, laboratory toxicity tests involved exposing organisms to various concentrations of liquid CFT Legumine (5% rotenone) in static trials of 48-h to 72-h duration. Test organisms included: Mozambique tilapia, non-native guppy Poecilia reticulata, the non-native odonate Rambur’s forktail Ischnura ramburii, native feeble shrimp Palaemon debilis, and native ‘ōpae’ula shrimp Halocaridina rubra. All organisms and water used in tests were obtained from ‘Aimakapā (12.6–12.7 ppt salinity), or, for H. rubra, an anchialine pool (15.0–15.2 ppt salinity). Survival analyses indicated CFT Legumine concentrations >3 ppm (>0.15 mg/L rotenone) achieved 100% mortality of tilapia and 93% of guppies within 24 h, with most tilapia killed by 6 h and most guppies by 2 h. Little or no mortality was observed among invertebrate exposed to 1 to 5 mg/L CFT Legumine: 0% mortality for ‘ōpae’ula shrimp, 4% for feeble shrimp; and 16% for odonate larvae. The 48 h LC50 values for Mozambique tilapia and guppy were 0.06 and 0.11 mg/L rotenone, respectively. Results demonstrate rotenone’s potential for non-native fish eradication in brackish-water habitats, with benefit of low mortality to certain macro-invertebrates. High rotenone tolerance displayed by ‘ōpae’ula shrimp is noteworthy. Invasive fish are common in anchialine pools, threatening existence of shrimp and other invertebrate fauna. Although rotenone’s effects on freshwater organisms have been well studied, our research represents one of only a few controlled laboratory experiments quantitatively assessing rotenone tolerance of brackish or marine fauna.

Monitoring wheat mitochondrial compositional and respiratory changes using Fourier transform mid-infrared spectroscopy in response to agrochemical treatments

USDA-ARS?s Scientific Manuscript database

Fungicides and plant growth regulators can impact plant growth outside of their effects on fungal pathogens. Although many of these chemicals are inhibitors of mitochondrial oxygen uptake, information remains limited as to whether they are able tomodify other mitochondrial constituents. Fourier tran...

Curcumin ameliorates dopaminergic neuronal oxidative damage via activation of the Akt/Nrf2 pathway.

PubMed

Cui, Qunli; Li, Xin; Zhu, Hongcan

2016-02-01

Parkinson's disease (PD) is an age-related complex neurodegenerative disease that affects ≤ 80% of dopaminergic neurons in the substantia nigra pars compacta (SNpc). It has previously been suggested that mitochondrial dysfunction, oxidative stress and oxidative damage underlie the pathogenesis of PD. Curcumin, which is a major active polyphenol component extracted from the rhizomes of Curcuma longa (Zingiberaceae), has been reported to exert neuroprotective effects on an experimental model of PD. The present study conducted a series of in vivo experiments, in order to investigate the effects of curcumin on behavioral deficits, oxidative damage and related mechanisms. The results demonstrated that curcumin was able to significantly alleviate motor dysfunction and increase suppressed tyrosine hydroxylase (TH) activity in the SNpc of rotenone (ROT)-injured rats. Biochemical measurements indicated that rats pretreated with curcumin exhibited increased glutathione (GSH) levels, and reduced reactive oxygen species activity and malondialdehyde content. Mechanistic studies demonstrated that curcumin significantly restored the expression levels of heme oxygenase-1 and quinone oxidoreductase 1, thus ameliorating ROT-induced damage in vivo, via the phosphorylation of Akt and nuclear factor erythroid 2-related factor 2 (Nrf2). Further studies indicated that the Akt/Nrf2 signaling pathway was associated with the protective role of curcumin in ROT-treated rats. Inhibiting the Akt/Nrf2 pathway using a lentiviral vector containing Nrf2-specific short hairpin RNA, or the phosphoinositide 3-kinase inhibitor LY294002, markedly reduced the expression levels of TH and GSH, ultimately attenuating the neuroprotective effects of curcumin against oxidative damage. These results indicated that curcumin was able to significantly ameliorate ROT-induced dopaminergic neuronal oxidative damage in the SNpc of rats via activation of the Akt/Nrf2 signaling pathway.

Calcium-dependent mitochondrial cAMP production enhances aldosterone secretion.

PubMed

Katona, Dávid; Rajki, Anikó; Di Benedetto, Giulietta; Pozzan, Tullio; Spät, András

2015-09-05

Glomerulosa cells secrete aldosterone in response to agonists coupled to Ca(2+) increases such as angiotensin II and corticotrophin, coupled to a cAMP dependent pathway. A recently recognized interaction between Ca(2+) and cAMP is the Ca(2+)-induced cAMP formation in the mitochondrial matrix. Here we describe that soluble adenylyl cyclase (sAC) is expressed in H295R adrenocortical cells. Mitochondrial cAMP formation, monitored with a mitochondria-targeted fluorescent sensor (4mtH30), is enhanced by HCO3(-) and the Ca(2+) mobilizing agonist angiotensin II. The effect of angiotensin II is inhibited by 2-OHE, an inhibitor of sAC, and by RNA interference of sAC, but enhanced by an inhibitor of phosphodiesterase PDE2A. Heterologous expression of the Ca(2+) binding protein S100G within the mitochondrial matrix attenuates angiotensin II-induced mitochondrial cAMP formation. Inhibition and knockdown of sAC significantly reduce angiotensin II-induced aldosterone production. These data provide the first evidence for a cell-specific functional role of mitochondrial cAMP. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

In Vivo Determination of Mitochondrial Function Using Luciferase-Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced Dysfunction.

PubMed

Luz, Anthony L; Lagido, Cristina; Hirschey, Matthew D; Meyer, Joel N

2016-08-01

Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase-expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady-state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short-term inhibitor exposure indicate toxicant-induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant-induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Toxicity of rotenone to giant river freshwater prawn Macrobrachium rosenbergii

USDA-ARS?s Scientific Manuscript database

Aquaculturists have often suffered predation losses in the production of freshwater giant river prawn Macrobrachium rosenbergii due to the presence of wild fish species in culture ponds. The piscicide rotenone is widely used to remove undesirable fish species from ponds. Although evidence in the t...

Rotanone analogs: method of preparation and use

DOEpatents

VanBrocklin, Henry F; O& #x27; Neil, James P; Gibbs, Andrew R; Erathodiyil, Nandanan

2013-10-08

The present invention provides rotenone analogs and methods of making and using them. Labeled with single photon and positron emitting isotopes, the rotenone analogs of the present invention are useful in, for example, clinical imaging applications as tracers to measure cardiac blood flow and detect regions of ischemia.

PGC-1α, A Potential Therapeutic Target for Early Intervention in Parkinson’s Disease

PubMed Central

Zheng, Bin; Liao, Zhixiang; Locascio, Joseph J.; Lesniak, Kristen A.; Roderick, Sarah S.; Watt, Marla L.; Eklund, Aron C.; Zhang-James, Yanli; Kim, Peter D.; Hauser, Michael A.; Grünblatt, Edna; Moran, Linda B.; Mandel, Silvia A.; Riederer, Peter; Miller, Renee M.; Federoff, Howard J.; Wüllner, Ullrich; Papapetropoulos, Spyridon; Youdim, Moussa B.; Cantuti-Castelvetri, Ippolita; Young, Anne B.; Vance, Jeffery M.; Davis, Richard L.; Hedreen, John C.; Adler, Charles H.; Beach, Thomas G.; Graeber, Manuel B.; Middleton, Frank A.; Rochet, Jean-Christophe; Scherzer, Clemens R.

2011-01-01

Parkinson’s disease affects 5 million people worldwide, but the molecular mechanisms underlying its pathogenesis are still unclear. Here, we report a genome-wide meta-analysis of gene sets (groups of genes that encode the same biological pathway or process) in 410 samples from patients with symptomatic Parkinson’s and subclinical disease and healthy controls. We analyzed 6.8 million raw data points from nine genome-wide expression studies, and 185 laser-captured human dopaminergic neuron and substantia nigra transcriptomes, followed by two-stage replication on three platforms. We found 10 gene sets with previously unknown associations with Parkinson’s disease. These gene sets pinpoint defects in mitochondrial electron transport, glucose utilization, and glucose sensing and reveal that they occur early in disease pathogenesis. Genes controlling cellular bioenergetics that are expressed in response to peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) are underexpressed in Parkinson’s disease patients. Activation of PGC-1α results in increased expression of nuclear-encoded subunits of the mitochondrial respiratory chain and blocks the dopaminergic neuron loss induced by mutant α-synuclein or the pesticide rotenone in cellular disease models. Our systems biology analysis of Parkinson’s disease identifies PGC-1α as a potential therapeutic target for early intervention. PMID:20926834

A lipid switch unlocks Parkinson’s disease-associated ATP13A2

PubMed Central

Holemans, Tine; Sørensen, Danny Mollerup; van Veen, Sarah; Martin, Shaun; Hermans, Diane; Kemmer, Gerdi Christine; Van den Haute, Chris; Baekelandt, Veerle; Günther Pomorski, Thomas; Agostinis, Patrizia; Wuytack, Frank; Palmgren, Michael; Eggermont, Jan; Vangheluwe, Peter

2015-01-01

ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor–Rakeb syndrome and Parkinson’s disease (PD), providing protection against α-synuclein, Mn2+, and Zn2+ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders. PMID:26134396

c-Jun N-terminal kinase 3 (JNK3) Mediates Paraquat- and Rotenone-Induced Dopaminergic Neuron Death

PubMed Central

Choi, Won Seok; Abel, Glen; Klintworth, Heather; Flavell, Richard A.; Xia, Zhengui

2011-01-01

Mechanistic studies underlying dopaminergic neuron death may identify new drug targets for the treatment of Parkinson disease (PD). Epidemiological studies have linked pesticide exposure to increased risk for sporadic PD. Here, we investigated the role of c-Jun N-terminal kinase 3 (JNK3), a neural-specific JNK isoform, in dopaminergic neuron death induced by the pesticides rotenone and paraquat. The role of JNK3 was evaluated using RNA silencing and gene deletion to block JNK3 signaling. Using an antibody that recognizes all isoforms of activated JNKs, we found that paraquat and rotenone stimulate JNK phosphorylation in primary cultured dopaminergic neurons. In cultured neurons transfected with Jnk3-specific siRNA and in neurons from Jnk3−/− mice, JNK phosphorylation was nearly abolished, suggesting that JNK3 is the main JNK isoform activated in dopaminergic neurons by these pesticides. Paraquat- and rotenone-induced death of dopaminergic neurons was also significantly reduced by Jnk3 siRNA or Jnk3 gene deletion and deletion of the Jnk3 gene completely attenuated paraquat-induced dopaminergic neuron death and motor-deficits in vivo. Our data identify JNK3 as a common and critical mediator of dopaminergic neuron death induced by paraquat and rotenone, suggesting that it is a potential drug target for PD treatment. PMID:20418776

Mitochondrial Division Inhibitor 1 (mdivi-1) Protects Neurons against Excitotoxicity through the Modulation of Mitochondrial Function and Intracellular Ca2+ Signaling.

PubMed

Ruiz, Asier; Alberdi, Elena; Matute, Carlos

2018-01-01

Excessive dynamin related protein 1 (Drp1)-triggered mitochondrial fission contributes to apoptosis under pathological conditions and therefore it has emerged as a promising therapeutic target. Mitochondrial division inhibitor 1 (mdivi-1) inhibits Drp1-dependent mitochondrial fission and is neuroprotective in several models of brain ischemia and neurodegeneration. However, mdivi-1 also modulates mitochondrial function and oxidative stress independently of Drp1, and consequently the mechanisms through which it protects against neuronal injury are more complex than previously foreseen. In this study, we have analyzed the effects of mdivi-1 on mitochondrial dynamics, Ca 2+ signaling, mitochondrial bioenergetics and cell viability during neuronal excitotoxicity in vitro . Time-lapse fluorescence microscopy revealed that mdivi-1 blocked NMDA-induced mitochondrial fission but not that triggered by sustained AMPA receptor activation, showing that mdivi-1 inhibits excitotoxic mitochondrial fragmentation in a source specific manner. Similarly, mdivi-1 strongly reduced NMDA-triggered necrotic-like neuronal death and, to a lesser extent, AMPA-induced toxicity. Interestingly, neuroprotection provided by mdivi-1 against NMDA, but not AMPA, correlated with a reduction in cytosolic Ca 2+ ([Ca 2+ ] cyt ) overload and calpain activation indicating additional cytoprotective mechanisms. Indeed, mdivi-1 depolarized mitochondrial membrane and depleted ER Ca 2+ content, leading to attenuation of mitochondrial [Ca 2+ ] increase and enhancement of the integrated stress response (ISR) during NMDA receptor activation. Finally, lentiviral knockdown of Drp1 did not rescue NMDA-induced mitochondrial fission and toxicity, indicating that neuroprotective activity of mdivi-1 is Drp1-independent. Together, these results suggest that mdivi-1 induces a Drp1-independent protective phenotype that prevents predominantly NMDA receptor-mediated excitotoxicity through the modulation of mitochondrial function and intracellular Ca 2+ signaling.

PARP Inhibitors Sensitize Ewing Sarcoma Cells to Temozolomide-Induced Apoptosis via the Mitochondrial Pathway.

PubMed

Engert, Florian; Schneider, Cornelius; Weiβ, Lilly Magdalena; Probst, Marie; Fulda, Simone

2015-12-01

Ewing sarcoma has recently been reported to be sensitive to poly(ADP)-ribose polymerase (PARP) inhibitors. Searching for synergistic drug combinations, we tested several PARP inhibitors (talazoparib, niraparib, olaparib, veliparib) together with chemotherapeutics. Here, we report that PARP inhibitors synergize with temozolomide (TMZ) or SN-38 to induce apoptosis and also somewhat enhance the cytotoxicity of doxorubicin, etoposide, or ifosfamide, whereas actinomycin D and vincristine show little synergism. Furthermore, triple therapy of olaparib, TMZ, and SN-38 is significantly more effective compared with double or monotherapy. Mechanistic studies revealed that the mitochondrial pathway of apoptosis plays a critical role in mediating the synergy of PARP inhibition and TMZ. We show that subsequent to DNA damage-imposed checkpoint activation and G2 cell-cycle arrest, olaparib/TMZ cotreatment causes downregulation of the antiapoptotic protein MCL-1, followed by activation of the proapoptotic proteins BAX and BAK, mitochondrial outer membrane permeabilization (MOMP), activation of caspases, and caspase-dependent cell death. Overexpression of a nondegradable MCL-1 mutant or BCL-2, knockdown of NOXA or BAX and BAK, or the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) all significantly reduce olaparib/TMZ-mediated apoptosis. These findings emphasize the role of PARP inhibitors for chemosensitization of Ewing sarcoma with important implications for further (pre)clinical studies. ©2015 American Association for Cancer Research.

4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic Acid (C75), an Inhibitor of Fatty-acid Synthase, Suppresses the Mitochondrial Fatty Acid Synthesis Pathway and Impairs Mitochondrial Function*

PubMed Central

Chen, Cong; Han, Xiao; Zou, Xuan; Li, Yuan; Yang, Liang; Cao, Ke; Xu, Jie; Long, Jiangang; Liu, Jiankang; Feng, Zhihui

2014-01-01

4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) is a synthetic fatty-acid synthase (FASN) inhibitor with potential therapeutic effects in several cancer models. Human mitochondrial β-ketoacyl-acyl carrier protein synthase (HsmtKAS) is a key enzyme in the newly discovered mitochondrial fatty acid synthesis pathway that can produce the substrate for lipoic acid (LA) synthesis. HsmtKAS shares conserved catalytic domains with FASN, which are responsible for binding to C75. In our study, we explored the possible effect of C75 on HsmtKAS and mitochondrial function. C75 treatment decreased LA content, impaired mitochondrial function, increased reactive oxygen species content, and reduced cell viability. HsmtKAS but not FASN knockdown had an effect that was similar to C75 treatment. In addition, an LA supplement efficiently inhibited C75-induced mitochondrial dysfunction and oxidative stress. Overexpression of HsmtKAS showed cellular protection against low dose C75 addition, whereas there was no protective effect upon high dose C75 addition. In summary, the mitochondrial fatty acid synthesis pathway has a vital role in mitochondrial function. Besides FASN, C75 might also inhibit HsmtKAS, thereby reducing LA production, impairing mitochondrial function, and potentially having toxic effects. LA supplements sufficiently ameliorated the toxicity of C75, showing that a combination of C75 and LA may be a reliable cancer treatment. PMID:24784139

Transcriptional upregulation of mitochondrial uncoupling protein 2 protects against oxidative stress-associated neurogenic hypertension.

PubMed

Chan, Samuel H H; Wu, Chiung-Ai; Wu, Kay L H; Ho, Ying-Hao; Chang, Alice Y W; Chan, Julie Y H

2009-10-23

Mitochondrial uncoupling proteins (UCPs) belong to a superfamily of mitochondrial anion transporters that uncouple ATP synthesis from oxidative phosphorylation and mitigates mitochondrial reactive oxygen species production. We assessed the hypothesis that UCP2 participates in central cardiovascular regulation by maintaining reactive oxygen species homeostasis in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons that maintain vasomotor tone located. We also elucidated the molecular mechanisms that underlie transcriptional upregulation of UCP2 in response to oxidative stress in RVLM. In Sprague-Dawley rats, transcriptional upregulation of UCP2 in RVLM by rosiglitazone, an activator of its transcription factor peroxisome proliferator-activated receptor (PPAR)gamma, reduced mitochondrial hydrogen peroxide level in RVLM and systemic arterial pressure. Oxidative stress induced by microinjection of angiotensin II into RVLM augmented UCP2 mRNA or protein expression in RVLM, which was antagonized by comicroinjection of NADPH oxidase inhibitor (diphenyleneiodonium chloride), superoxide dismutase mimetic (tempol), or p38 mitogen-activated protein kinase inhibitor (SB203580) but not by extracellular signal-regulated kinase 1/2 inhibitor (U0126). Angiotensin II also induced phosphorylation of the PPARgamma coactivator, PPARgamma coactivator (PGC)-1alpha, and an increase in formation of PGC-1alpha/PPARgamma complexes in a p38 mitogen-activated protein kinase-dependent manner. Intracerebroventricular infusion of angiotensin II promoted an increase in mitochondrial hydrogen peroxide production in RVLM and chronic pressor response, which was potentiated by gene knockdown of UCP2 but blunted by rosiglitazone. These results suggest that transcriptional upregulation of mitochondrial UCP2 in response to an elevation in superoxide plays an active role in feedback regulation of reactive oxygen species production in RVLM and neurogenic hypertension associated with chronic oxidative stress.

Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia.

PubMed

Moshal, Karni S; Tipparaju, Srinivas M; Vacek, Thomas P; Kumar, Munish; Singh, Mahavir; Frank, Iluiana E; Patibandla, Phani K; Tyagi, Neetu; Rai, Jayesh; Metreveli, Naira; Rodriguez, Walter E; Tseng, Michael T; Tyagi, Suresh C

2008-08-01

Cardiomyocyte N-methyl-d-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (-dL/dt), and maximal rate of relaxation (+dL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, -dL/dt, and +dL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1.

Activation of Akt rescues endoplasmic reticulum stress-impaired murine cardiac contractile function via glycogen synthase kinase-3β-mediated suppression of mitochondrial permeation pore opening.

PubMed

Zhang, Yingmei; Xia, Zhi; La Cour, Karissa H; Ren, Jun

2011-11-01

The present study was designed to examine the impact of chronic Akt activation on endoplasmic reticulum (ER) stress-induced cardiac mechanical anomalies, if any, and the underlying mechanism involved. Wild-type and transgenic mice with cardiac-specific overexpression of the active mutant of Akt (Myr-Akt) were subjected to the ER stress inducer tunicamycin (1 or 3 mg/kg). ER stress led to compromised echocardiographic (elevated left ventricular end-systolic diameter and reduced fractional shortening) and cardiomyocyte contractile function, intracellular Ca(2+) mishandling, and cell survival in wild-type mice associated with mitochondrial damage. In vitro ER stress induction in murine cardiomyocytes upregulated the ER stress proteins Gadd153, GRP78, and phospho-eIF2α, and promoted reactive oxygen species production, carbonyl formation, apoptosis, mitochondrial membrane potential loss, and mitochondrial permeation pore (mPTP) opening associated with overtly impaired cardiomyocyte contractile and intracellular Ca(2+) properties. Interestingly, these anomalies were mitigated by chronic Akt activation or the ER chaperon tauroursodeoxycholic acid (TUDCA). Treatment with tunicamycin also dephosphorylated Akt and its downstream signal glycogen synthase kinase 3β (GSK3β) (leading to activation of GSK3β), the effect of which was abrogated by Akt activation and TUDCA. The ER stress-induced cardiomyocyte contractile and mitochondrial anomalies were obliterated by the mPTP inhibitor cyclosporin A, GSK3β inhibitor SB216763, and ER stress inhibitor TUDCA. This research reported the direct relationship between ER stress and cardiomyocyte contractile and mitochondrial anomalies for the first time. Taken together, these data suggest that ER stress may compromise cardiac contractile and intracellular Ca(2+) properties, possibly through the Akt/GSK3β-dependent impairment of mitochondrial integrity.

D-Lactate transport and metabolism in rat liver mitochondria.

PubMed

de Bari, Lidia; Atlante, Anna; Guaragnella, Nicoletta; Principato, Giovanni; Passarella, Salvatore

2002-07-15

In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize D-lactate. We found the following: (1) externally added D-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation)=2] and membrane potential (Delta(psi)) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by alpha-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (D-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate D-lactate traffic across the mitochondrial inner membrane: the D-lactate/H(+) symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the D-lactate/oxoacid antiporter (which mediates both the D-lactate/pyruvate and D-lactate/oxaloacetate exchanges) and D-lactate/malate antiporter studied by monitoring photometrically the appearance of the D-lactate counteranions outside mitochondria. The D-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the V(max) values and in the inhibition and pH profiles and were shown to regulate mitochondrial D-lactate metabolism in vitro. The D-lactate translocators and the D-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for D-lactate-dependent gluconeogenesis.

Lumbee traditional medicine: Neuroprotective activities of medicinal plants used to treat Parkinson's disease-related symptoms.

PubMed

de Rus Jacquet, Aurélie; Timmers, Michael; Ma, Sin Ying; Thieme, Andrew; McCabe, George P; Vest, Jay Hansford C; Lila, Mary Ann; Rochet, Jean-Christophe

2017-07-12

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta and the presence in surviving neurons of Lewy body inclusions enriched with aggregated forms of the presynaptic protein α-synuclein (aSyn). Although current therapies provide temporary symptomatic relief, they do not slow the underlying neurodegeneration in the midbrain. In this study, we analyzed contemporary herbal medicinal practices used by members of the Lumbee tribe to treat PD-related symptoms, in an effort to identify safe and effective herbal medicines to treat PD. The aims of this study were to (i) document medicinal plants used by Lumbee Indians to treat PD and PD-related symptoms, and (ii) characterize a subset of plant candidates in terms of their ability to alleviate neurotoxicity elicited by PD-related insults and their potential mechanisms of neuroprotection. Interviews of Lumbee healers and local people were carried out in Pembroke, North Carolina, and in surrounding towns. Plant samples were collected and prepared as water extracts for subsequent analysis. Extracts were characterized in terms of their ability to induce activation of the nuclear factor E2-related factor 2 (Nrf2) antioxidant response in cortical astrocytes. An extract prepared from Sambucus caerulea flowers (elderflower extract) was further examined for the ability to induce Nrf2-mediated transcription in induced pluripotent stem cell (iPSC)-derived astrocytes and primary midbrain cultures, to ameliorate mitochondrial dysfunction, and to alleviate rotenone- or aSyn-mediated neurotoxicity. The ethnopharmacological interviews resulted in the documentation of 32 medicinal plants used to treat PD-related symptoms and 40 plants used to treat other disorders. A polyphenol-rich extract prepared from elderflower activated the Nrf2-mediated antioxidant response in cortical astrocytes, iPSC-derived astrocytes, and primary midbrain cultures, apparently via the inhibition of Nrf2 degradation mediated by the ubiquitin proteasome system. Furthermore, the elderflower extract rescued mitochondrial functional deficits in a neuronal cell line and alleviated neurotoxicity elicited by rotenone and aSyn in primary midbrain cultures. These results highlight potential therapeutic benefits of botanical extracts used in traditional Lumbee medicine, and they provide insight into mechanisms by which an elderflower extract could suppress neurotoxicity elicited by environmental and genetic PD-related insults. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, So Jung; Park, Young Jun; Shin, Ji Hyun

2011-05-13

Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactivemore » chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.« less

Iron alters cell survival in a mitochondria-dependent pathway in ovarian cancer cells

PubMed Central

Bauckman, Kyle; Haller, Edward; Taran, Nicholas; Rockfield, Stephanie; Ruiz-Rivera, Abigail; Nanjundan, Meera

2015-01-01

The role of iron in the development of cancer remains unclear. We previously reported that iron reduces cell survival in a Ras/mitogen-activated protein kinase (MAPK)-dependent manner in ovarian cells; however, the underlying downstream pathway leading to reduced survival was unclear. Although levels of intracellular iron, ferritin/CD71 protein and reactive oxygen species did not correlate with iron-induced cell survival changes, we identified mitochondrial damage (via TEM) and reduced expression of outer mitochondrial membrane proteins (translocase of outer membrane: TOM20 and TOM70) in cell lines sensitive to iron. Interestingly, Ru360 (an inhibitor of the mitochondrial calcium uniporter) reversed mitochondrial changes and restored cell survival in HEY ovarian carcinoma cells treated with iron. Further, cells treated with Ru360 and iron also had reduced autophagic punctae with increased lysosomal numbers, implying cross-talk between these compartments. Mitochondrial changes were dependent on activation of the Ras/MAPK pathway since treatment with a MAPK inhibitor restored expression of TOM20/TOM70 proteins. Although glutathione antioxidant levels were reduced in HEY treated with iron, extracellular glutamate levels were unaltered. Strikingly, oxalomalate (inhibitor of aconitase, involved in glutamate production) reversed iron-induced responses in a similar manner to Ru360. Collectively, our results implicate iron in modulating cell survival in a mitochondria-dependent manner in ovarian cancer cells. PMID:25697096

Protective role of apigenin on rotenone induced rat model of Parkinson's disease: Suppression of neuroinflammation and oxidative stress mediated apoptosis.

PubMed

Anusha, Chandran; Sumathi, Thangarajan; Joseph, Leena Dennis

2017-05-01

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra which is associated with oxidative stress, neuroinflammation and apoptosis. Apigenin (AGN), a non-mutagenic flavone found in fruits and vegetables, exhibits a variety of biological effects including anti-apoptotic, anti-inflammatory, and free radical scavenging activities. The current study was aimed to investigate the neuroprotective effects and molecular mechanisms of AGN in a rat model of PD induced by rotenone (ROT). Unilateral stereotaxic intranigral infusion of ROT caused the loss of tyrosine hydroxylase (TH) immunoreactivity in striatum and substantia nigra. AGN treatment (10 and 20 mg/kg, i.p.) showed a significant improvement in behavioral, biochemical and mitochondrial enzyme activities as compared to ROT exposed rats. The mRNA expression of inflammatory markers and neurotrophic factors was quantified by reverse transcriptase polymerase chain reaction (RT-PCR). Administration of AGN significantly attenuated the upregulation of NF-κB gene expression in ROT induced group and prevented the neuroinflammation in substantia nigra pars compacta (SNpc). Further, AGN inhibited the release of pro-inflammatory cytokines TNF- α, IL-6 and pro-inflammatory enzyme iNOS-1 induced by ROT. Additionally, AGN prevents the reduction of neurotrophic factors BDNF and GDNF mRNA expression in ROT lesioned rats. Immunoblot results illustrated that AGN treatment downregulated α-synuclein aggregation and upregulated the TH protein expression as well as dopamine D2 receptor (D2R) expression in ROT lesioned rats. Thus, the present findings collectively suggest that AGN exerts its neuroprotection in ROT model of PD and may act as an effective agent for treatment of PD. Copyright © 2017 Elsevier B.V. All rights reserved.

VPS41, a protein involved in lysosomal trafficking, is protective in C. elegans and mammalian cellular models of Parkinson's disease

PubMed Central

Ruan, Qingmin; Harrington, Adam J.; Caldwell, Kim A.; Caldwell, Guy A.; Standaert, David G.

2009-01-01

VPS41 is a protein identified as a potential therapeutic target for Parkinson's disease (PD) as a result of a high-throughput RNAi screen in C. elegans. VPS41 has a plausible mechanistic link to the pathogenesis of PD, as in yeast it is known to participate in trafficking of proteins to the lysosomal system and several recent lines of evidence have pointed to the importance of lysosomal system dysfunction in the neurotoxicity of alpha-synuclein (α-syn). We found that expression of the human form of VPS41 (hVPS41) prevents dopamine (DA) neuron loss induced by α-syn overexpression and 6-hydroxydopamine (6-OHDA) neurotoxicity in C. elegans. In SH-SY5Y neuroblastoma cell lines stably transfected with hVPS41, we determined that presence of this protein conferred protection against the neurotoxins 6-OHDA and rotenone. Overexpression of hVPS41 did not alter the mitochondrial membrane depolarization induced by these neurotoxins. hVPS41 did, however, block downstream events in the apoptotic cascade including activation of caspase-9 and caspase-3, and PARP cleavage. We also observed that hVPS41 reduced the accumulation of insoluble high molecular weight forms of α-syn in SH-SY5Y cells after treatment with rotenone. These data show that hVPS41 is protective against both α-syn and neurotoxic-mediated injury in invertebrate and cellular models of PD. These protective functions may be related to enhanced clearance of misfolded or aggregated protein, including α-syn. Our studies indicate that hVPS41 may be a useful target for developing therapeutic strategies for human PD. PMID:19850127

N-acetylcysteine and vitamin E rescue animal longevity and cellular oxidative stress in pre-clinical models of mitochondrial complex I disease.

PubMed

Polyak, Erzsebet; Ostrovsky, Julian; Peng, Min; Dingley, Stephen D; Tsukikawa, Mai; Kwon, Young Joon; McCormack, Shana E; Bennett, Michael; Xiao, Rui; Seiler, Christoph; Zhang, Zhe; Falk, Marni J

2018-04-01

Oxidative stress is a known contributing factor in mitochondrial respiratory chain (RC) disease pathogenesis. Yet, no efficient means exists to objectively evaluate the comparative therapeutic efficacy or toxicity of different antioxidant compounds empirically used in human RC disease. We postulated that pre-clinical comparative analysis of diverse antioxidant drugs having suggested utility in primary RC disease using animal and cellular models of RC dysfunction may improve understanding of their integrated effects and physiologic mechanisms, and enable prioritization of lead antioxidant molecules to pursue in human clinical trials. Here, lifespan effects of N-acetylcysteine (NAC), vitamin E, vitamin C, coenzyme Q10 (CoQ10), mitochondrial-targeted CoQ10 (MS010), lipoate, and orotate were evaluated as the primary outcome in a well-established, short-lived C. elegans gas-1(fc21) animal model of RC complex I disease. Healthspan effects were interrogated to assess potential reversal of their globally disrupted in vivo mitochondrial physiology, transcriptome profiles, and intermediary metabolic flux. NAC or vitamin E fully rescued, and coenzyme Q, lipoic acid, orotic acid, and vitamin C partially rescued gas-1(fc21) lifespan toward that of wild-type N2 Bristol worms. MS010 and CoQ10 largely reversed biochemical pathway expression changes in gas-1(fc21) worms. While nearly all drugs normalized the upregulated expression of the "cellular antioxidant pathway", they failed to rescue the mutant worms' increased in vivo mitochondrial oxidant burden. NAC and vitamin E therapeutic efficacy were validated in human fibroblast and/or zebrafish complex I disease models. Remarkably, rotenone-induced zebrafish brain death was preventable partially with NAC and fully with vitamin E. Overall, these pre-clinical model animal data demonstrate that several classical antioxidant drugs do yield significant benefit on viability and survival in primary mitochondrial disease, where their major therapeutic benefit appears to result from targeting global cellular, rather than intramitochondria-specific, oxidative stress. Clinical trials are needed to evaluate whether the two antioxidants, NAC and vitamin E, that show greatest efficacy in translational model animals significantly improve the survival, function, and feeling of human subjects with primary mitochondrial RC disease. Copyright © 2018 Elsevier Inc. All rights reserved.

Synergistic Anticancer Action of Lysosomal Membrane Permeabilization and Glycolysis Inhibition.

PubMed

Kosic, Milica; Arsikin-Csordas, Katarina; Paunovic, Verica; Firestone, Raymond A; Ristic, Biljana; Mircic, Aleksandar; Petricevic, Sasa; Bosnjak, Mihajlo; Zogovic, Nevena; Mandic, Milos; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

2016-10-28

We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Combined Inhibition of the Renin-Angiotensin System and Neprilysin Positively Influences Complex Mitochondrial Adaptations in Progressive Experimental Heart Failure

PubMed Central

Reinders, Jörg; Schröder, Josef; Dietl, Alexander; Schmid, Peter M.; Jungbauer, Carsten; Resch, Markus; Maier, Lars S.; Luchner, Andreas; Birner, Christoph

2017-01-01

Background Inhibitors of the renin angiotensin system and neprilysin (RAS-/NEP-inhibitors) proved to be extraordinarily beneficial in systolic heart failure. Furthermore, compelling evidence exists that impaired mitochondrial pathways are causatively involved in progressive left ventricular (LV) dysfunction. Consequently, we aimed to assess whether RAS-/NEP-inhibition can attenuate mitochondrial adaptations in experimental heart failure (HF). Methods and Results By progressive right ventricular pacing, distinct HF stages were induced in 15 rabbits, and 6 animals served as controls (CTRL). Six animals with manifest HF (CHF) were treated with the RAS-/NEP-inhibitor omapatrilat. Echocardiographic studies and invasive blood pressure measurements were undertaken during HF progression. Mitochondria were isolated from LV tissue, respectively, and further worked up for proteomic analysis using the SWATH technique. Enzymatic activities of citrate synthase and the electron transfer chain (ETC) complexes I, II, and IV were assessed. Ultrastructural analyses were performed by transmission electron microscopy. During progression to overt HF, intricate expression changes were mainly detected for proteins belonging to the tricarboxylic acid cycle, glucose and fat metabolism, and the ETC complexes, even though ETC complex I, II, or IV enzymatic activities were not significantly influenced. Treatment with a RAS-/NEP-inhibitor then reversed some maladaptive metabolic adaptations, positively influenced the decline of citrate synthase activity, and altered the composition of each respiratory chain complex, even though this was again not accompanied by altered ETC complex enzymatic activities. Finally, ultrastructural evidence pointed to a reduction of autophagolytic and degenerative processes with omapatrilat-treatment. Conclusions This study describes complex adaptations of the mitochondrial proteome in experimental tachycardia-induced heart failure and shows that a combined RAS-/NEP-inhibition can beneficially influence mitochondrial key pathways. PMID:28076404

Ameliorative Effect of Quercetin on Neurochemical and Behavioral Deficits in Rotenone Rat Model of Parkinson's Disease: Modulating Autophagy (Quercetin on Experimental Parkinson's Disease).

PubMed

El-Horany, Hemat E; El-Latif, Rania N Abd; ElBatsh, Maha M; Emam, Marwa N

2016-07-01

Autophagy is necessary for neuronal homeostasis and its dysfunction has been implicated in Parkinson's disease (PD) as it can exacerbate endoplasmic reticulum (ER) stress and ER stress-induced apoptosis. Quercetin is a flavonoid known for its neuroprotective and antioxidant effects. The present study investigated the protective, autophagy-modulating effects of quercetin in the rotenone rat model of PD. Rotenone was intraperitoneally injected at dose of 2 ml/kg/day for 4 weeks. Simultaneous intraperitoneal injection of quercetin was given at a dose of 50 mg/kg/day also for 4 weeks. Neurobehavioral changes were studied. Oxidative/antioxidant status, C/EBP homologous protein (CHOP), Beclin-1, and dopamine levels were assessed. DNA fragmentation and histopathological changes were evaluated. This research work revealed that quercetin significantly attenuated rotenone-induced behavioral impairment, augmented autophagy, ameliorated ER stress- induced apoptosis with attenuated oxidative stress. From the current study, quercetin can act as an autophagy enhancer in PD rat model and modulates the microenvironment that leads to neuronal death. © 2016 Wiley Periodicals, Inc.

Suicide attempt by ingestion of rotenone-containing plant extracts: one case report in French Guiana.

PubMed

Chesneau, Pierre; Knibiehly, Marc; Tichadou, Lucia; Calvez, Mélanie; Joubert, Michel; Hayek-Lanthois, Maryvonne; De Haro, Luc

2009-09-01

Several species of plants in the Fabaceae family are traditionally used for poison fishing because they contain ichthyotoxic rotenoids. In French Guiana two species of Fabaceae belonging to Lonchocarpus genus with a toxic rotenone effect are used for such ancestral practices. Rotenone is of low toxicity for humans when it is diluted, but its neurotoxicity at higher concentrations is well known to users. The purpose of this article is to describe a case of self-poisoning by an 86-year-old woman who ingested a bowl of mashed ichthyotoxic plants. Despite early onset of severe symptoms, the patient regained consciousness and resumed normal breathing within a few hours with only symptomatic treatment. The clinical pattern observed in this patient (onset of digestive manifestations followed quickly by loss of conscience and respiratory insufficiency) is in agreement with the few poisonings reported in the literature involving other Fabaceae species containing rotenoids in Asia or involving concentrated rotenone used in insecticides. In patients, who survive the initial phase, symptoms usually regress quickly.

Activation of mPTP-dependent mitochondrial apoptosis pathway by a novel pan HDAC inhibitor resminostat in hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Meili; Shi, Wenhong; Li, Zhengling

Over-expression and aberrant activation of histone deacetylases (HDACs) are often associated with poor prognosis of hepatocellular carcinoma (HCC). Here, we evaluated the potential anti-hepatocellular carcinoma (HCC) cell activity by resminostat, a novel pan HDAC inhibitor (HDACi). We demonstrated that resminostat induced potent cytotoxic and anti-proliferative activity against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, resminostat treatment in HCC cells activated mitochondrial permeability transition pore (mPTP)-dependent apoptosis pathway, which was evidenced by physical association of cyclophilin-D and adenine nucleotide translocator 1 (ANT-1), mitochondrial depolarization, cytochrome C release and caspase-9 activation. Intriguingly, the mPTP blockers (sanglifehrinmore » A and cyclosporine A), shRNA knockdown of cyclophilin-D or the caspase-9 inhibitor dramatically attenuated resminostat-induced HCC cell apoptosis and cytotoxicity. Reversely, HCC cells with exogenous cyclophilin-D over-expression were hyper-sensitive to resminostat. Intriguingly, a low concentration of resminostat remarkably potentiated sorafenib-induced mitochondrial apoptosis pathway activation, leading to a profound cytotoxicity in HCC cells. The results of this preclinical study indicate that resminostat (or plus sorafenib) could be further investigated as a valuable anti-HCC strategy. - Highlights: • Resminostat inhibits human HCC cell survival and proliferation. • Resminostat activates mPTP-dependent mitochondrial apoptosis pathway in HCC cells. • Resminostat potentiates sorafenib-induced mitochondrial apoptosis pathway activation. • mPTP or caspase-9 inhibition attenuates apoptosis by resminostat or plus sorafenib.« less

Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca2+-Independent Mechanism

PubMed Central

Guidarelli, Andrea; Cerioni, Liana; Fiorani, Mara; Cantoni, Orazio

2017-01-01

Exposure of U937 cells to peroxynitrite promotes mitochondrial superoxide formation via a mechanism dependent on both inhibition of complex III and increased mitochondrial Ca2+ accumulation. Otherwise inactive concentrations of the oxidant produced the same maximal effects in the presence of either complex III inhibitors or agents mobilizing Ca2+ from the ryanodine receptor and enforcing its mitochondrial accumulation. l-Ascorbic acid (AA) produced similar enhancing effects in terms of superoxide formation, DNA strand scission and cytotoxicity. However, AA failed to enhance the intra-mitochondrial concentration of Ca2+ and the effects observed in cells supplemented with peroxinitrite, while insensitive to manipulations preventing the mobilization of Ca2+, or the mitochondrial accumulation of the cation, were also detected in human monocytes and macrophages, which do not express the ryanodine receptor. In all these cell types, mitochondrial permeability transition-dependent toxicity was detected in cells exposed to AA/peroxynitrite and, based on the above criteria, these responses also appeared Ca2+-independent. The enhancing effects of AA are therefore similar to those mediated by bona fide complex III inhibitors, although the vitamin failed to directly inhibit complex III, and in fact enhanced its sensitivity to the inhibitory effects of peroxynitrite. PMID:28767071

Oligomeric BAX induces mitochondrial permeability transition and complete cytochrome c release without oxidative stress.

PubMed

Li, Tsyregma; Brustovetsky, Tatiana; Antonsson, Bruno; Brustovetsky, Nickolay

2008-11-01

In the present study, we investigated the mechanism of cytochrome c release from isolated brain mitochondria induced by recombinant oligomeric BAX (BAX(oligo)). We found that BAX(oligo) caused a complete release of cytochrome c in a concentration- and time-dependent manner. The release was similar to those induced by alamethicin, which causes maximal mitochondrial swelling and eliminates barrier properties of the OMM. BAX(oligo) also produced large amplitude mitochondrial swelling as judged by light scattering assay and transmission electron microscopy. In addition, BAX(oligo) resulted in a strong mitochondrial depolarization. ATP or a combination of cyclosporin A and ADP, inhibitors of the mPT, suppressed BAX(oligo)-induced mitochondrial swelling and depolarization as well as cytochrome c release but did not influence BAX(oligo) insertion into the OMM. Both BAX(oligo)- and alamethicin-induced cytochrome c releases were accompanied by inhibition of ROS generation, which was assessed by measuring mitochondrial H(2)O(2) release with an Amplex Red assay. The mPT inhibitors antagonized suppression of ROS generation caused by BAX(oligo) but not by alamethicin. Thus, BAX(oligo) resulted in a complete cytochrome c release from isolated brain mitochondria in the mPT-dependent manner without involvement of oxidative stress by the mechanism requiring mitochondrial remodeling and permeabilization of the OMM.

Direct effect of Taxol on free radical formation and mitochondrial permeability transition.

PubMed

Varbiro, G; Veres, B; Gallyas, F; Sumegi, B

2001-08-15

To elucidate the potential role of mitochondria in Taxol-induced cytotoxicity, we studied its direct mitochondrial effects. In Percoll-gradient purified liver mitochondria, Taxol induced large amplitude swelling in a concentration-dependent manner in the microM range. Opening of the permeability pore was also confirmed by the access of mitochondrial matrix enzymes for membrane impermeable substrates in Taxol-treated mitochondria. Taxol induced the dissipation of mitochondrial membrane potential (DeltaPsi) determined by Rhodamine123 release and induced the release of cytochrome c from the intermembrane space. All these effects were inhibited by 2.5 microM cyclosporine A. Taxol significantly increased the formation of reactive oxygen species (ROS) in both the aqueous and the lipid phase as determined by dihydrorhodamine123 and resorufin derivative. Cytochrome oxidase inhibitor CN(-), azide, and NO abrogated the Taxol-induced mitochondrial ROS formation while inhibitors of the other respiratory complexes and cyclosporine A had no effect. We confirmed that the Taxol-induced collapse of DeltaPsi and the induction of ROS production occurs in BRL-3A cells. In conclusion, Taxol-induced adenine nucleotide translocase-cyclophilin complex mediated permeability transition, and cytochrome oxidase mediated ROS production. Because both cytochrome c release and mitochondrial ROS production can induce suicide pathways, the direct mitochondrial effects of Taxol may contribute to its cytotoxicity.

Thymidine kinase 2 enzyme kinetics elucidate the mechanism of thymidine-induced mitochondrial DNA depletion.

PubMed

Sun, Ren; Wang, Liya

2014-10-07

Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

Neuroprotective potential of spermidine against rotenone induced Parkinson's disease in rats.

PubMed

Sharma, Sunaina; Kumar, Puneet; Deshmukh, Rahul

2018-06-01

Parkinson's disease is a leading hypokinetic disorder characterized by selective loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) region of mid-brain. Degeneration of dopaminergic neurons is considered to be due to oxidative stress, neuroinflammation, disturbed calcium homeostasis and glutamate excitotoxicity etc. Spermidine is a polyamine which counteracts age associated cell death by scavenging free radical formation, activates authophagic machinery by enhancing formation of autophagosome, and antagonizes NMDA receptor. In the current study we investigated the neuroprotective potential of spermidine against rotenone induced PD in rats. Rats were treated subcutaneously with rotenone 1.5 mg/kg daily for 28 days. Spermidine 5&10 mg/kg was administered orally 1 h prior to rotenone administration from 15 to 28. Rotenone caused significant reduction in motor functioning and elevated levels of oxidative stress markers and proinflammatory cytokines levels (IL-1β, IL6 and TNF-α). The neurochemical analysis revealed a significant decrease in serotonin, norepinephrine, dopamine and their metabolites accompanied by a significant loss of dopaminergic neurons in the SNpc following ROT injection. However, treatment with spermidine rescued DAergic neurons in SNpc and nerve terminals in the striatum following ROT insult. Spermidine treatment also attenuated oxidative stress, neuroinflammation and restored striatal neurochemistry. Results of our study suggest that spermidine has promising neuroprotective effect against degenerative changes in experimental PD, and the protective effects are mediated through its antioxidant and anti-inflammatory properties. Copyright © 2018 Elsevier Ltd. All rights reserved.

The behavioural and neuropathological impact of intranigral AAV-α-synuclein is exacerbated by systemic infusion of the Parkinson's disease-associated pesticide, rotenone, in rats.

PubMed

Mulcahy, Pádraig; O'Doherty, Aideen; Paucard, Alexia; O'Brien, Timothy; Kirik, Deniz; Dowd, Eilís

2013-04-15

Despite the widely held belief that Parkinson's disease is caused by both underlying genetics and exposure to environmental risk factors, it is still widely modelled in preclinical models using a single genetic or neurotoxic insult. This single-insult approach has resulted in a variety of models that are limited with respect to their aetiological, construct, face and/or predictive validity. Thus, the aim of the current study was to investigate the interplay between genes and the environment as an alternative approach to modelling Parkinson's disease. To do so, rats underwent stereotaxic surgery for unilateral delivery of the Parkinson's disease-associated gene, α-synuclein, into the substantia nigra (using AAV vectors). This was followed 13 weeks later by subcutaneous implantation of an osmotic minipump delivering the Parkinson's disease-associated pesticide, rotenone (2.5mgkg(-1)day(-1) for 4 weeks). The effect of the genetic and environmental insults alone or in combination on lateralised motor performance (Corridor, Stepping and Whisker Tests), nigrostriatal integrity (tyrosine hydroxylase immunohistochemistry) and α-synucleinopathy (α-synuclein immunohistochemistry) was assessed. We found that exposing AAV-α-synuclein-treated rats to rotenone led to a model in which the classical Parkinson's disease triad of progressive motor dysfunction, nigrostriatal neurodegeneration and α-synucleinopathy was evident. However, delivering rotenone systemically was also associated with bilateral motor dysfunction and loss of body weight. Thus, although we have shown that Parkinson's disease can be modelled in experimental animals by combined exposure to both genetic and environmental risk factors, this approach is limited by systemic toxicity of the pesticide rotenone. Direct intracerebral delivery of rotenone may be more useful in longer-term studies as we have previously shown that it overcomes this limitation. Copyright © 2013 Elsevier B.V. All rights reserved.

Role of L-thyroxin in counteracting rotenone induced neurotoxicity in rats.

PubMed

Salama, Mohamed; Helmy, Basem; El-Gamal, Mohamed; Reda, Amr; Ellaithy, Amr; Tantawy, Dina; Mohamed, Mie; El-Gamal, Aya; Sheashaa, Hussein; Sobh, Mohamed

2013-03-01

A key feature of Parkinson's disease is the dopaminergic neuronal cell loss in the substantia nigra pars compacta. Many triggering pathways have been incriminated in the pathogenesis of this disease including inflammation, oxidative stress, excitotoxicity and apoptosis. Thyroid hormone is an essential agent for the growth and maturation of neurons; moreover, it has variable mechanisms for neuroprotection. So, we tested the efficacy of (L)-thyroxin as a neuroprotectant in rotenone model of Parkinson's disease in rats. Thirty Sprague Dawley rats aged 3 months were divided into 3 equal groups. The first received daily intraperitoneal injections of 0.5% carboxymethyl cellulose (CMC) 3 mL/Kg. The second group received rotenone suspended in 0.5% CMC intraperitoneally at a dose of 3 mg/kg, daily. The third group received the same rotenone regimen subcutaneous l-thyroxine at a dose of 7.5 μg daily. All animals were evaluated regarding locomotor disturbance through blinded investigator who monitored akinesia, catalepsy, tremors and performance in open field test. After 35 days the animals were sacrificed and their brains were immunostained against anti-tyrosine hydroxylase and iba-1. Photomicrographs for coronal sections of the substantia nigra and striatum were taken and analyzed using image J software to evaluate cell count in SNpc and striatal fibers density and number of microglia in the nigrostriatal system. The results were then analyzed statistically. Results showed selective protective effects of thyroxin against rotenone induced neurotoxicity in striatum, however, failed to exert similar protection on SN. Moreover, microglial elevated number in nigrostriatal system that was induced by rotenone injections was diminished selectively in striatum only in the l-thyroxin treated group. One of the possible mechanisms deduced from this work was the selective regulation of microglia in striatal tissues. Thus, this study provides an insight into thyroxin neuroprotection warranting further investigation as therapeutic option for Parkinson's disease patients. Copyright © 2013 Elsevier B.V. All rights reserved.

BID links ferroptosis to mitochondrial cell death pathways.

PubMed

Neitemeier, Sandra; Jelinek, Anja; Laino, Vincenzo; Hoffmann, Lena; Eisenbach, Ina; Eying, Roman; Ganjam, Goutham K; Dolga, Amalia M; Oppermann, Sina; Culmsee, Carsten

2017-08-01

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the X c - system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by X c - inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Mdivi-1, mitochondrial fission inhibitor, impairs developmental competence and mitochondrial function of embryos and cells in pigs

PubMed Central

YEON, Ji-Yeong; MIN, Sung-Hun; PARK, Hyo-Jin; KIM, Jin-Woo; LEE, Yong-Hee; PARK, Soo-Yong; JEONG, Pil-Soo; PARK, Humdai; LEE, Dong-Seok; KIM, Sun-Uk; CHANG, Kyu-Tae; KOO, Deog-Bon

2014-01-01

Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells. PMID:25501014

Peroxisomal fatty acid oxidation and inhibitors of the mitochondrial carnitine palmitoyltransferase I in isolated rat hepatocytes.

PubMed Central

Skorin, C; Necochea, C; Johow, V; Soto, U; Grau, A M; Bremer, J; Leighton, F

1992-01-01

Fatty acid oxidation was studied in the presence of inhibitors of carnitine palmitoyltransferase I (CPT I), in normal and in peroxisome-proliferated rat hepatocytes. The oxidation decreased in mitochondria, as expected, but in peroxisomes it increased. These two effects were seen, in variable proportions, with (+)-decanoylcarnitine, 2-tetradecylglycidic acid (TDGA) and etomoxir. The decrease in mitochondrial oxidation (ketogenesis) affected saturated fatty acids with 12 or more carbon atoms, whereas the increase in peroxisomal oxidation (H2O2 production) affected saturated fatty acids with 8 or more carbon atoms. The peroxisomal increase was sensitive to chlorpromazine, a peroxisomal inhibitor. To study possible mechanisms, palmitoyl-, octanoyl- and acetyl-carnitine acyltransferase activities were measured, in homogenates and in subcellular fractions from control and TDGA-treated cells. The palmitoylcarnitine acyltransferase was inhibited, as expected, but the octanoyltransferase activity also decreased. The CoA derivative of TDGA was synthesized and tentatively identified as being responsible for inhibition of the octanoylcarnitine acyltransferase. These results show that inhibitors of the mitochondrial CPT I may also inhibit the peroxisomal octanoyl transferase; they also support the hypothesis that the octanoyltransferase has the capacity to control or regulate peroxisomal fatty acid oxidation. PMID:1736904

A high-throughput screen for mitochondrial function reveals known and novel mitochondrial toxicants in a library of environmental agents

PubMed Central

Datta, Sandipan; Sahdeo, Sunil; Gray, Jennifer A.; Morriseau, Christophe; Hammock, Bruce D.; Cortopassi, Gino

2016-01-01

Mitochondrial toxicity is emerging as a major mechanism underlying serious human health consequences. This work performs a high-throughput screen (HTS) of 176 environmental chemicals for mitochondrial toxicity utilizing a previously reported biosensor platform. This established HTS confirmed known mitochondrial toxins and identified novel mitotochondrial uncouplers such as 2, 2′-Methylenebis(4-chlorophenol) and pentachlorophenol. It also identified a mitochondrial ‘structure activity relationship’ (SAR) in the sense that multiple environmental chlorophenols are mitochondrial inhibitors and uncouplers. This study demonstrates proof-of-concept that a mitochondrial HTS assay detects known and novel environmental mitotoxicants, and could be used to quickly evaluate human health risks from mitotoxicants in the environment. PMID:27717841

Proteasome inhibitor MG132 induces selective apoptosis in glioblastoma cells through inhibition of PI3K/Akt and NFkappaB pathways, mitochondrial dysfunction, and activation of p38-JNK1/2 signaling.

PubMed

Zanotto-Filho, Alfeu; Braganhol, Elizandra; Battastini, Ana Maria Oliveira; Moreira, José Cláudio Fonseca

2012-12-01

Proteasome inhibitors are emerging as a new class of anticancer agents. In this work, we examined the mechanisms underlying cytotoxicity, selectivity and adjuvant potential of the proteasome inhibitor MG132 in a panel of glioblastoma (GBM) cells (U138MG, C6, U87 and U373) and in normal astrocytes. MG132 markedly inhibited GBM cells growth irrespective of the p53 or PTEN mutational status of the cells whereas astrocytic viability was not affected, suggesting a selective toxicity of MG132 to cancerous glial cells. Mechanistically, MG132 arrested cells in G2/M phase of the cell cycle and increased p21(WAF1) protein immunocontent. Following cell arrest, cells become apoptotic as shown by annexin-V binding, caspase-3 activation, chromatin condensation and formation of sub-G1 apoptotic cells. MG132 promoted mitochondrial depolarization and decreased the mitochondrial antiapoptotic protein bcl-xL; it also induced activation of JNK and p38, and inhibition of NFkappaB and PI3K/Akt survival pathways. Pre-treatment of GBMs with the mitochondrial permeability transition pore inhibitor, bongkrekic acid, or pharmacological inhibitors of JNK1/2 and p38, SP600125 and SB203580, attenuated MG132-induced cell death. Besides its apoptotic effect alone, MG132 also enhanced the antiglioma effect of the chemotherapeutics cisplatin, taxol and doxorubicin in C6 and U138MG cells, indicating an adjuvant/chemosensitizer potential. In summary, MG132 exerted profound and selective toxicity in GBMs, being a potential agent for further testing in animal models of the disease.

Catecholamine secretion by chemical hypoxia in guinea-pig, but not rat, adrenal medullary cells: differences in mitochondria.

PubMed

Harada, K; Endo, Y; Warashina, A; Inoue, M

2015-08-20

The effects of mitochondrial inhibitors (CN(-), a complex IV inhibitor and CCCP, protonophore) on catecholamine (CA) secretion and mitochondrial function were explored functionally and biochemically in rat and guinea-pig adrenal chromaffin cells. Guinea-pig chromaffin cells conspicuously secreted CA in response to CN(-) or CCCP, but rat cells showed a little, if any, secretory response to either of them. The resting metabolic rates in rat adrenal medullae did not differ from those in guinea-pig adrenal medullae. On the other hand, the time course of depolarization of the mitochondrial membrane potential (ΔΨm) in guinea-pig chromaffin cells in response to CN(-) was slower than that in rat chromaffin cells, and this difference was abolished by oligomycin, an F1F0-ATPase inhibitor. The extent of CCCP-induced decrease in cellular ATP in guinea-pig chromaffin cells, which was indirectly measured using a Mg(2+) indicator, was smaller than that in rat chromaffin cells. Relative expression levels of F1F0-ATPase inhibitor factor in guinea-pig adrenal medullae were smaller than in rat adrenal medullae, and the opposite was true for F1F0-ATPase α subunit. The present results indicate that guinea-pig chromaffin cells secrete more CA in response to a mitochondrial inhibitor than rat chromaffin cells and this higher susceptibility in the former is accounted for by a larger extent of reversed operation of F1F0-ATPase with the consequent decrease in ATP under conditions where ΔΨm is depolarized. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

In Vitro Effects of Cognitives and Nootropics on Mitochondrial Respiration and Monoamine Oxidase Activity.

PubMed

Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

2017-10-01

Impairment of mitochondrial metabolism, particularly the electron transport chain (ETC), as well as increased oxidative stress might play a significant role in pathogenesis of Alzheimer's disease (AD). Some effects of drugs used for symptomatic AD treatment may be related to their direct action on mitochondrial function. In vitro effects of pharmacologically different cognitives (galantamine, donepezil, rivastigmine, 7-MEOTA, memantine) and nootropic drugs (latrepirdine, piracetam) were investigated on selected mitochondrial parameters: activities of ETC complexes I, II + III, and IV, citrate synthase, monoamine oxidase (MAO), oxygen consumption rate, and hydrogen peroxide production of pig brain mitochondria. Complex I activity was decreased by galantamine, donepezil, and memantine; complex II + III activity was increased by galantamine. None of the tested drugs caused significant changes in the rate of mitochondrial oxygen consumption, even at high concentrations. Except galantamine, all tested drugs were selective MAO-A inhibitors. Latrepirdine, donepezil, and 7-MEOTA were found to be the most potent MAO-A inhibitors. Succinate-induced mitochondrial hydrogen peroxide production was not significantly affected by the drugs tested. The direct effect of cognitives and nootropics used in the treatment of AD on mitochondrial respiration is relatively small. The safest drugs in terms of disturbing mitochondrial function appear to be piracetam and rivastigmine. The MAO-A inhibition by cognitives and nootropics may also participate in mitochondrial neuroprotection. The results support the future research aimed at measuring the effects of currently used drugs or newly synthesized drugs on mitochondrial functioning in order to understand their mechanism of action.

Sevoflurane postconditioning improves myocardial mitochondrial respiratory function and reduces myocardial ischemia-reperfusion injury by up-regulating HIF-1

PubMed Central

Yang, Long; Xie, Peng; Wu, Jianjiang; Yu, Jin; Yu, Tian; Wang, Haiying; Wang, Jiang; Xia, Zhengyuan; Zheng, Hong

2016-01-01

Background: Sevoflurane postconditioning (SPostC) can exert myocardial protective effects similar to ischemic preconditioning. However, the exact myocardial protection mechanism by SPostC is unclear. Studies indicate that hypoxia-inducible factor-1 (HIF-1) maintains cellular respiration homeostasis by regulating mitochondrial respiratory chain enzyme activity under hypoxic conditions. This study investigated whether SPostC could regulate the expression of myocardial HIF-1α and to improve mitochondrial respiratory function, thereby relieving myocardial ischemia-reperfusion injury in rats. Methods: The myocardial ischemia-reperfusion rat model was established using the Langendorff isolated heart perfusion apparatus. Additionally, postconditioning was performed using sevoflurane alone or in combination with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). The changes in hemodynamic parameters, HIF-1α protein expression levels, mitochondrial respiratory function and enzyme activity, mitochondrial reactive oxygen species (ROS) production rates, and mitochondrial ultrastructure were measured or observed. Results: Compared to the ischemia-reperfusion (I/R) group, HIF-1α expression in the SPostC group was significantly up-regulated. Additionally, cardiac function indicators, mitochondrial state 3 respiratory rate, respiratory control ratio (RCR), cytochrome C oxidase (CcO), NADH oxidase (NADHO), and succinate oxidase (SUCO) activities, mitochondrial ROS production rate, and mitochondrial ultrastructure were significantly better than those in the I/R group. However, these advantages were completely reversed by the HIF-1α specific inhibitor 2ME2 (P<0.05). Conclusion: The myocardial protective function of SPostC might be associated with the improvement of mitochondrial respiratory function after up-regulation of HIF-1α expression. PMID:27830025

Sevoflurane postconditioning improves myocardial mitochondrial respiratory function and reduces myocardial ischemia-reperfusion injury by up-regulating HIF-1.

PubMed

Yang, Long; Xie, Peng; Wu, Jianjiang; Yu, Jin; Yu, Tian; Wang, Haiying; Wang, Jiang; Xia, Zhengyuan; Zheng, Hong

2016-01-01

Sevoflurane postconditioning (SPostC) can exert myocardial protective effects similar to ischemic preconditioning. However, the exact myocardial protection mechanism by SPostC is unclear. Studies indicate that hypoxia-inducible factor-1 (HIF-1) maintains cellular respiration homeostasis by regulating mitochondrial respiratory chain enzyme activity under hypoxic conditions. This study investigated whether SPostC could regulate the expression of myocardial HIF-1α and to improve mitochondrial respiratory function, thereby relieving myocardial ischemia-reperfusion injury in rats. The myocardial ischemia-reperfusion rat model was established using the Langendorff isolated heart perfusion apparatus. Additionally, postconditioning was performed using sevoflurane alone or in combination with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). The changes in hemodynamic parameters, HIF-1α protein expression levels, mitochondrial respiratory function and enzyme activity, mitochondrial reactive oxygen species (ROS) production rates, and mitochondrial ultrastructure were measured or observed. Compared to the ischemia-reperfusion (I/R) group, HIF-1α expression in the SPostC group was significantly up-regulated. Additionally, cardiac function indicators, mitochondrial state 3 respiratory rate, respiratory control ratio (RCR), cytochrome C oxidase (C c O), NADH oxidase (NADHO), and succinate oxidase (SUCO) activities, mitochondrial ROS production rate, and mitochondrial ultrastructure were significantly better than those in the I/R group. However, these advantages were completely reversed by the HIF-1α specific inhibitor 2ME2 ( P <0.05). The myocardial protective function of SPostC might be associated with the improvement of mitochondrial respiratory function after up-regulation of HIF-1α expression.

Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy.

PubMed

Fiesel, Fabienne C; James, Elle D; Hudec, Roman; Springer, Wolfdieter

2017-12-05

Loss-of-function mutations in PINK1 or PARKIN are associated with early-onset Parkinson's disease. Upon mitochondrial stress, PINK1 and Parkin together mediate a response that protects cells from the accumulation of harmful, damaged mitochondria. PINK1, the upstream kinase accumulates on the mitochondrial surface and recruits the E3 ubiquitin ligase Parkin on site to ubiquitylate substrate proteins. The joint activity of both to generate phosphorylated poly-ubiquitin chains on the mitochondrial surface induces the recruitment of autophagy receptors and eventually whole organelles are cleared by autophagy. While this pathway is generally accepted to occur upon chemical uncoupling of mitochondria, the (patho-) physiologic relevance has been questioned. However, few studies have indicated that PINK1 and Parkin are also activated upon accumulation of misfolded proteins in the mitochondrial lumen upon overexpression of ΔOTC (Ornithine transcarbamylase). Here, we used the mitochondrial targeted HSP90 inhibitor Gamitrinib-triphenylphosphonium (G-TPP), an anti-cancer agent, to chemically interfere with mitochondrial protein folding. G-TPP treatment induced PINK1 accumulation, ubiquitin phosphorylation at Ser65, Parkin activation and its recruitment to mitochondria was specific for mitochondrial HSP90 inhibition and largely independent of mitochondrial membrane depolarization. Mitophagy induction was observed by monitoring autophagy receptor recruitment and the mitoKeima reporter. Importantly, mitophagy was not only induced in cancer cells but also in primary human fibroblasts and thereof converted neurons. G-TPP treatment might represent a novel strategy to study PINK1 and Parkin-mediated mitochondrial quality control using a more physiologically relevant stress.

Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1.

PubMed

Tsai, Ming-Feng; Jiang, Dawei; Zhao, Linlin; Clapham, David; Miller, Christopher

2014-01-01

The leucine zipper, EF hand-containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca(2+) and K(+) homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca(2+) fluorophore and (45)Ca(2+)-based assays, we demonstrate directly that Letm1 is a Ca(2+) transporter, with apparent affinities of cations in the sequence of Ca(2+) ≈ Mn(2+) > Gd(3+) ≈ La(3+) > Sr(2+) > Ba(2+), Mg(2+), K(+), Na(+). Kinetic analysis yields a Letm1 turnover rate of 2 Ca(2+)/s and a Km of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca(2+)/2 H(+) antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H(+)-dependent Ca(2+) transport mechanism identified in intact mitochondria.

Chickamauga Reservoir 1992 fisheries monitoring cove rotenone results

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kerley, B.L.

1993-06-01

The Tennessee Valley Authority (TVA) is required by the National Pollutant Discharge Elimination System (NPDES) Permit for Sequoyah Nuclear Plant (SQN) to conduct and report annually a nonradiological operational monitoring program to evaluate potential effects of SQN on Chickamauga Reservoir. This monitoring program was initially designed to identify potential changes in water quality and biological communities in Chickamauga Reservoir resulting from operation of SQU. Chickamauga Reservoir cove rotenone sampling has also been conducted as part of the preoperational monitoring program for Watts Bar Nuclear Plant (WBN) to evaluate the combined effects of operating two nuclear facilities on one reservoir oncemore » WBU becomes operational. The purpose of this report is to present results of cove rotenone sampling conducted on Chickamauga Reservoir in 1992.« less

Production of reactive oxygen species in mitochondria of HeLa cells under oxidative stress.

PubMed

Chernyak, Boris V; Izyumov, Denis S; Lyamzaev, Konstantin G; Pashkovskaya, Alina A; Pletjushkina, Olga Y; Antonenko, Yuri N; Sakharov, Dmitrii V; Wirtz, Karel W A; Skulachev, Vladimir P

2006-01-01

Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed ("dark") accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H2O2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.

Naringin Ameliorates HIV-1 Nucleoside Reverse Transcriptase Inhibitors- Induced Mitochondrial Toxicity.

PubMed

Oluwafeyisetan, Adebiyi; Olubunmi, Adebiyi; Peter, Owira

2016-01-01

Mitochondrial reactive oxygen species (ROS) generation and defective oxidative phosphorylation (OXPHOS) have been proposed as possible mechanisms underlying the development of nucleoside reverse transcriptase inhibitors (NRTIs)-induced mitochondrial toxicities. Available options in managing these complications have, so far, produced controversial results, thus necessitating further research into newer agents with promise. Antioxidant and free-radical scavenging effects of naringin, a plant-derived flavonoid, have previously been demonstrated. This study was designed to investigate the effects of naringin on NRTIs-induced mitochondrial toxicity. Wistar rats were randomly divided into Zidovudine (AZT)-only (100 mg/kg body weight BW); AZT+Naringin (100+50 mg/kg BW); AZT+Vitamin E (100+100 mg/kg BW); Stavudine (d4T)- only (50 mg/kg BW); d4T+Naringin (50+50 mg/kg BW); d4T+Vitamin E (50+100 mg/kg BW) and Vehicle (3.0 mL/kg BW)-treated groups, respectively. After 56 days of oral daily dosing, rats were euthanized by halothane overdose, blood collected by cardiac puncture and livers promptly excised for further biochemical and ultrastructural analyses. </p> Results: AZT- or d4T-only caused significant mitochondrial dysfunction and mitochondrial ultrastructural damage compared to controls, while either naringin or vitamin E reversed indices of mitochondrial dysfunction evidenced by significantly reduced mitochondrial malondialdehyde (MDA) and blood lactate concentrations, increased liver manganese superoxide dismutase (MnSOD) activity and upregulate expression of mitochondrial-encoded subunit of electron transport chain (ETC) complex IV protein compared to AZT- or d4T-only treated rats. Furthermore, naringin or vitamin E, respectively, ameliorated mitochondrial damage observed in AZT- or d4T-only treated rats. Naringin ameliorated oxidative stress and NRTI-induced mitochondrial damage and might, therefore, be beneficial in managing toxicities and complications arising from NRTI use.

Inhibition of mitochondrial fission prevents hypoxia-induced metabolic shift and cellular proliferation of pulmonary arterial smooth muscle cells.

PubMed

Parra, Valentina; Bravo-Sagua, Roberto; Norambuena-Soto, Ignacio; Hernández-Fuentes, Carolina P; Gómez-Contreras, Andrés G; Verdejo, Hugo E; Mellado, Rosemarie; Chiong, Mario; Lavandero, Sergio; Castro, Pablo F

2017-11-01

Chronic hypoxia exacerbates proliferation of pulmonary arterial smooth muscle cells (PASMC), thereby reducing the lumen of pulmonary arteries. This leads to poor blood oxygenation and cardiac work overload, which are the basis of diseases such as pulmonary artery hypertension (PAH). Recent studies revealed an emerging role of mitochondria in PAH pathogenesis, as key regulators of cell survival and metabolism. In this work, we assessed whether hypoxia-induced mitochondrial fragmentation contributes to the alterations of both PASMC death and proliferation. In previous work in cardiac myocytes, we showed that trimetazidine (TMZ), a partial inhibitor of lipid oxidation, stimulates mitochondrial fusion and preserves mitochondrial function. Thus, here we evaluated whether TMZ-induced mitochondrial fusion can prevent human PASMC proliferation in an in vitro hypoxic model. Using confocal fluorescence microscopy, we showed that prolonged hypoxia (48h) induces mitochondrial fragmentation along with higher levels of the mitochondrial fission protein DRP1. Concomitantly, both mitochondrial potential and respiratory rates decreased, indicative of mitochondrial dysfunction. In accordance with a metabolic shift towards non-mitochondrial ATP generation, mRNA levels of glycolytic markers HK2, PFKFB2 and GLUT1 increased during hypoxia. Incubation of PASMC with TMZ, prior to hypoxia, prevented all these changes and precluded the increase in PASMC proliferation. These findings were also observed using Mdivi-1 (a pharmacological DRP1 inhibitor) or a dominant negative DRP1 K38A as pre-treatments. Altogether, our data indicate that TMZ exerts a protective role against hypoxia-induced PASMC proliferation, by preserving mitochondrial function, thus highlighting DRP1-dependent morphology as a novel therapeutic approach for diseases such as PAH. Copyright © 2017 Elsevier B.V. All rights reserved.

CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger, protects neurons from excitotoxicity by blocking voltage-gated Ca2+ channels.

PubMed

Ruiz, A; Alberdi, E; Matute, C

2014-04-10

Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (NCLX) by CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca(2+) homeostasis. However, the Ca(2+) signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca(2+) levels are modulated by CGP37157 (10 μM) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of CGP37157 on neuronal Ca(2+) homeostasis using cameleon-based mitochondrial Ca(2+) and cytosolic Ca(2+) ([Ca(2+)]i) live imaging. We observed that NCLX-driven mitochondrial Ca(2+) exchange occurs in cortical neurons under basal conditions as CGP37157 induced a decrease in [Ca(2)]i concomitant with a Ca(2+) accumulation inside the mitochondria. In turn, CGP37157 also inhibited mitochondrial Ca(2+) efflux after the stimulation of acetylcholine receptors. In contrast, CGP37157 strongly prevented depolarization-induced [Ca(2+)]i increase by blocking voltage-gated Ca(2+) channels (VGCCs), whereas it did not induce depletion of ER Ca(2+) stores. Moreover, mitochondrial Ca(2+) overload was reduced as a consequence of diminished Ca(2+) entry through VGCCs. The decrease in cytosolic and mitochondrial Ca(2+) overload by CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity CGP37157 modulates cytosolic and mitochondrial Ca(2+) dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs.

CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger, protects neurons from excitotoxicity by blocking voltage-gated Ca2+ channels

PubMed Central

Ruiz, A; Alberdi, E; Matute, C

2014-01-01

Inhibition of the mitochondrial Na+/Ca2+ exchanger (NCLX) by CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca2+ homeostasis. However, the Ca2+ signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca2+ levels are modulated by CGP37157 (10 μM) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of CGP37157 on neuronal Ca2+ homeostasis using cameleon-based mitochondrial Ca2+ and cytosolic Ca2+ ([Ca2+]i) live imaging. We observed that NCLX-driven mitochondrial Ca2+ exchange occurs in cortical neurons under basal conditions as CGP37157 induced a decrease in [Ca2]i concomitant with a Ca2+ accumulation inside the mitochondria. In turn, CGP37157 also inhibited mitochondrial Ca2+ efflux after the stimulation of acetylcholine receptors. In contrast, CGP37157 strongly prevented depolarization-induced [Ca2+]i increase by blocking voltage-gated Ca2+ channels (VGCCs), whereas it did not induce depletion of ER Ca2+ stores. Moreover, mitochondrial Ca2+ overload was reduced as a consequence of diminished Ca2+ entry through VGCCs. The decrease in cytosolic and mitochondrial Ca2+ overload by CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity CGP37157 modulates cytosolic and mitochondrial Ca2+ dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs. PMID:24722281

Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, TongFa; Gao, DaQuan; Fang, Zheng-yu, E-mail: fangzhengyu158@sina.com

In this study, we showed that PF-543, a novel sphingosine kinase 1 (SphK1) inhibitor, exerted potent anti-proliferative and cytotoxic effects against a panel of established (HCT-116, HT-29 and DLD-1) and primary human colorectal cancer (CRC) cells. Its sensitivity was negatively associated with SphK1 expression level in the CRC cells. Surprisingly, PF-543 mainly induced programmed necrosis, but not apoptosis, in the CRC cells. CRC cell necrotic death was detected by lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP) collapse and mitochondrial P53-cyclophilin-D (Cyp-D) complexation. Correspondingly, the necrosis inhibitor necrostatin-1 largely attenuated PF-543-induced cytotoxicity against CRC cells. Meanwhile, the Cyp-D inhibitors (sanglifehrinmore » A and cyclosporin A), or shRNA-mediated knockdown of Cyp-D, remarkably alleviated PF-543-induced CRC cell necrotic death. Reversely, over-expression of wild-type Cyp-D in HCT-116 cells significantly increased PF-543's sensitivity. In vivo, PF-543 intravenous injection significantly suppressed HCT-116 xenograft growth in severe combined immunodeficient (SCID) mice, whiling remarkably improving the mice survival. The in vivo activity by PF-543 was largely attenuated when combined with the Cyp-D inhibitor cyclosporin A. Collectively, our results demonstrate that PF-543 exerts potent anti-CRC activity in vitro and in vivo. Mitochondrial programmed necrosis pathway is likely the key mechanism responsible for PF-543's actions in CRC cells. - Highlights: • PF-543 is anti-proliferative and cytotoxic to established and primary CRC cells. • PF-543 induces programmed necrosis, but not apoptosis, in CRC cells. • Modulation of mitochondrial protein cyclophilin-D alters PF-543's sensitivity. • PF-543 inhibits HCT-116 xenograft growth in SCID mice, improving mice survival. • Co-administration of cyclophilin-D inhibitor CsA inhibits PF-543's activity in vivo.« less

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zaja, Ivan; Bai, Xiaowen, E-mail: xibai@mcw.edu; Liu, Yanan

Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1more » (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of mitochondrial fission; and (2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.« less

Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria

PubMed Central

Briston, Thomas; Lewis, Sian; Koglin, Mumta; Mistry, Kavita; Shen, Yongchun; Hartopp, Naomi; Katsumata, Ryosuke; Fukumoto, Hironori; Duchen, Michael R.; Szabadkai, Gyorgy; Staddon, James M.; Roberts, Malcolm; Powney, Ben

2016-01-01

Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca2+ uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca2+-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca2+-induced membrane depolarisation and Ca2+ retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature. PMID:27886240

Environmental and Metabolic Transformations of the Piscicide Antimycin A

EPA Science Inventory

Antimycin A is a natural occurring product of bacterial fermentation and is an extremely potent inhibitor of mitochondrial respiration. It is toxic to all organisms that depend on mitochondrial respiration; however, there is a large divergence in toxicity, even between closely re...

Alterations in Energy/Redox Metabolism Induced by Mitochondrial and Environmental Toxins: A Specific Role for Glucose-6-Phosphate-Dehydrogenase and the Pentose Phosphate Pathway in Paraquat Toxicity

PubMed Central

2015-01-01

Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression. PMID:24937102

c-Jun N-terminal kinase modulates oxidant stress and peroxynitrite formation independent of inducible nitric oxide synthase in acetaminophen hepatotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saito, Chieko; Lemasters, John J.; Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425

Acetaminophen (APAP) overdose, which causes liver injury in animals and humans, activates c-jun N-terminal kinase (JNK). Although it was shown that the JNK inhibitor SP600125 effectively reduced APAP hepatotoxicity, the mechanisms of protection remain unclear. C57Bl/6 mice were treated with 10 mg/kg SP600125 or vehicle (8% dimethylsulfoxide) 1 h before 600 mg/kg APAP administration. APAP time-dependently induced JNK activation (detected by JNK phosphorylation). SP600125, but not the vehicle, reduced JNK activation, attenuated mitochondrial Bax translocation and prevented the mitochondrial release of apoptosis-inducing factor at 4-12 h. Nuclear DNA fragmentation, nitrotyrosine staining, tissue GSSG levels and liver injury (plasma ALT releasemore » and necrosis) were partially attenuated by the vehicle (- 65%) and completely eliminated by SP600125 (- 98%) at 6 and 12 h. Furthermore, SP600125 attenuated the increase of inducible nitric oxide synthase (iNOS) mRNA and protein. However, APAP did not enhance plasma nitrite + nitrate levels (NO formation); SP600125 had no effect on this parameter. The iNOS inhibitor L-NIL did not reduce NO formation or injury after APAP but prevented NO formation caused by endotoxin. Since SP600125 completely eliminated the increase in hepatic GSSG levels, an indicator of mitochondrial oxidant stress, it is concluded that the inhibition of peroxynitrite was mainly caused by reduced superoxide formation. Our data suggest that the JNK inhibitor SP600125 protects against APAP-induced liver injury in part by attenuation of mitochondrial Bax translocation but mainly by preventing mitochondrial oxidant stress and peroxynitrite formation and thereby preventing the mitochondrial permeability transition pore opening, a key event in APAP-induced cell necrosis.« less

Neuroprotective role of chrysin in attenuating loss of dopaminergic neurons and improving motor, learning and memory functions in rats.

PubMed

Ahmed, Muhammad Rashid; Shaikh, Masood Ahmed; Ul Haq, Syed Hafiz Imran; Nazir, Shakila

2018-01-01

Selective degeneration of dopaminergic neurons is the pathological hallmark of Parkinson disease (PD). Enhanced oxidative stress, lipid peroxidation and susceptibility of dopaminergic neurons to apoptotic cellular death are the leading pathogenetic mechanisms. Chrysin is an active flavonoid. Its neuroprotective effects have been reported. This study examined the neuroprotective effects of chrysin in ameliorating the dopaminergic neuronal degeneration and motor behavioral changes in rotenone model of PD. Thirty Sprague-Dawley rats were assigned into three groups: Control, rotenone-treated, and rotenone+chrysin treated groups. Rotenone was given at a dose of 3 mg/kg daily intraperitoneally, and chrysin was given at a dose of 50 mg/kg daily intraperitoneally for 4 weeks. Using five neurobehavioral assessment tests, evaluation was done weekly to record the motor behavioral changes. After 4 weeks, animals were sacrificed, brains were removed, and section from striatum and substantia nigra were stained using hematoxylin and eosin and cresyl violet stains. Immunohistochemical sections were also prepared using anti-tyrosine hydroxylase (TH) antibody. Rotenone-induced Parkinson like changes were evident from deteriorating motor behavior. These animals showed extensive loss of dopaminergic neurons, decreased immunoreactivity against anti-TH antibodies and number of TH positive dopaminergic neurons in the nigrostriatal region. Chrysin treated animals showed a significant reduction in motor behavioral changes, degeneration and loss of nigrostriatal dopaminergic neurons and increased immunoreactivity to anti-TH antibody. This study concludes that chrysin confers neuroprotection in rat model of PD. It attenuates the degeneration of the nigrostriatal dopaminergic neurons and motor behavioral abnormalities.

Rotenone and paraquat perturb dopamine metabolism: a computational analysis of pesticide toxicity

PubMed Central

Qi, Zhen; Miller, Gary W.; Voit, Eberhard O.

2014-01-01

Pesticides, such as rotenone and paraquat, are suspected in the pathogenesis of Parkinson’s disease (PD), whose hallmark is the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Thus, compounds expected to play a role in the pathogenesis of PD will likely impact the function of dopaminergic neurons. To explore the relationship between pesticide exposure and dopaminergic toxicity, we developed a custom-tailored mathematical model of dopamine metabolism and utilized it to infer potential mechanisms underlying the toxicity of rotenone and paraquat, asking how these pesticides perturb specific processes. We performed two types of analyses, which are conceptually different and complement each other. The first analysis, a purely algebraic reverse engineering approach, analytically and deterministically computes the altered profile of enzyme activities that characterize the effects of a pesticide. The second method consists of large-scale Monte Carlo simulations that statistically reveal possible mechanisms of pesticides. The results from the reverse engineering approach show that rotenone and paraquat exposures lead to distinctly different flux perturbations. Rotenone seems to affect all fluxes associated with dopamine compartmentalization, whereas paraquat exposure perturbs fluxes associated with dopamine and its breakdown metabolites. The statistical results of the Monte-Carlo analysis suggest several specific mechanisms. The findings are interesting, because no a priori assumptions are made regarding specific pesticide actions, and all parameters characterizing the processes in the dopamine model are treated in an unbiased manner. Our results show how approaches from computational systems biology can help identify mechanisms underlying the toxicity of pesticide exposure. PMID:24269752

Levetiracetam attenuates rotenone-induced toxicity: A rat model of Parkinson's disease.

PubMed

Erbaş, Oytun; Yılmaz, Mustafa; Taşkıran, Dilek

2016-03-01

Levetiracetam (LEV), a second-generation anti-epileptic drug, is used for treatment of both focal and generalized epilepsy. Growing body of evidence suggests that LEV may have neuroprotective effects. The present study was undertaken to investigate the neuroprotective effects of LEV on rotenone-induced Parkinson's disease (PD) in rats. Twenty-four adult Sprague-Dawley rats were infused with rotenone (3 μg/μl in DMSO) or vehicle (1 μl DMSO) into the left substantia nigra pars compacta (SNc) under stereotaxic surgery. PD model was assessed by rotational test ten days after drug infusion. The valid PD rats were randomly distributed into two groups; Group 1 (n=8) and Group 2 (n=8) were administered saline (1 ml/kg/day, i.p.) and LEV (600 mg/kg/day, i.p.) through 21 days, respectively. The effects of LEV treatment were evaluated by behavioral (rotation score), biochemical (brain homovalinic acid level and oxidant/antioxidant status) and immunohistochemical (tyrosine hydroxylase) parameters. Apomorphine-induced rotations in PD rats were significantly suppressed by LEV treatment. While unilateral rotenone lesion induced a dramatic loss of dopaminergic neurons both in the striatum and SNc, LEV treatment significantly attenuated the degenerative changes in dopaminergic neurons. Furthermore, LEV significantly decreased lipid peroxide levels, a marker of lipid peroxidation, and induced glutathione levels, catalase and superoxide dismutase activity in PD rats compared with saline group. We conclude that LEV may have beneficial effects on dopaminergic neurons against rotenone-induced injury. The underlying mechanism may be associated with the attenuation of oxidative stress. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of endoplasmic reticulum stress is involved in the neuroprotective effects of candesartan cilexitil in the rotenone rat model of Parkinson's disease.

PubMed

Wu, Liang; Tian, You-Yong; Shi, Jing-Ping; Xie, Wei; Shi, Jian-Quan; Lu, Jie; Zhang, Ying-Dong

2013-08-26

Recent studies indicated that angiotensin II (Ang II) receptor blockers could reduce neurotoxins-induced dopaminergic (DA) cell death, but the underlying mechanisms are still unclear. Given that endoplasmic reticulum (ER) stress plays a major role in rotenone-induced neuronal apoptosis, we investigated whether candesartan cilexetil, a selective and high-affinity Ang II receptor antagonist, could protect the DA neuron via reducing ER stress in a chronic rotenone rat model for Parkinson's disease (PD). Our data showed that candesartan cilexetil could ameliorate the descent latency in catalepsy tests, and decrease rotenone-induced DA neuron apoptosis. Moreover, candesartan cilexetil has been found to play a protective role via down-regulating the expression of activating transcription factor 4 (ATF4), the CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP), and p53 upregulated modulator of apoptosis (Puma). Thus, our experiments strongly suggest that administration of candesartan cilexetil protects DA neuron involving blocking ER stress, possibly via inhibiting activation of the ATF4-CHOP-Puma pathway, which could provide new insight into clinical therapeutics for PD. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Neuroprotective effect of curcumin-loaded lactoferrin nano particles against rotenone induced neurotoxicity.

PubMed

Bollimpelli, V Satish; Kumar, Prashant; Kumari, Sonali; Kondapi, Anand K

2016-05-01

Curcumin is known to have neuroprotective role and possess antioxidant, anti-inflammatory activities. Rotenone, a flavonoid induced neurotoxicity in dopaminergic cells is being widely studied in Parkinson's Disease (PD) research. In the present study, curcumin loaded lactoferrin nano particles prepared by sol-oil chemistry were used to protect dopaminergic cell line SK-N-SH against rotenone induced neurotoxicity. These curcumin loaded nano particles were of 43-60 nm diameter size and around 100 nm hydrodynamic size as assessed by transmission electron microscopy, atomic force microscopy and dynamic light scattering analysis respectively. The encapsulation efficiency was 61.3% ± 2.4%. Cellular uptake of curcumin through these nano particles was confirmed by confocal imaging and spectrofluorimetric analysis. The curcumin loaded lactoferrin nanoparticles showed greater intracellular drug uptake, sustained retention and greater neuroprotection than soluble counterpart. Neuroprotective activity was characterized through viability assays and by estimating ROS levels. Furthermore rotenone induced PD like features were characterized by decrease in tyrosine hydroxylase expression and increase in α-synuclein expression. Taken together curcumin loaded lactoferrin nanoparticles could be a promising drug delivery strategy against neurotoxicity in dopaminergic neurons. Copyright © 2016 Elsevier Ltd. All rights reserved.

Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.

PubMed

Blum, Roy; Jacob-Hirsch, Jasmine; Rechavi, Gideon; Kloog, Yoel

2006-09-01

The Ras inhibitor farnesylthiosalicylic acid (FTS) has been shown to induce apoptosis in glioblastoma multiforme, but its mechanism of action was unknown. We show that FTS or dominant-negative Ras, by deregulating extracellular signal-regulated kinase and Akt signaling, decreases survivin gene transcripts in U87 glioblastoma multiforme, leading to disappearance of survivin protein and cell death. FTS affected both Ras-controlled regulators of survivin transcription and Ras-regulated survival signals. Thus, Ras inhibition by FTS resulted in release of the survivin "brake" on apoptosis and in activation of the mitochondrial apoptotic pathway: dephosphorylation of Bad, activation of Bax, release of cytochrome c, and caspase activation. FTS-induced apoptosis of U87 cells was strongly attenuated by forced expression of survivin or by caspase inhibitors. These results show that resistance to apoptosis in glioblastoma multiforme can be abolished by a single Ras inhibitor, which targets both survivin, a critical inhibitor of apoptosis, and the intrinsic mitochondrial apoptotic machinery.

Mitochondrial Dysfunction and Its Relationship with mTOR Signaling and Oxidative Damage in Autism Spectrum Disorders.

PubMed

Yui, Kunio; Sato, Atsushi; Imataka, George

2015-01-01

Mitochondria are organelles that play a central role in processes related to cellular viability, such as energy production, cell growth, cell death via apoptosis, and metabolism of reactive oxygen species (ROS). We can observe behavioral abnormalities relevant to autism spectrum disorders (ASDs) and their recovery mediated by the mTOR inhibitor rapamycin in mouse models. In Tsc2(+/-) mice, the transcription of multiple genes involved in mTOR signaling is enhanced, suggesting a crucial role of dysregulated mTOR signaling in the ASD model. This review proposes that the mTOR inhibitor may be useful for the pharmacological treatment of ASD. This review offers novel insights into mitochondrial dysfunction and the related impaired glutathione synthesis and lower detoxification capacity. Firstly, children with ASD and concomitant mitochondrial dysfunction have been reported to manifest clinical symptoms similar to those of mitochondrial disorders, and it therefore shows that the clinical manifestations of ASD with a concomitant diagnosis of mitochondrial dysfunction are likely due to these mitochondrial disorders. Secondly, the adenosine triphosphate (ATP) production/oxygen consumption pathway may be a potential candidate for preventing mitochondrial dysfunction due to oxidative stress, and disruption of ATP synthesis alone may be related to impaired glutathione synthesis. Finally, a decrease in total antioxidant capacity may account for ASD children who show core social and behavioral impairments without neurological and somatic symptoms.

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells.

PubMed

Jiao, Jiao; Sun, Ling; Zhou, Benguo; Gao, Zhengliang; Hao, Yu; Zhu, Xiaoping; Liang, Yuancun

2014-08-15

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H2O2) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H2O2 accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H2O2 production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H2O2 accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Constitutive Activation of PINK1 Protein Leads to Proteasome-mediated and Non-apoptotic Cell Death Independently of Mitochondrial Autophagy*

PubMed Central

Akabane, Shiori; Matsuzaki, Kohei; Yamashita, Shun-ichi; Arai, Kana; Okatsu, Kei; Kanki, Tomotake; Matsuda, Noriyuki; Oka, Toshihiko

2016-01-01

Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), a Ser/Thr kinase, and PARKIN, a ubiquitin ligase, are causal genes for autosomal recessive early-onset parkinsonism. Multiple lines of evidence indicate that PINK1 and PARKIN cooperatively control the quality of the mitochondrial population via selective degradation of damaged mitochondria by autophagy. Here, we report that PINK1 and PARKIN induce cell death with a 12-h delay after mitochondrial depolarization, which differs from the time profile of selective autophagy of mitochondria. This type of cell death exhibited definite morphologic features such as plasma membrane rupture, was insensitive to a pan-caspase inhibitor, and did not involve mitochondrial permeability transition. Expression of a constitutively active form of PINK1 caused cell death in the presence of a pan-caspase inhibitor, irrespective of the mitochondrial membrane potential. PINK1-mediated cell death depended on the activities of PARKIN and proteasomes, but it was not affected by disruption of the genes required for autophagy. Furthermore, fluorescence and electron microscopic analyses revealed that mitochondria were still retained in the dead cells, indicating that PINK1-mediated cell death is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play critical roles in selective cell death in which damaged mitochondria are retained, independent of mitochondrial autophagy. PMID:27302064

FFA-ROS-P53-mediated mitochondrial apoptosis contributes to reduction of osteoblastogenesis and bone mass in type 2 diabetes mellitus.

PubMed

Li, Jun; He, Wang; Liao, Bo; Yang, Jingyue

2015-07-31

This study evaluated the association between free fatty acid (FFA), ROS generation, mitochondrial dysfunction and bone mineral density (BMD) in type 2 diabetic patients and investigated the molecular mechanism. db/db and high fat (HF)-fed mice were treated by Etomoxir, an inhibitor of CPT1, MitoQ, and PFT-α, an inhibitor of P53. Bone metabolic factors were assessed and BMSCs were isolated and induced to osteogenic differentiation. FFA, lipid peroxidation and mtDNA copy number were correlated with BMD in T2DM patients. Etomoxir, MitoQ and PFT-α significantly inhibited the decrease of BMD and bone breaking strength in db/db and HF-fed mice and suppressed the reduction of BMSCs-differentiated osteoblasts. Etomoxir and MitoQ, but not PFT-α, inhibited the increase of mitochondrial ROS generation in db/db and HF-fed mice and osteoblasts. In addition, Etomoxir, MitoQ and PFT-α significantly inhibited mitochondrial dysfunction in osteoblasts. Moreover, mitochondrial apoptosis was activated in osteoblasts derived from db/db and HF-fed mice, which was inhibited by Etomoxir, MitoQ and PFT-α. Furthermore, mitochondrial accumulation of P53 recruited Bax and initiated molecular events of apoptotic events. These results demonstrated that fatty acid oxidation resulted in ROS generation, activating P53/Bax-mediated mitochondrial apoptosis, leading to reduction of osteogenic differentiation and bone loss in T2DM.

ROS Modifiers and NOX4 Affect the Expression of the Survivin-Associated Radio-Adaptive Response.

PubMed

Murley, Jeffrey S; Arbiser, Jack L; Weichselbaum, Ralph R; Grdina, David J

2018-04-13

The survivin-associated radio-adaptive response can be induced following exposure to ionizing radiation in the dose range from 5 to 100 mGy, and its magnitude of expression is dependent upon the TP53 mutational status of cells and ROS signaling. The purpose of the study was to investigate the potential role of ROS in the development of the survivin-associated adaptive response. Utilizing human colon carcinoma HCT116 TP53 wild type (WT) and HCT116 isogenic TP53 null mutant (Mut) cell cultures, the roles of inter- and intracellular ROS signaling on expression of the adaptive response as evidenced by changes in intracellular translocation of survivin measured by ELISA, and cell survival determined by a standard colony forming assay were investigated using ROS modifying agents that include emodin, N-acetyl-l-cysteine (NAC), fulvene-5, honokiol, metformin and rotenone. The role of NADPH oxidase 4 (NOX4) in the survivin-associated adaptive response was investigated by transfecting HCT116 cells, both WT and Mut, with two different NOX4 siRNA oligomers and Western blotting. A dose of 5 mGy or a 15min exposure to 50µM of the ROS producing drug emodin were equally effective in inducing a pro-survival adaptive response in TP53 WT and a radio-sensitization adaptive response in TP53 Mut HCT116 cells. Each response was associated with a corresponding translocation of survivin into the cytoplasm or nucleus, respectively. Exposure to 10mM NAC completely inhibited both responses. Exposure to 10µM honokiol induced responses similar to those observed following NAC exposure in TP53 WT and Mut cells. The mitochondrial complex 1 inhibitor rotenone was effective in reducing both cytoplasmic and nuclear survivin levels, but was ineffective in altering the expression of the adaptive response in either TP53 WT or Mut cells. In contrast, both metformin and fulvene-5, inhibitors of NOX4, facilitated the reversal of TP53 WT and Mut adaptive responses from pro-survival to radio-sensitization and vice versa, respectively. These changes were accompanied by corresponding reversals in the translocation of survivin to the nuclei of TP53 WT and to the cytoplasm of TP53 Mut cells. The potential role of NOX4 in the expression of the survivin-associated adaptive response was investigated by transfecting HCT116 cells with NOX4 siRNA oligomers to inhibit NOX4 expression. Under these conditions NOX4 expression was inhibited by about 50%, resulting in a reversal in the expression of the TP53 WT and Mut survivin-associated adaptive responses as was observed following metformin and fulvene-5 treatment. Exposure to 5 mGy resulted in enhanced NOX4 expression by about 40% in both TP53 WT and Mut cells, in contrast to only a 1 to 2% increase following a 2Gy only exposure. Utilizing mixed cultures of HCT116 TP53 WT and isogenic null Mut cells, as few as 10% TP53 Mut cells were sufficient to control the expression of the remaining 90% WT cells and resulted in an overall radio-sensitization response accompanied by the nuclear translocation of survivin characteristic of homogeneous TP53 Mut populations. Copyright © 2018. Published by Elsevier Inc.

Calcineurin Regulates Myocardial Function during Acute Endotoxemia

PubMed Central

Joshi, Mandar S.; Julian, Mark W.; Huff, Jennifer E.; Bauer, John A.; Xia, Yong; Crouser, Elliott D.

2006-01-01

Rationale: Cyclosporin A (CsA) is known to preserve cardiac contractile function during endotoxemia, but the mechanism is unclear. Increased nitric oxide (NO) production and altered mitochondrial function are implicated as mechanisms contributing to sepsis-induced cardiac dysfunction, and CsA has the capacity to reduce NO production and inhibit mitochondrial dysfunction relating to the mitochondrial permeability transition (MPT). Objectives: We hypothesized that CsA would protect against endotoxin-mediated cardiac contractile dysfunction by attenuating NO production and preserving mitochondrial function. Methods: Left ventricular function was measured continuously over 4 h in cats assigned as follows: control animals (n = 7); LPS alone (3 mg/kg, n = 8); and CsA (6 mg/kg, n = 7), a calcineurin inhibitor that blocks the MPT, or tacrolimus (FK506, 0.1 mg/kg, n = 7), a calcineurin inhibitor lacking MPT activity, followed in 30 min by LPS. Myocardial tissue was then analyzed for NO synthase-2 expression, tissue nitration, protein carbonylation, and mitochondrial morphology and function. Measurements and Main Results: LPS treatment resulted in impaired left ventricular contractility, altered mitochondrial morphology and function, and increased protein nitration. As hypothesized, CsA pretreatment normalized cardiac performance and mitochondrial respiration and reduced myocardial protein nitration. Unexpectedly, FK506 pretreatment had similar effects, normalizing both cardiac and mitochondrial parameters. However, CsA and FK506 pretreatments markedly increased protein carbonylation in the myocardium despite elevated manganese superoxide dismutase activity during endotoxemia. Conclusions: Our data indicate that calcineurin is a critical regulator of mitochondrial respiration, tissue nitration, protein carbonylation, and contractile function in the heart during acute endotoxemia. PMID:16424445

Cancer metabolism, stemness and tumor recurrence: MCT1 and MCT4 are functional biomarkers of metabolic symbiosis in head and neck cancer.

PubMed

Curry, Joseph M; Tuluc, Madalina; Whitaker-Menezes, Diana; Ames, Julie A; Anantharaman, Archana; Butera, Aileen; Leiby, Benjamin; Cognetti, David M; Sotgia, Federica; Lisanti, Michael P; Martinez-Outschoorn, Ubaldo E

2013-05-01

Here, we interrogated head and neck cancer (HNSCC) specimens (n = 12) to examine if different metabolic compartments (oxidative vs. glycolytic) co-exist in human tumors. A large panel of well-established biomarkers was employed to determine the metabolic state of proliferative cancer cells. Interestingly, cell proliferation in cancer cells, as marked by Ki-67 immunostaining, was strictly correlated with oxidative mitochondrial metabolism (OXPHOS) and the uptake of mitochondrial fuels, as detected via MCT1 expression (p < 0.001). More specifically, three metabolic tumor compartments were delineated: (1) proliferative and mitochondrial-rich cancer cells (Ki-67+/TOMM20+/COX+/MCT1+); (2) non-proliferative and mitochondrial-poor cancer cells (Ki-67-/TOMM20-/COX-/MCT1-); and (3) non-proliferative and mitochondrial-poor stromal cells (Ki-67-/TOMM20-/COX-/MCT1-). In addition, high oxidative stress (MCT4+) was very specific for cancer tissues. Thus, we next evaluated the prognostic value of MCT4 in a second independent patient cohort (n = 40). Most importantly, oxidative stress (MCT4+) in non-proliferating epithelial cancer cells predicted poor clinical outcome (tumor recurrence; p < 0.0001; log-rank test), and was functionally associated with FDG-PET avidity (p < 0.04). Similarly, oxidative stress (MCT4+) in tumor stromal cells was specifically associated with higher tumor stage (p < 0.03), and was a highly specific marker for cancer-associated fibroblasts (p < 0.001). We propose that oxidative stress is a key hallmark of tumor tissues that drives high-energy metabolism in adjacent proliferating mitochondrial-rich cancer cells, via the paracrine transfer of mitochondrial fuels (such as L-lactate and ketone bodies). New antioxidants and MCT4 inhibitors should be developed to metabolically target "three-compartment tumor metabolism" in head and neck cancers. It is remarkable that two "non-proliferating" populations of cells (Ki-67-/MCT4+) within the tumor can actually determine clinical outcome, likely by providing high-energy mitochondrial "fuels" for proliferative cancer cells to burn. Finally, we also show that in normal mucosal tissue, the basal epithelial "stem cell" layer is hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and is metabolically programmed to use mitochondrial fuels (MCT1+), such as ketone bodies and L-lactate. Thus, oxidative mitochondrial metabolism (OXPHOS) is a common feature of both (1) normal stem cells and (2) proliferating cancer cells. As such, we should consider metabolically treating cancer patients with mitochondrial inhibitors (such as Metformin), and/or with a combination of MCT1 and MCT4 inhibitors, to target "metabolic symbiosis."

Cancer metabolism, stemness and tumor recurrence

PubMed Central

Curry, Joseph M.; Tuluc, Madalina; Whitaker-Menezes, Diana; Ames, Julie A.; Anantharaman, Archana; Butera, Aileen; Leiby, Benjamin; Cognetti, David M.; Sotgia, Federica; Lisanti, Michael P.; Martinez-Outschoorn, Ubaldo E.

2013-01-01

Here, we interrogated head and neck cancer (HNSCC) specimens (n = 12) to examine if different metabolic compartments (oxidative vs. glycolytic) co-exist in human tumors. A large panel of well-established biomarkers was employed to determine the metabolic state of proliferative cancer cells. Interestingly, cell proliferation in cancer cells, as marked by Ki-67 immunostaining, was strictly correlated with oxidative mitochondrial metabolism (OXPHOS) and the uptake of mitochondrial fuels, as detected via MCT1 expression (p < 0.001). More specifically, three metabolic tumor compartments were delineated: (1) proliferative and mitochondrial-rich cancer cells (Ki-67+/TOMM20+/COX+/MCT1+); (2) non-proliferative and mitochondrial-poor cancer cells (Ki-67−/TOMM20−/COX−/MCT1−); and (3) non-proliferative and mitochondrial-poor stromal cells (Ki-67−/TOMM20−/COX−/MCT1−). In addition, high oxidative stress (MCT4+) was very specific for cancer tissues. Thus, we next evaluated the prognostic value of MCT4 in a second independent patient cohort (n = 40). Most importantly, oxidative stress (MCT4+) in non-proliferating epithelial cancer cells predicted poor clinical outcome (tumor recurrence; p < 0.0001; log-rank test), and was functionally associated with FDG-PET avidity (p < 0.04). Similarly, oxidative stress (MCT4+) in tumor stromal cells was specifically associated with higher tumor stage (p < 0.03), and was a highly specific marker for cancer-associated fibroblasts (p < 0.001). We propose that oxidative stress is a key hallmark of tumor tissues that drives high-energy metabolism in adjacent proliferating mitochondrial-rich cancer cells, via the paracrine transfer of mitochondrial fuels (such as L-lactate and ketone bodies). New antioxidants and MCT4 inhibitors should be developed to metabolically target “three-compartment tumor metabolism” in head and neck cancers. It is remarkable that two “non-proliferating” populations of cells (Ki-67−/MCT4+) within the tumor can actually determine clinical outcome, likely by providing high-energy mitochondrial “fuels” for proliferative cancer cells to burn. Finally, we also show that in normal mucosal tissue, the basal epithelial “stem cell” layer is hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and is metabolically programmed to use mitochondrial fuels (MCT1+), such as ketone bodies and L-lactate. Thus, oxidative mitochondrial metabolism (OXPHOS) is a common feature of both (1) normal stem cells and (2) proliferating cancer cells. As such, we should consider metabolically treating cancer patients with mitochondrial inhibitors (such as Metformin), and/or with a combination of MCT1 and MCT4 inhibitors, to target “metabolic symbiosis.” PMID:23574725

The leading role of mitochondrial depolarization in the mechanism of glutamate-induced disruptions in Ca2+ homeostasis.

PubMed

Khodorov, B I; Storozhevykh, T P; Surin, A M; Yuryavichyus, A I; Sorokina, E G; Borodin, A V; Vinskaya, N P; Khaspekov, L G; Pinelis, V G

2002-01-01

Data obtained in studies of the nature of the correlation which we have previously observed [10,17] between mitochondrial depolarization and the level of disruption of Ca2+ homeostasis in cultivated brain neuronsare summarized. Experiments were performed on cultured cerebellar granule cells loaded with Fura-2-AM or rhodamine 123 to measure changes in cytoplasmic Ca2+ and mitochondrial potential during pathogenic treatments of the cells. Prolonged exposure to 100 microM glutamate induced a reversible increase in [Ca2+]i, which was accompanied by only a small degree of mitochondrial depolarization. A sharp increase in this mitochondrial depolarization, induced by addition of 3 mM NaCN or 300 microM dinitrophenol (DNP) to the glutamate-containing solution, resulted in further increase in [Ca2+]i, due to blockade of electrophoretic mitochondrial Ca2+ uptake. Prolonged exposure to CN- or DNP in the post-glutamate period maintained [Ca2+]i at a high level until the metabolic inhibitors were removed. In most cells, this plateau was characterized by low sensitivity to removal of external Ca2+, demonstrating that the mechanisms of Ca2+ release from neurons were disrupted. Addition of oligomycin, a blocker of mitochondrial ATP synthase/ATPase, to the solution containing glutamate and CN- or DNP eliminated the post-glutamate plateau. Parallel experiments with direct measurements of intracellular ATP levels ([ATP]) showed that profound mitochondrial depolarization induced by CN- or DNP sharply enhanced the drop in ATP due to glutamate, while oligomycin significantly weakened this effect of the metabolic inhibitors. Analysis of these data led to the conclusion that blockade of mitochondrial Ca2+ uptake and inhibition of ATP synthesis resulted from mitochondrial depolarization and plays a key role in the mechanism disrupting [Ca2+]i homeostasis after toxic exposure to glutamate.

Calcium transport across the inner mitochondrial membrane: molecular mechanisms and pharmacology

PubMed Central

Csordás, György; Várnai, Peter; Golenár, Tünde; Sheu, Shey-Shing; Hajnóczky, György

2011-01-01

Growing evidence supports that mitochondrial calcium uptake is important for cell metabolism, signaling and survival. However, both the molecular nature of the mitochondrial Ca2+ transport sites and the calcium signals they respond to remained elusive. Recent RNA interference studies have identified new candidate proteins for Ca2+ uptake across the inner mitochondrial membrane, including LETM1, MCU, MICU1 and NCLX. The sensitivity of these factors to several drugs has been tested and in parallel, some new inhibitors of mitochondrial Ca2+ uptake have been described. This paper provides an update on the pharmacological aspects of the molecular mechanisms of the inner mitochondrial membrane Ca2+ transport. PMID:22123069

Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa.

PubMed

Ozaki, Taku; Nakazawa, Mitsuru; Yamashita, Tetsuro; Sorimachi, Hiroyuki; Hata, Shoji; Tomita, Hiroshi; Isago, Hitomi; Baba, Ayaka; Ishiguro, Sei-Ichi

2012-11-01

Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP. Copyright © 2012 Elsevier B.V. All rights reserved.

Detection of Redox Imbalance in Normal Lymphocytes with Induced Mitochondrial Dysfunction - EPR Study.

PubMed

Georgieva, Ekaterina; Zhelev, Zhivko; Aoki, Ichio; Bakalova, Rumiana; Higashi, Tatsuya

2016-10-01

The present study describes a new approach for direct imaging of redox status in live cells using paramagnetic spin-probes, which allows evaluation of the level of oxidative stress due to overproduction of superoxide. The method is based on redox cycling of cell/mitochondria-penetrating nitroxide radicals (e.g. mito-TEMPO) and their electron-paramagnetic resonance (EPR) contrast, which makes them useful molecular sensors for analysis of redox status and oxidative stress in cells and tissues. Oxidative stress was induced in normal human lymphocytes by treatment with 2-methoxyestradiol and rotenone (ME/Rot) at different concentrations. This combination provokes mitochondrial dysfunction, which is accompanied by overproduction of superoxide. The EPR measurements were performed in dynamics on X-Band spectrometer after addition of mito-TEMPO to cell suspensions. The intensity of the EPR signal in untreated cells decreased significantly, which indicates a conversion of paramagnetic mito-TEMPO to its non-contrast diamagnetic form (hydroxylamine - mito-TEMPOH) due to reduction. In ME/Rot-treated cells, the signal decreased more slowly and to a lower level with increasing the concentration of ME/Rot. These data indicate an induction of oxidative stress in the cells in a concentration-dependent manner. A very good positive correlation between the intensity of EPR signal of mito-TEMPO and the intracellular level of superoxide was found, analyzed by conventional dihydroethidium test (R=0.9143, p<0.001). In conclusion, our study demonstrated that cell-penetrating paramagnetic spin-probes, such as mito-TEMPO, are valuable tools for EPR imaging of the superoxide level in live cells, as well as for EPR imaging of mitochondrial dysfunction and metabolic activity, accompanied by superoxide imbalance. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Sulforaphane is anticonvulsant and improves mitochondrial function.

PubMed

Carrasco-Pozo, Catalina; Tan, Kah Ni; Borges, Karin

2015-12-01

The nuclear factor erythroid 2-related factor 2 pathway (Nrf2) has been previously identified to protect the brain against various impacts. Here, we investigated the effect of the Nrf2 activator sulforaphane in various seizure models and hippocampal mitochondrial bioenergetics. We found that daily injections of sulforaphane for 5 days elevated the seizure thresholds to 6 Hz stimulation and fluorothyl-, but not pentylenetetrazole-induced tonic seizures and protected mice against pilocarpine-induced status epilepticus (SE). Also, sulforaphane increased the antioxidant defences within hippocampal formations and blood plasma. In addition, sulforaphane treatment reduced the extent of hippocampal lipid peroxidation 24 h post-SE and protected hippocampal mitochondria against SE-induced reduction in state 2 and uncoupler-stimulated state 3 respiration. SE-mediated partial loss of rotenone-sensitive and complex II-driven respiration was reduced, consistent with the enhanced activities of complexes I and II in sulforaphane-treated SE mice. In mitochondria isolated from both no SE and SE mice, sulforaphane increased state 3 respiration and respiration linked to ATP synthesis, which may contribute to its anticonvulsant and antioxidant effects by providing more ATP for cellular vital and protective functions. However, sulforaphane did not prevent SE-induced hippocampal cell death. In conclusion, sulforaphane and/or Nrf2 activation are viable anticonvulsant strategies, which are antioxidant and enhance mitochondrial function, especially the ability to produce ATP. Sulforaphane was anticonvulsant in two acute mouse models of epilepsy and protected mice against pilocarpine-induced status epilepticus (SE). We also found antioxidant effects of sulforaphane in mouse plasma and hippocampal formations, exhibited by increased catalase and superoxide dismutase (SOD) activity, as well as increased abilities of hippocampal mitochondria to produce ATP. These effects likely underlie sulforaphane's anticonvulsant mechanisms of action. © 2015 International Society for Neurochemistry.

Mitochondria-targeted antioxidant (MitoQ) ameliorates age-related arterial endothelial dysfunction in mice.

PubMed

Gioscia-Ryan, Rachel A; LaRocca, Thomas J; Sindler, Amy L; Zigler, Melanie C; Murphy, Michael P; Seals, Douglas R

2014-06-15

Age-related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age-related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria-targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ∼30% in old (∼27 months) compared with young (∼8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium-independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age-related increases in total and mitochondria-derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age-related increase in endothelial susceptibility to acute mitochondrial damage (rotenone-induced impairment in EDD). Our results suggest that mitochondria-derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria-targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age-related CVD. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

Mitochondria-targeted antioxidant (MitoQ) ameliorates age-related arterial endothelial dysfunction in mice

PubMed Central

Gioscia-Ryan, Rachel A; LaRocca, Thomas J; Sindler, Amy L; Zigler, Melanie C; Murphy, Michael P; Seals, Douglas R

2014-01-01

Age-related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age-related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria-targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ∼30% in old (∼27 months) compared with young (∼8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium-independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age-related increases in total and mitochondria-derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age-related increase in endothelial susceptibility to acute mitochondrial damage (rotenone-induced impairment in EDD). Our results suggest that mitochondria-derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria-targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age-related CVD. PMID:24665093

Hypoxic augmentation of Ca2+ channel currents requires a functional electron transport chain.

PubMed

Brown, Stephen T; Scragg, Jason L; Boyle, John P; Hudasek, Kristin; Peers, Chris; Fearon, Ian M

2005-06-10

The incidence of Alzheimer disease is increased following ischemic episodes, and we previously demonstrated that following chronic hypoxia (CH), amyloid beta (Abeta) peptide-mediated increases in voltage-gated L-type Ca(2+) channel activity contribute to the Ca(2+) dyshomeostasis seen in Alzheimer disease. Because in certain cell types mitochondria are responsible for detecting altered O(2) levels we examined the role of mitochondrial oxidant production in the regulation of recombinant Ca(2+) channel alpha(1C) subunits during CH and exposure to Abeta-(1-40). In wild-type (rho(+)) HEK 293 cells expressing recombinant L-type alpha(1C) subunits, Ca(2+) currents were enhanced by prolonged (24 h) exposure to either CH (6% O(2)) or Abeta-(1-40) (50 nm). By contrast the response to CH was absent in rho(0) cells in which the mitochondrial electron transport chain (ETC) was depleted following long term treatment with ethidium bromide or in rho(+) cells cultured in the presence of 1 microm rotenone. CH was mimicked in rho(0) cells by the exogenous production of O2(-.). by xanthine/xanthine oxidase. Furthermore Abeta-(1-40) enhanced currents in rho(0) cells to a degree similar to that seen in cells with an intact ETC. The antioxidants ascorbate (200 microm) and Trolox (500 microm) ablated the effect of CH in rho(+) cells but were without effect on Abeta-(1-40)-mediated augmentation of Ca(2+) current in rho(0) cells. Thus oxidant production in the mitochondrial ETC is a critical factor, acting upstream of amyloid beta peptide production in the up-regulation of Ca(2+) channels in response to CH.

GABA-BZD Receptor Modulating Mechanism of Panax quinquefolius against 72-h Sleep Deprivation Induced Anxiety like Behavior: Possible Roles of Oxidative Stress, Mitochondrial Dysfunction and Neuroinflammation

PubMed Central

Chanana, Priyanka; Kumar, Anil

2016-01-01

Rationale: Panax quinquefolius (American Ginseng) is known for its therapeutic potential against various neurological disorders, but its plausible mechanism of action still remains undeciphered. GABA (Gamma Amino Butyric Acid) plays an important role in sleep wake cycle homeostasis. Thus, there exists rationale in exploring the GABA-ergic potential of Panax quinquefolius as neuroprotective strategy in sleep deprivation induced secondary neurological problems. Objective: The present study was designed to explore the possible GABA-ergic mechanism in the neuro-protective effect of Panax quinquefolius against 72-h sleep deprivation induced anxiety like behavior, oxidative stress, mitochondrial dysfunction, HPA-axis activation and neuroinflammation. Materials and Methods: Male laca mice were sleep deprived for 72-h by using Grid suspended over water method. Panax quinquefolius (American Ginseng 50, 100, and 200 mg/kg) was administered alone and in combination with GABA modulators (GABA Cl− channel inhibitor, GABA-benzodiazepine receptor inhibitor and GABAA agonist) for 8 days, starting 5 days prior to 72-h sleep deprivation period. Various behavioral (locomotor activity, mirror chamber test), biochemical (lipid peroxidation, reduced glutathione, catalase, nitrite levels), mitochondrial complexes, neuroinflammation marker (Tumor Necrosis Factor, TNF-alpha), serum corticosterone, and histopathological sections of brains were assessed. Results: Seventy two hours sleep deprivation significantly impaired locomotor activity, caused anxiety-like behavior, conditions of oxidative stress, alterations in mitochondrial enzyme complex activities, raised serum corticosterone levels, brain TNFα levels and led to neuroinflammation like signs in discrete brain areas as compared to naive group. Panax quinquefolius (100 and 200 mg/kg) treatment restored the behavioral, biochemical, mitochondrial, molecular and histopathological alterations. Pre-treatment of GABA Cl− channel inhibitor as well as GABA-benzodiazepine receptor inhibitor, significantly reversed the protective effect of P. quinquefolius (100 mg/kg) in 72-h sleep deprived animals (P < 0.05). However, pretreatment with GABAA agonist, potentiated Panax quinquefolius's protective effect which was significant as compared to their effect per se (p < 0.05). Conclusion: GABA-ergic mechanism could be involved in the neuroprotective effect of P.quinquefolius against sleep deprivation induced anxiety-like behavior, oxidative stress, mitochondrial dysfunction, HPA axis activation and neuroinflammation. PMID:27013946

The role of mitochondria in carbon catabolite repression in yeast.

PubMed

Haussmann, P; Zimmermann, F K

1976-10-18

The role of mitochondria in carbon catabolite repression in Saccharomyces cerevisiae was investigated by comparing normal, respiratory competent (RHO) strains with their mitochondrially inherited, respiratory deficient mutant derivatives (rho). Formation of maltase and invertase was used as an indicator system for the effect of carbon catabolite repression on carbon catabolic reactions. Fermentation rates for glucose, maltose and sucrose were the same in RHO and rho strains. Specific activities of maltase and invertase were usually higher in the rho-mutants. A very pronounced difference in invertase levels was observed when cells were grown on maltose; rho-mutants had around 30 times more invertase than their RHO parent strains. The fact that rho-mutants were much less sensitive to carbon catabolite repression of invertase synthesis than their RHO parents was used to search for the mitochondrial factor(s) or function(s) involved in carbon catabolite repression. A possible metabolic influence of mitochondria on this system of regulation was tested after growth of RHO strains under anaerobic conditions (no respiration nor oxidative phosphorylation), in the presence of KCN (respiration inhibited), dinitrophenol (uncoupling of oxidative phosphorylation) and of both inhibitors anaerobic conditions and dinitrophenol had no effect on the extent of invertase repression. KCN reduced the degree of repression but not to the level found in rho-mutants. A combination of both inhibitors gave the same results as with KCN alone. Erythromycin and chloramphenicol were used as specific inhibitors of mitochondrial protein synthesis. Erythromycin prevented the formation of mitochondrial respiratory systems but did not induce rho-mutants under the conditions used. However, repression of invertase was as strong as in the absence of the inhibitor. Chloramphenicol led only to a slight reduction of the respiratory systems and did not affect invertase levels. A combination of both antibiotics had about the same effect as growth in the presence of KCN. The results showed that mitochondria are involved in carbon catabolite repression and they cause an increase in the degree of repression. These effects cannot be due to mere metabolic activities nor to factors made on the mitochondrial protein synthesizing machinery. This regulatory role of mitochondria is observed as long as an intact mitochondrial genome is maintained.

Enzymatic properties of the membrane-bound NADH oxidase system in the aerobic respiratory chain of Bacillus cereus.

PubMed

Kim, Man Suk; Kim, Young Jae

2004-11-30

Membranes prepared from Bacillus cereus KCTC 3674, grown aerobically on a complex medium, oxidized NADH exclusively, whereas deamino-NADH was little oxidized. The respiratory chain-linked NADH oxidase exhibited an apparent K(m) value of approximately 65 microM for NADH. The maximum activity of the NADH oxidase was obtained at about pH 8.5 in the presence of 0.1 M KCl (or NaCl). Respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited the activity of the NADH oxidase by about 90% at a concentration of 40 microM. Interestingly, rotenone and capsaicin inhibited the activity of the NADH oxidase by about 60% at a concentration of 40 microM and the activity was also highly sensitive to Ag(+).

Desensitizing Mitochondrial Permeability Transition by ERK-Cyclophilin D Axis Contributes to the Neuroprotective Effect of Gallic Acid against Cerebral Ischemia/Reperfusion Injury

PubMed Central

Sun, Jing; Ren, Da-Dui; Wan, Jin-Yi; Chen, Chen; Chen, Dong; Yang, Huan; Feng, Chun-Lai; Gao, Jing

2017-01-01

Ischemic stroke is a devastating disease with complex pathophysiology. Much evidence confirms that opening of the mitochondrial permeability transition pore (MPTP) is related with mitochondrial dysfunction to apoptosis in ischemic stroke, thus elucidating its signaling mechanism and screening novel MPTP inhibitor is therefore of paramount importance. Our earlier studies identified that gallic acid (GA), a naturally occurring plant phenol, endows with effect on inhibition of mitochondrial dysfunction, which has significant neuroprotective effect in cerebral ischemia/reperfusion injury. However, its molecular mechanisms regulating mitochondrial dysfunction remain elusive. Here, we uncover a role of GA in protecting mitochondria via MPTP inhibition. In addition to inhibit CypD binding to adenine nucleotide translocator, GA potentiates extracellular signal-regulated kinases (ERK) phosphorylation, leading to a decrease in cyclophilin D (CypD) expression, resulting in a desensitization to induction of MPTP, thus inhibiting caspase activation and ultimately giving rise to cellular survival. Our study firstly identifies ERK-CypD axis is one of the cornerstones of the cell death pathways following ischemic stroke, and confirms GA is a novel inhibitor of MPTP, which inhibits apoptosis depending on regulating the ERK-CypD axis. PMID:28428752

DRP-1 is required for BH3 mimetic-mediated mitochondrial fragmentation and apoptosis

PubMed Central

Milani, Mateus; Byrne, Dominic P; Greaves, Georgia; Butterworth, Michael; Cohen, Gerald M; Eyers, Patrick A; Varadarajan, Shankar

2017-01-01

The concept of using BH3 mimetics as anticancer agents has been substantiated by the efficacy of selective drugs, such as Navitoclax and Venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend on MCL-1 for survival, which is highly amplified in multiple cancers and a major factor determining chemoresistance. Most MCL-1 inhibitors that have been generated so far, while demonstrating early promise in vitro, fail to exhibit specificity and potency in a cellular context. To address the lack of standardised assays for benchmarking the in vitro binding of putative inhibitors before analysis of their cellular effects, we developed a rapid differential scanning fluorimetry (DSF)-based assay, and used it to screen a panel of BH3 mimetics. We next contrasted their binding signatures with their ability to induce apoptosis in a MCL-1 dependent cell line. Of all the MCL-1 inhibitors tested, only A-1210477 induced rapid, concentration-dependent apoptosis, which strongly correlated with a thermal protective effect on MCL-1 in the DSF assay. In cells that depend on both MCL-1 and BCL-XL, A-1210477 exhibited marked synergy with A-1331852, a BCL-XL specific inhibitor, to induce cell death. Despite this selectivity and potency, A-1210477 induced profound structural changes in the mitochondrial network in several cell lines that were not phenocopied following MCL-1 RNA interference or transcriptional repression, suggesting that A-1210477 induces mitochondrial fragmentation in an MCL-1-independent manner. However, A-1210477-induced mitochondrial fragmentation was dependent upon DRP-1, and silencing expression levels of DRP-1 diminished not just mitochondrial fragmentation but also BH3 mimetic-mediated apoptosis. These findings provide new insights into MCL-1 ligands, and the interplay between DRP-1 and the anti-apoptotic BCL-2 family members in the regulation of apoptosis. PMID:28079887

Amyloid β-induced impairments on mitochondrial dynamics, hippocampal neurogenesis, and memory are restored by phosphodiesterase 7 inhibition.

PubMed

Bartolome, Fernando; de la Cueva, Macarena; Pascual, Consuelo; Antequera, Desiree; Fernandez, Tamara; Gil, Carmen; Martinez, Ana; Carro, Eva

2018-02-20

The phosphodiesterase (PDE) 7 inhibitor S14 is a cell-permeable small heterocyclic molecule that is able to cross the blood-brain barrier. We previously found that intraperitoneal treatment with S14 exerted neuroprotection in an Alzheimer's disease (AD) model (in APP/PS1 mice). The objective of this study was to investigate the neurogenic and cellular effects of oral administration of S14 on amyloid β (Aβ) overload. We orally administered the PDE7 inhibitor S14 (15 mg/kg/day) or vehicle in 6-month-old APP/PS1 mice. After 5 weeks of S14 treatment, we evaluated cognitive functions and brain tissues. We also assessed the effects of S14 on the Aβ-treated human neuroblastome SH-SY5Y cell line. Targeting the cyclic adenosine monophosphate (cAMP)/cAMP-response element binding protein (CREB) pathway, S14 rescued cognitive decline by improving hippocampal neurogenesis in APP/PS1 transgenic mice. Additionally, S14 treatment reverted the Aβ-induced reduction in mitochondrial mass in APP/PS1 mice and in the human neuroblastoma SH-SY5Y cells co-exposed to Aβ. The restoration of the mitochondrial mass was found to be a dual effect of S14: a rescue of the mitochondrial biogenesis formerly slowed down by Aβ overload, and a reduction in the Aβ-increased mitochondrial clearance mechanism of mitophagy. Here, we show new therapeutic effects of the PDE7 inhibitor, confirming S14 as a potential therapeutic drug for AD.

PPARβ/δ Agonist Provides Neuroprotection by Suppression of IRE1α-Caspase-12-Mediated Endoplasmic Reticulum Stress Pathway in the Rotenone Rat Model of Parkinson's Disease.

PubMed

Tong, Qiang; Wu, Liang; Gao, Qing; Ou, Zhou; Zhu, Dongya; Zhang, Yingdong

2016-08-01

Two recent studies demonstrated that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) agonists exerted neuroprotective effects in mouse model of Parkinson's disease (PD). However, the underlying mechanisms remain unknown. Endoplasmic reticulum (ER) stress plays a major role in rotenone-induced dopaminergic neuronal degeneration. In the present study, we explored whether GW501516, a selective and high-affinity PPARβ/δ agonist, could protect the dopaminergic neurons against degeneration and improve PD behavior via suppressing the ER stress in the rotenone rat model of PD. GW501516 was administered intracerebroventricular infusion. Catalepsy and open field tests were used to test catalepsy and locomotor activities. The levels of dopamine and its metabolites were determined using high-performance liquid chromatography. Western blot and immunohistochemistry analysis were performed to assess dopaminergic neuronal degeneration. Quantitative real-time RT-PCR and Western blot analysis were executed to detect ER stress. TUNEL and immunohistochemistry assays were used to detect ER stress-mediated apoptosis. Our results showed that GW501516 ameliorated the catalepsy symptom and increased locomotor activity. Meanwhile, GW501516 partially reversed the loss of dopaminergic neurons. Moreover, GW501516 suppressed the activation of ER stress markers including inositol-requiring enzyme 1α (IRE1α) and caspase-12. Furthermore, GW501516 inhibited caspase-12-mediated neuronal apoptosis. These findings suggest that GW501516 conferred neuroprotection of not only biochemical and pathological attenuation but also behavioral improvement in the rotenone rat model of PD. More importantly, we demonstrated for the first time that suppressing IRE1α-caspase-12-mediated ER stress pathway may represent one potential mechanism underlying the neuroprotective effects of PPARβ/δ agonist in the rotenone rat model of PD.

β-Caryophyllene, a phytocannabinoid attenuates oxidative stress, neuroinflammation, glial activation, and salvages dopaminergic neurons in a rat model of Parkinson disease.

PubMed

Ojha, Shreesh; Javed, Hayate; Azimullah, Sheikh; Haque, M Emdadul

2016-07-01

Parkinson disease (PD) is a neurodegenerative disease characterized by progressive dopaminergic neurodegeneration in the substantia nigra pars compacta (SNc) area. The present study was undertaken to evaluate the neuroprotective effect of β-caryophyllene (BCP) against rotenone-induced oxidative stress and neuroinflammation in a rat model of PD. In the present study, BCP was administered once daily for 4 weeks at a dose of 50 mg/kg body weight prior to a rotenone (2.5 mg/kg body weight) challenge to mimic the progressive neurodegenerative nature of PD. Rotenone administration results in oxidative stress as evidenced by decreased activities of superoxide dismutase, catalase, and depletion of glutathione with a concomitant rise in lipid peroxidation product, malondialdehyde. Rotenone also significantly increased pro-inflammatory cytokines in the midbrain region and elevated the inflammatory mediators such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the striatum. Further, immunohistochemical analysis revealed loss of dopaminergic neurons in the SNc area and enhanced expression of ionized calcium-binding adaptor molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP), indicators of microglia activation, and astrocyte hypertrophy, respectively, as an index of inflammation. However, treatment with BCP rescued dopaminergic neurons and decreased microglia and astrocyte activation evidenced by reduced Iba-1 and GFAP expression. BCP in addition to attenuation of pro-inflammatory cytokines and inflammatory mediators such as COX-2 and iNOS, also restored antioxidant enzymes and inhibited lipid peroxidation as well as glutathione depletion. The findings demonstrate that BCP provides neuroprotection against rotenone-induced PD and the neuroprotective effects can be ascribed to its potent antioxidant and anti-inflammatory activities.

The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development

USDA-ARS?s Scientific Manuscript database

The mitochondrial alternative oxidase (AOX) is a non-energy conserving ubiquinol oxidase found in most fungal genomes studied to date. With the development of fungicides containing cytochrome-dependent respiratory chain (CRC) inhibitors, a strong interest in studying AOX functions in phytopathogenic...

Assignment of two mitochondrially synthesized polypeptides to human mitochondrial DNA and their use in the study of intracellular mitochondrial interaction.

PubMed Central

Oliver, N A; Wallace, D C

1982-01-01

Two mitochondrially synthesized marker polypeptides, MV-1 and MV-2, were found in human HeLa and HT1080 cells. These were assigned to the mitochondrial DNA in HeLa-HT1080 cybrids and hybrids by demonstrating their linkage to cytoplasmic genetic markers. These markers include mitochondrial DNA restriction site polymorphisms and resistance to chloramphenicol, an inhibitor of mitochondrial protein synthesis. In the absence of chloramphenicol, the expression of MV-1 and MV-2 in cybrids and hybrids was found to be directly proportional to the ratio of the parental mitochondrial DNAs. In the presence of chloramphenicol, the marker polypeptide linked to the chloramphenicol-sensitive mitochondrial DNA continued to be expressed. This demonstrated that resistant and sensitive mitochondrial DNAs can cooperate within a cell for gene expression and that the CAP-resistant allele was dominant or codominant to sensitive. Such cooperation suggests that mitochondrial DNAs can be exchanged between mitochondria. Images PMID:6955589

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

PubMed

Reuther, C; Ganjam, G K; Dolga, A M; Culmsee, C

2014-11-01

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Dynamin-related protein inhibitor downregulates reactive oxygen species levels to indirectly suppress high glucose-induced hyperproliferation of vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maimaitijiang, Alimujiang; Zhuang, Xinyu; Jiang, Xiaofei

Hyperproliferation of vascular smooth muscle cells is a pathogenic mechanism common in diabetic vascular complications and is a putatively important therapeutic target. This study investigated multiple levels of biology, including cellular and organellar changes, as well as perturbations in protein synthesis and morphology. Quantitative and qualitative analysis was utilized to assess the effect of mitochondrial dynamic changes and reactive oxygen species(ROS) levels on high-glucose-induced hyperproliferation of vascular smooth muscle cells. The data demonstrated that the mitochondrial fission inhibitor Mdivi-1 and downregulation of ROS levels both effectively inhibited the high-glucose-induced hyperproliferation of vascular smooth muscle cells. Downregulation of ROS levels playedmore » a more direct role and ROS levels were also regulated by mitochondrial dynamics. Increased ROS levels induced excessive mitochondrial fission through dynamin-related protein (Drp 1), while Mdivi-1 suppressed the sensitivity of Drp1 to ROS levels, thus inhibiting excessive mitochondrial fission under high-glucose conditions. This study is the first to propose that mitochondrial dynamic changes and ROS levels interact with each other and regulate high-glucose-induced hyperproliferation of vascular smooth muscle cells. This finding provides novel ideas in understanding the pathogenesis of diabetic vascular remodeling and intervention. - Highlights: • Mdivi-1 inhibits VSMC proliferation by lowering ROS level in high-glucose condition. • ROS may be able to induce mitochondrial fission through Drp1 regulation. • Mdivi-1 can suppress the sensitivity of Drp1 to ROS.« less

Inhibiting prenylation augments chemotherapy efficacy in renal cell carcinoma through dual inhibition on mitochondrial respiration and glycolysis.

PubMed

Huang, Jiangrong; Yang, Xiaoyu; Peng, Xiaochun; Huang, Wei

2017-11-18

Prenylation is a posttranslational lipid modification required for the proper functions of a number of proteins involved in cell regulation. Here, we show that prenylation inhibition is important for renal cell carcinoma (RCC) growth, survival and response to chemotherapy, and its underlying mechanism may be contributed to mitochondrial dysfunction. We first demonstrated that a HMG-CoA reductase inhibitor pitavastatin inhibited mevalonate pathway and thereby prenylation in RCC cells. In addition, pitavastatin is effective in inhibiting growth and inducing apoptosis in a panel of RCC cell lines. Combination of pitavastatin and paclitaxel is significantly more effective than pitavastatin or paclitaxel alone as shown by both in vitro cell culture system and in vivo RCC xenograft model. Importantly, pitavastatin treatment inhibits mitochondrial respiration via suppressing mitochondrial complex I and II enzyme activities. Interestingly, different from mitochondrial inhibitor phenformin that inhibits mitochondrial respiration but activates glycolytic rate in RCC cells, pitavastatin significantly decreases glycolytic rate. The dual inhibitory action of pitavastatin on mitochondrial respiration and glycolysis results in remarkable energy depletion and oxidative stress in RCC cells. In addition, inhibition of prenylation by depleting Isoprenylcysteine carboxylmethyltransferase (Icmt) also mimics the inhibitory effects of pitavastatin in RCC cells. Our work demonstrates the previously unappreciated association between prenylation inhibition and energy metabolism in RCC, which can be therapeutically exploited, likely in tumors that largely rely on energy metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

Environmental Assessment for Watershed Enhancements at Joint Base Elmendorf-Richardson

DTIC Science & Technology

2013-07-03

Potassium permanganate would be utilized to prevent lethal dose of rotenone migrating beyond the largest beaver dam on Otter Creek. Lowering the lake level...Finding of No Significant Impact JBER Joint Base Elmendorf-Richardson KMnO4 potassium permanganate MOA Municipality of Anchorage NEPA National...Potassium permanganate would be utilized to prevent lethal dose of rotenone migrating beyond the largest beaver dam on Otter Creek. Lowering the lake