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Sample records for inhibits 6-hydroxydopamine-induced cytochrome

  1. Inhibition of Mitochondrial Clearance and Cu/Zn-SOD Activity Enhance 6-Hydroxydopamine-Induced Neuronal Apoptosis.

    PubMed

    In, Sua; Hong, Chang-Won; Choi, Boyoung; Jang, Bong-Geum; Kim, Min-Ju

    2016-01-01

    Parkinson's disease (PD) is a common movement disorder among neurodegenerative diseases, involving neuronal cell death in the substantia nigra of the midbrain. Although mechanisms of cell death in PD have been studied, the exact molecular pathogenesis is still unclear. Here, we explore the relationship between two types of cell death, autophagy and apoptosis, which have been studied separately in parkinsonian mimetic model of 6-hydroxydopamine (6-OHDA). 6-OHDA induced autophagy firstly and then later inhibition of autophagy flux occurred with apoptosis. The apoptosis was prevented by treatment of pan-caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-VAD-fluoromethylketone (zVAD)), or early phase inhibitor of autophagy, 3-methyladenine (3-MA), indicating that autophagic induction was followed by the apoptosis. Interestingly, late step inhibitor of autophagy, bafilomycin A1 (BafA), aggravated 6-OHDA-induced apoptosis. This was associated with mitochondrial abnormality such as the inhibition of damaged mitochondrial clearance and aberrant increase of extracellular oxygen consumption. Furthermore, treatment of BafA did not inhibit 6-OHDA-mediated superoxide formation but strongly reduced the hydrogen peroxide production to below basal levels, indicating failure from superoxide to hydrogen peroxide. These results were accompanied by a lowered expression and activity of copper/zinc superoxide dismutase (Cu/Zn-SOD) but not of manganese SOD (MnSOD) and catalase. Thus, the present study suggests that crosstalk among apoptosis, autophagy, and oxidative stress is a causative factor of 6-OHDA-induced neuronal death and provides a mechanistic understanding of PD pathogenesis.

  2. Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents 6-hydroxydopamine induced apoptosis in SH-SY5Y cells.

    PubMed

    Tian, Lin-Lin; Wang, Xue-Jun; Sun, Yu-Ning; Li, Chun-Rong; Xing, Ya-Ling; Zhao, Hai-Bao; Duan, Ming; Zhou, Zhe; Wang, Sheng-Qi

    2008-01-01

    Oxidative stress caused by dopamine may play an important role in the pathogenesis of Parkinson's disease. Salvianolic acid B is an antioxidant derived from the Chinese herb, Salvia miltiorrhiza. In this study, we investigated the neuroprotective effect of salvianolic acid B against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with salvianolic acid B significantly reduced 6-hydroxydopamine-induced generation of reactive oxygen species, and prevented 6-hydroxydopamine-induced increases in intracellular calcium. Our data demonstrated that 6-hydroxydopamine-induced apoptosis was reversed by salvianolic acid B treatment. Salvianolic acid B reduced the 6-hydroxydopamine-induced increase of caspase-3 activity, and reduced cytochrome C translocation into the cytosol from mitochondria. The 6-hydroxydopamine-induced decrease in the Bcl-x/Bax ratio was prevented by salvianolic acid B. Additionally, salvianolic acid B decreased the activation of extracellular signal-regulated kinase and induced the activation of 6-hydroxydopamine-suppressed protein kinase C. These results indicate that the protective function of salvianolic acid B is dependent upon its antioxidative potential. Our results strongly suggest that salvianolic acid B may be effective in treating neurodegenerative diseases associated with oxidative stress.

  3. 6-HYDROXYDOPAMINE INDUCES MITOCHONDRIAL ERK ACTIVATION

    PubMed Central

    Kulich, Scott M.; Horbinski, Craig; Patel, Manisha; Chu, Charleen T.

    2007-01-01

    Reactive oxygen species (ROS) are implicated in 6-hydroxydopamine (6-OHDA) injury to catecholaminergic neurons; however, the mechanism(s) are unclear. In addition to ROS generated during autoxidation, 6-OHDA may initiate secondary cellular sources of ROS that contribute to toxicity. Using a neuronal cell line, we found that catalytic metalloporphyrin antioxidants conferred protection if added 1 hour after exposure to 6-OHDA, whereas the hydrogen peroxide scavenger catalase failed to protect if added more than 15 min after 6-OHDA. There was a temporal correspondence between loss of protection and loss of the ability of the antioxidant to inhibit 6-OHDA-induced ERK phosphorylation. Time course studies of aconitase inactivation, as an indicator of intracellular superoxide, and MitoSOX red, a mitochondria targeted ROS indicator, demonstrate early intracellular ROS followed by a delayed phase of mitochondrial ROS production, associated with phosphorylation of a mitochondrial pool of ERK. Furthermore, upon initiation of mitochondrial ROS and ERK activation, 6-OHDA-injured cells became refractory to rescue by metalloporphyrin antioxidants. Together with previous studies showing that inhibition of the ERK pathway confers protection from 6-OHDA toxicity, and that phosphorylated ERK accumulates in mitochondria of degenerating human Parkinson’s disease neurons, these studies implicate mitochondrial ERK activation in Parkinsonian oxidative neuronal injury. PMID:17602953

  4. Baicalein prevents 6-hydroxydopamine-induced mitochondrial dysfunction in SH-SY5Y cells via inhibition of mitochondrial oxidation and up-regulation of DJ-1 protein expression.

    PubMed

    Wang, Yue-Hua; Yu, Hai-Tao; Pu, Xiao-Ping; Du, Guan-Hua

    2013-11-27

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons at the substantia nigra. Mitochondrial dysfunction is involved in the mechanism of cell damage in Parkinson's disease (PD). 6-Hydroxydopamine (6-OHDA) is a dopamine analog which specifically damages dopaminergic neurons. Baicalein has been previously reported to have potential in the treatment of PD. The purpose of the present study was to investigate the mechanism of action of baicalein against 6-OHDA injury in SH-SY5Y cells. The results showed that baicalein significantly alleviated alterations of mitochondrial redox activity and mitochondrial membrane potential induced by 6-OHDA in a dose-dependent manner in SH-SY5Y cells compared with vehicle group. Futhermore, baicalein decreased the production of ROS and upregulated the DJ-1 protein expression in SH-SY5Y cells. In addition, baicalein also inhibited ROS production and lipid peroxidation (IC50 = 6.32 ± 0.03 μM) in rat brain mitochondia. In summary, the underlying mechanisms of baicalein against 6-OHDA-induced mitochondrial dysfunction may involve inhibition of mitochondrial oxidation and upregulation of DJ-1 protein expression.

  5. Lonicera japonica THUNB. protects 6-hydroxydopamine-induced neurotoxicity by inhibiting activation of MAPKs, PI3K/Akt, and NF-κB in SH-SY5Y cells.

    PubMed

    Kwon, Seung-Hwan; Hong, Sa-Ik; Jung, Yang-Hee; Kim, Min-Jung; Kim, Sun-Yeou; Kim, Hyoung-Chun; Lee, Seok-Yong; Jang, Choon-Gon

    2012-03-01

    In this study, we investigated the neuroprotective effects of Lonicera japonica THUNB. extract (LJ) on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells. We found that LJ significantly increased cell viability decrease, lactate dehydrogenase release (LDH), morphological changes, nuclear condensation, fragmentation, and reactive oxygen species (ROS) production induced by 6-OHDA in SH-SY5Y cells. The cytoprotection afforded by pretreatment with LJ was associated with increases of the glutathione (GSH) level, superoxide dismutase (SOD) activity, and catalase (CAT) activity in 6-OHDA-induced SH-SY5Y cells. In addition, LJ strikingly inhibited 6-OHDA-induced mitochondrial dysfunctions including reduction of mitochondria membrane potential (MMP) and activation of cleaved poly-ADP-ribose polymerase (PARP), cleaved caspase-3, cleaved caspase-9, increased Bax, as well as decreased Bcl-2 and Bcl-xL. Additionally, LJ dramatically attenuated 6-OHDA-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK 1/2), and phosphoinositide 3-kinase (PI3K)/Akt. Meanwhile, LJ counteracted nuclear factor-κB (NF-κB) activation by blocking its translocation to the nucleus. These findings suggest that LJ has a potent anti-parkinsonism; this effect was mediated, at least in part, by inhibition of neurotoxicity, apoptotic cascade events, and oxidative stress via activation of MAPKs, PI3K/Akt, and NF-κB. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Involvement of Mu Opioid Receptor Signaling in the Protective Effect of Opioid against 6-Hydroxydopamine-Induced SH-SY5Y Human Neuroblastoma Cells Apoptosis

    PubMed Central

    Eftekhar-Vaghefi, Shahrzad; Esmaeili-Mahani, Saeed; Elyasi, Leila; Abbasnejad, Mehdi

    2015-01-01

    Introduction: The neuroprotective role of opioid morphine against 6-hydroxydopamine-induced cell death has been demonstrated. However, the exact mechanism(s) underlying such neuroprotection, especially the role of subtype receptors, has not yet been fully clarified. Methods: Here, we investigated the effects of different opioid agonists on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson’s disease. Cell damage was induced by 150 μM 6-OHDA and the cells viability was examined by MTT assay. Intracellular calcium, reactive oxygen species and mitochondrial membrane potential were assessed by fluorescence spectrophotometry method. Immunoblot technique was used to evaluate cytochrome-c and activated caspase-3 as biochemical markers of apoptosis induction. Results: The data showed that 6-OHDA caused significant cell damage, loss of mitochondrial membrane potential and increase in intracellular reactive oxygen species and calcium levels as well as activated caspase-3 and cytochrome-c release. Incubation of SH-SY5Y cells with μ-opioid agonists, morphine and DAMGO, but not with δ-opioid agonist, DADLE, elicited protective effect and reduced biochemical markers of cell damage and death. Discussion: The results suggest that μ-opioid receptors signaling participate in the opioid neuroprotective effects against 6-OHDA-induced neurotoxicity. PMID:26904174

  7. Isoliquiritigenin isolated from licorice Glycyrrhiza uralensis prevents 6-hydroxydopamine-induced apoptosis in dopaminergic neurons.

    PubMed

    Hwang, Cheol Kyu; Chun, Hong Sung

    2012-01-01

    Licorice (Glycyrrhiza uralensis) is a medicinal herb containing various bioactive components implicated in antioxidative, anti-inflammatory, antiviral, and neuroprotective effects, but the effects of licorice against Parkinson's disease (PD)-related dopaminergic cell death have not been studied. In this study, we investigated the protective effects of isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in a dopaminergic cell line, SN4741. ISL (1 µM) significantly attenuated 6-OHDA (50 µM)-induced reactive oxygen species (ROS) and nitric oxide (NO) generation and apoptotic cell death. ISL pretreatment effectively suppressed 6-OHDA-mediated upregulation of Bax, p-c-Jun N-terminal kinase (JNK), p-p38 mitogen-activated protein (MAP) kinase, cytochrome c release, and caspase 3 activation. In addition, ISL significantly attenuated 6-OHDA-induced Bcl-2, brain-derived neurotrophic factor (BDNF), and mitochondrial membrane potential (MMP) reduction. Pharmacological inhibitors of the phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway reversed ISL-mediated neuroprotection against 6-OHDA toxicity in SN4741 cells. These results provide the first evidence that ISL can protect dopaminergic cells under oxidative stress conditions by regulating the apoptotic process.

  8. Neuroprotective effect of trans-cinnamaldehyde on the 6-hydroxydopamine-induced dopaminergic injury.

    PubMed

    Pyo, Ji-Hi; Jeong, You-Kyung; Yeo, Sujung; Lee, Je-Hyun; Jeong, Mi-Young; Kim, Sung-Hoon; Choi, Yeong-Gon; Lim, Sabina

    2013-01-01

    The anti-inflammatory and neuroprotective effects of trans-cinnamaldehyde (TCA) were investigated on the inflammatory cells and the dopaminergic degeneration in mice. TCA inhibited the up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-induced inflammatory BV2 microglial cells. To investigate the TCA efficacy on the 6-hydroxydopamine (6-OHDA)-induced dopaminergic degeneration in mice, an intracerebroventricular injection of 6-OHDA was given to the mice, and TCA (30 mg/kg) was intraperitoneally administered. At 7 d after the 6-OHDA injection, 6-OHDA led to a severe loss of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the striatum and substantia nigra (SN). On the other hand, TCA dramatically maintained the number of TH-positive dopaminergic neurons in the striatum and SN regions of the 6-OHDA-treated mice, which indicates that TCA is able to inhibit the 6-OHDA-induced reduction of TH expression in the dopaminergic neurons in the striatum and SN regions. TCA also inhibited the induction of iNOS and COX-2 in the 6-OHDA model, similarly as shown in the LPS-induced inflammatory BV2 microglial cells. These results indicate that TCA has a neuroprotective effect on dopaminergic neurons and that this effect may be associated with the inhibition of inflammatory responses. These findings suggest that TCA may be a therapeutic candidate for the prevention of inflammation-mediated neurodegenerative diseases.

  9. Effect of acupuncture on 6-hydroxydopamine-induced nigrostratal dopaminergic neuronal cell death in rats.

    PubMed

    Kim, Yeung-Kee; Lim, Hyung-Ho; Song, Yun-Kyung; Lee, Hee-Hyuk; Lim, Sabina; Han, Seung-Moo; Kim, Chang-Ju

    In this study, we investigated the effect of acupuncture at the Zusanli acupoint (ST36) on the nigrostriatal dopaminergic neuronal cell death in the rats with Parkinson's disease. Two weeks after unilateral injection of 6-hydroxydopamine (6-OHDA) into the striatum, an apomorphine-induced rotational behavior test showed significant rotational asymmetry in the rats with Parkinson's disease. Immunostaining for tyrosine hydroxylase demonstrated a dopaminergic neuronal loss in the substantia nigra and dopaminergic fiber loss in the striatum. Acupuncture at the ST36 for 14 days significantly inhibited rotational asymmetry in the rats with Parkinson's disease, and also protected against 6-OHDA-induced nigrostriatal dopaminergic neuronal loss. These effects of acupuncture were not observed for the non-acupoint (hip) acupuncture. The present study shows that acupuncture at the ST36 acupoint can be used as a useful strategy for the treatment of Parkinson's disease.

  10. The flavanoide caffeic acid phenethyl ester blocks 6-hydroxydopamine-induced neurotoxicity.

    PubMed

    Noelker, Carmen; Bacher, Michael; Gocke, Petra; Wei, Xing; Klockgether, Thomas; Du, Yansheng; Dodel, Richard

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons of the substantia nigra pars compacta. 6-Hydroxydopamine (6-OHDA) is specific to dopaminergic neurons in intrastriatal rodent models. It induces neuronal death either via uncoupling mitochondrial oxidative phosphorylation resulting in energy deprivation or alternatively, is associated with its ability to produce hydrogen peroxide, hydroxyl and superoxide radicals. Caffeic acid phenethyl ester (CAPE), an antioxidant flavanoid, has antiviral, anti-inflammatory, antioxidant, and immunomodulatory properties. Recent studies have shown that CAPE has also a neuroprotective effects in ischemia and low potassium-induced neuronal apoptotic models. In cerebellar granule neurons CAPE significantly blocks 6-OHDA mediated cell death (70 microM) in a dose-dependent manner. Furthermore, CAPE was able to modulate the Ca(2+)-induced release of cyctochrome c in isolated liver mitochondria. Caspase-3 activation following 6-OHDA treatment was markedly inhibited in the presence of CAPE. Although the molecular mechanisms associated with CAPE's neuroprotective effects remain to be elucidated in more detail, our results clearly demonstrate a considerable neuroprotective effect of CAPE. Since a mitochondrial insult is a major cause for the degeneration of nigral neurons in PD, we hypothesize that propolis derivatives, in particular CAPE, may have a neuroprotective effect on those cells and may be a promising drug candidate to be taken into in vivo models of PD.

  11. Cortex Fraxini (Qingpi) Protects Rat Pheochromocytoma Cells against 6-Hydroxydopamine-Induced Apoptosis

    PubMed Central

    Li, Jing-Jie; Zhou, Shi-Ya; Zhang, Huan; Lam, Kim-Hung; Lee, Simon Ming-Yuen; Yu, Peter Hoi-Fu; Chan, Shun-Wan

    2015-01-01

    Parkinson's disease (PD) is a chronic neurodegenerative disorder having close relationship with oxidative stress induced by reactive oxygen species (ROS). Cortex Fraxini (QP) is a kind of traditional Chinese medicinal herb with antioxidant properties. It may be a potential candidate for preventing the development of chronic neurodegenerative diseases. Thus, the key objective of the current study was to investigate the neuroprotective effect of QP water extract on 6-hydroxydopamine (6-OHDA) induced apoptosis in rat pheochromocytoma (PC12) cells. It was found that QP water extract possesses strong antioxidant property with SC50 = 0.15 mg/mL. Total phenolic content of QP water extract was found to be 200.78 ± 2.65 mg GAE/g. QP water extract's free radical scavenging capacity was demonstrated by reversing the increased level of intracellular ROS induced by 6-OHDA, using 2′,7′-dichlorodihydrofluorescein diacetate. Moreover, QP water extract (0.5 mg/mL) could remarkably increase the viability of PC12 cells treated with 6-OHDA. The protective effect of QP water extract was found to be via inhibiting MEK/ERK pathway and reversing PI3-K/Akt/GSK3β pathway. The current results suggest that QP might be a potential candidate for preventing the development of neurodegenerative diseases, such as PD. PMID:26347850

  12. Neuromodulatory effects of Calyptranthes grandifolia extracts against 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells.

    PubMed

    Kich, Débora Mara; Bitencourt, Shanna; Alves, Celso; Silva, Joana; Pinteus, Susete; Pedrosa, Rui; Laufer, Stefan; de Souza, Claucia Fernanda Volken; Goettert, Márcia Inês

    2016-12-01

    Alzheimer's and Parkinson's diseases are neurodegenerative disorders characterized by progressive neuronal dysfunction. Previous studies revealed that some natural products have neuroprotective properties, including species of the Myrtaceae family. However, the neuromodulatory potential of Calyptranthes grandifolia is not clear. In the present study, we examined the ability of the ethanol and hexane leaf extracts of C. grandifolia to prevent 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. Initially, we investigated the potential of the extracts to inhibit the neurodegenerative-related enzymes c-Jun N-terminal kinase 3 (JNK3) and acetylcholinesterase (AChE). In addition, SH-SY5Y cell viability was assessed by MTT assay after 100μM 6-OHDA-induced cell damage. In order to verify the possible effects of both extracts on 6-OHDA-induced cell death, hydrogen peroxide generation, mitochondrial potential and caspases-3 activity were assessed. Our findings revealed that ethanol extract exhibited inhibitory activity against JNK3 and AChE. In addition, when co-treating SH-SY5Y cells with 6-OHDA and the extracts, oxidative stress was inhibited by both extracts through a decrease of mitochondrial depolarization and caspases-3 activity. In summary, ethanol and hexane extracts of C. grandifolia have some suppressive property against neurotoxicity induced by 6-OHDA. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Peganum Harmala L. Extract Reduces Oxidative Stress and Improves Symptoms in 6-Hydroxydopamine-Induced Parkinson's Disease in Rats.

    PubMed

    Rezaei, Maryam; Nasri, Sima; Roughani, Mehrdad; Niknami, Zeinab; Ziai, Seyed Ali

    2016-01-01

    Parkinson's disease is one of the most common neurodegenerative disorders. There are many documents about the effects of oxidative stress in Parkinson's disease etiology. Angiotensin II activates NADPH dependent oxidases and causes superoxides formation. Peganum harmala L. extract, which has angiotensin converting enzyme (ACE) inhibitory effect, is considered to evaluate oxidative stress inhibition and Parkinson's disease improvement. Male rats weighting 200-250 g were divided into 5 groups: Control, Neurotoxin (injection of 6-hydroxydopamine into left hemisphere substantia nigra), Peganum harmala's seeds aqueous extract (10 mg/kg) and captopril (5 mg/kg). Peganum harmala and captopril were injected intraperitonealy -144, -120, -96, -72, -48, -24, -2, 4 and 24 h relative to 6-hydroxydopamine injection time. Muscle stiffness, apomorphine induced unilateral rotation, amount of brain's protein oxidation and lipid peroxidation, ACE activity and histology of substantia nigra were assayed in all groups. Peganum harmala improved Muscle stiffness and one-direction rotation behavior significantly. It also reduced brain's lipid and protein oxidation levels in neurotoxin-injected rats significantly. In Peganum harmala group compared to control group, brain's ACE activity was significantly inhibited. In histological study, Peganum harmala prevented degeneration of dopaminergic neurons, too. In conclusion, aqueous extract of Peganum harmala could prevent symptoms and reduced oxidative stress markers in rats with Parkinson's disease induced by 6-hydroxydopamine.

  14. Human Albumin Prevents 6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and In Vivo

    PubMed Central

    Zhang, Li-Juan; Xue, Yue-Qiang; Yang, Chun; Yang, Wei-Hua; Chen, Long; Zhang, Qian-Jin; Qu, Ting-Yu; Huang, Shile; Zhao, Li-Ru; Wang, Xiao-Min; Duan, Wei-Ming

    2012-01-01

    Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis. PMID:22815976

  15. Inhibition of 6-hydroxydopamine-induced PC12 cell apoptosis by olive (Olea europaea L.) leaf extract is performed by its main component oleuropein.

    PubMed

    Pasban-Aliabadi, Hamzeh; Esmaeili-Mahani, Saeed; Sheibani, Vahid; Abbasnejad, Mehdi; Mehdizadeh, Anahita; Yaghoobi, Mohammad Mehdi

    2013-04-01

    Parkinson disease (PD) is the most common progressive neurodegenerative disorder characterized by progressive death of midbrain dopaminergic neurons. Most neurodegenerative disease treatments are, at present, palliative. However, some natural herbal products have been shown to rescue neurons from death and apoptosis in some of neurodegenerative diseases. Not only Olea europaea L. olive oil, but also the leaves of this plant have been used for medical purposes. Olive leaf extract (OLE) is being used by people as a drink across the world and as an integral ingredient in their desire to maintain and improve their health. Here, we investigated the effects of OLE and its main phenolic component oleuropein on 6-hydroxydopamine (6-OHDA)-induced toxicity in rat adrenal pheochromocytoma (PC12) cells as an in vitro model of PD. Cell damage was induced by 150 μM 6-OHDA. The cell survival rate was examined by MTT assay. Generation of intra-cellular reactive oxygen species (ROS) was studied using fluorescence spectrophotometry. Immunoblotting and DNA analysis were also employed to determine the levels of biochemical markers of apoptosis in the cells. The data showed that 6-OHDA could decrease the viability of the cells. In addition, intra-cellular ROS, activated caspase 3, Bax/Bcl-2 ratio, as well as DNA fragmentation were significantly increased in 6-OHDA-treated cells. Incubation of cells with OLE (400 and 600 μg/mL) and oleuropein (20 and 25 μg/mL) could decrease cell damage and reduce biochemical markers of cell death. The results suggest that OLE and oleuropein have anti-oxidant protective effects against 6-OHDA-induced PC12 cell damage. The protective effects of OLE and oleuropein are correlative with their anti-oxidative and anti-apoptotic properties and suggest their therapeutic potential in the treatment of PD.

  16. Peganum Harmala L. Extract Reduces Oxidative Stress and Improves Symptoms in 6-Hydroxydopamine-Induced Parkinson’s Disease in Rats

    PubMed Central

    Rezaei, Maryam; Nasri, Sima; Roughani, Mehrdad; Niknami, Zeinab; Ziai, Seyed Ali

    2016-01-01

    Parkinson’s disease is one of the most common neurodegenerative disorders. There are many documents about the effects of oxidative stress in Parkinson’s disease etiology. Angiotensin II activates NADPH dependent oxidases and causes superoxides formation. Peganum harmala L. extract, which has angiotensin converting enzyme (ACE) inhibitory effect, is considered to evaluate oxidative stress inhibition and Parkinson's disease improvement. Male rats weighting 200-250 g were divided into 5 groups: Control, Neurotoxin (injection of 6-hydroxydopamine into left hemisphere substantia nigra), Peganum harmala's seeds aqueous extract (10 mg/kg) and captopril (5 mg/kg). Peganum harmala and captopril were injected intraperitonealy -144, -120, -96, -72, -48, -24, -2, 4 and 24 h relative to 6-hydroxydopamine injection time. Muscle stiffness, apomorphine induced unilateral rotation, amount of brain's protein oxidation and lipid peroxidation, ACE activity and histology of substantia nigra were assayed in all groups. Peganum harmala improved Muscle stiffness and one-direction rotation behavior significantly. It also reduced brain's lipid and protein oxidation levels in neurotoxin-injected rats significantly. In Peganum harmala group compared to control group, brain's ACE activity was significantly inhibited. In histological study, Peganum harmala prevented degeneration of dopaminergic neurons, too. In conclusion, aqueous extract of Peganum harmala could prevent symptoms and reduced oxidative stress markers in rats with Parkinson’s disease induced by 6-hydroxydopamine. PMID:27610168

  17. Gelatin nanoparticle-mediated intranasal delivery of substance P protects against 6-hydroxydopamine-induced apoptosis: an in vitro and in vivo study

    PubMed Central

    Lu, Cui-Tao; Jin, Rong-Rong; Jiang, Yi-Na; Lin, Qian; Yu, Wen-Ze; Mao, Kai-Li; Tian, Fu-Rong; Zhao, Ya-Ping; Zhao, Ying-Zheng

    2015-01-01

    Background The aim of this study was to investigate the protective role of intranasally administered substance P-loaded gelatin nanoparticles (SP-GNPs) against 6-hydroxydopamine (6-OHDA)-induced apoptosis in vitro and in vivo, and to provide a new strategy for treating brain pathology, such as Parkinson’s disease. Methods SP-GNPs were prepared by a water-in-water emulsion method, and their stability, encapsulating efficiency, and loading capacity were evaluated. PC-12 cells were used to examine the enhancement of growth and inhibition of apoptosis by SP-GNPs in vitro using MTT assays. In the in vivo study, hemiparkinsonian rats were created by intracerebroventricular injection of 6-OHDA. The rats then received intranasal SP-GNPs daily for 2 weeks. Functional improvement was assessed by quantifying rotational behavior, and the degree of apoptosis was assessed by immunohistochemical staining for caspase-3 in the substantia nigra region. Results PC-12 cells with 6-OHDA-induced disease treated with SP-GNPs showed higher cell viability than their untreated counterparts, and cell viability increased as the concentration of substance P (SP) increased, indicating that SP could enhance cell growth and inhibit the cell apoptosis induced by 6-OHDA. Rats with 6-OHDA-induced hemiparkinsonism treated with SP-GNPs made fewer rotations and showed less staining for caspase-3 than their counterparts not treated with SP, indicating that SP protects rats with 6-OHDA-induced hemiparkinsonism from apoptosis and therefore demonstrates their functional improvement. Conclusion Intranasal delivery of SP-GNPs protects against 6-OHDA-induced apoptosis both in vitro and in vivo. PMID:25897205

  18. An In Vivo Microdialysis Study of FLZ Penetration through the Blood-Brain Barrier in Normal and 6-Hydroxydopamine Induced Parkinson's Disease Model Rats

    PubMed Central

    Hou, Jinfeng; Liu, Qian; Li, Yingfei; Sun, Hua; Zhang, Jinlan

    2014-01-01

    FLZ (N-[2-(4-hydroxy-phenyl)-ethyl]-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide) is a novel synthetic squamosamide derivative and a potential anti-Parkinson's disease (PD) agent. The objective of the present study was to investigate the penetration of free FLZ across the BBB and the effects of P-gp inhibition on FLZ transport in normal and 6-hydroxydopamine (6-OHDA) induced PD model rats. In vivo microdialysis was used to collect FLZ containing brain and blood dialysates following intravenous (i.v.) drug administration either with or without pretreatment with the specific P-gp inhibitor, zosuquidar trihydrochloride (zosuquidar·3HCl). A sensitive, rapid, and reliable ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique was developed and validated to quantitate free FLZ levels in the dialysates. No significant differences were observed in the brain/blood FLZ area under the concentration-time curve (AUC) ratio between normal and PD model rats. However, pretreatment with zosuquidar·3HCl markedly increased the AUC ratio in both rat models. In addition, FLZ penetration was similar in zosuquidar·3HCl-pretreated normal and PD rats. These results suggest that P-gp inhibition increases BBB permeability to FLZ, thereby supporting the hypothesis that P-gp normally restricts FLZ transfer to the brain. These findings could provide reference data for future clinical trials and may aid investigation of the BBB permeability of other CNS-active substances. PMID:25045708

  19. Effects of (-)-sesamin on 6-hydroxydopamine-induced neurotoxicity in PC12 cells and dopaminergic neuronal cells of Parkinson's disease rat models.

    PubMed

    Park, Hyun Jin; Zhao, Ting Ting; Lee, Kyung Sook; Lee, Seung Ho; Shin, Keon Sung; Park, Keun Hong; Choi, Hyun Sook; Lee, Myung Koo

    2015-01-01

    The present study investigated the effects of (-)-sesamin on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity using PC12 cells and dopaminergic neuronal cells of 6-OHDA-lesioned rat model of Parkinson's disease (PD). In PC12 cells, treatment with (-)-sesamin (25 µM) reduced 6-OHDA (100 µM)-induced cell death and induced transient extracellular signal-regulated kinase (ERK1/2) phosphorylation and Bad phosphorylation at Ser112 (BadSer112). In contrast, sustained ERK1/2 phosphorylation, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK1/2) phosphorylation, and cleaved-caspase-3 activity, all of which were induced by 6-OHDA (100 µM), were inhibited by treatment with (-)-sesamin (25 µM). Furthermore, co-treatment with (-)-sesamin (30 mg/kg, p.o.) once a day for 28 days significantly increased the number of tyrosine hydroxylase-immunopositive neuronal cells and the levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid in the substantia nigra-striatum of 6-OHDA-lesioned rat model of PD with or without L-DOPA treatment. These results suggest that (-)-sesamin protects 6-OHDA-induced cytotoxicity via the activation of transient ERK1/2-BadSer112 system and the inhibition of sustained ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells. (-)-Sesamin also shows protective effects on long-term L-DOPA therapy in dopaminergic neuronal cells of PD rat models. (-)-Sesamin may serve as adjuvant therapeutics in PD.

  20. An in vivo microdialysis study of FLZ penetration through the blood-brain barrier in normal and 6-hydroxydopamine induced Parkinson's disease model rats.

    PubMed

    Hou, Jinfeng; Liu, Qian; Li, Yingfei; Sun, Hua; Zhang, Jinlan

    2014-01-01

    FLZ (N-[2-(4-hydroxy-phenyl)-ethyl]-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide) is a novel synthetic squamosamide derivative and a potential anti-Parkinson's disease (PD) agent. The objective of the present study was to investigate the penetration of free FLZ across the BBB and the effects of P-gp inhibition on FLZ transport in normal and 6-hydroxydopamine (6-OHDA) induced PD model rats. In vivo microdialysis was used to collect FLZ containing brain and blood dialysates following intravenous (i.v.) drug administration either with or without pretreatment with the specific P-gp inhibitor, zosuquidar trihydrochloride (zosuquidar·3HCl). A sensitive, rapid, and reliable ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique was developed and validated to quantitate free FLZ levels in the dialysates. No significant differences were observed in the brain/blood FLZ area under the concentration-time curve (AUC) ratio between normal and PD model rats. However, pretreatment with zosuquidar·3HCl markedly increased the AUC ratio in both rat models. In addition, FLZ penetration was similar in zosuquidar·3HCl-pretreated normal and PD rats. These results suggest that P-gp inhibition increases BBB permeability to FLZ, thereby supporting the hypothesis that P-gp normally restricts FLZ transfer to the brain. These findings could provide reference data for future clinical trials and may aid investigation of the BBB permeability of other CNS-active substances.

  1. RA Differentiation Enhances Dopaminergic Features, Changes Redox Parameters, and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

    PubMed

    Lopes, Fernanda M; da Motta, Leonardo Lisbôa; De Bastiani, Marco A; Pfaffenseller, Bianca; Aguiar, Bianca W; de Souza, Luiz F; Zanatta, Geancarlo; Vargas, Daiani M; Schönhofen, Patrícia; Londero, Giovana F; de Medeiros, Liana M; Freire, Valder N; Dafre, Alcir L; Castro, Mauro A A; Parsons, Richard B; Klamt, Fabio

    2017-05-01

    Research on Parkinson's disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.

  2. Adaptive down-regulation of the serotonin transporter in the 6-hydroxydopamine-induced rat model of preclinical stages of Parkinson's disease and after chronic pramipexole treatment.

    PubMed

    Berghauzen-Maciejewska, K; Wardas, J; Kosmowska, B; Domin, H; Śmiałowska, M; Głowacka, U; Ossowska, K

    2016-02-09

    Our recent study has indicated that a moderate lesion induced by bilateral 6-hydroxydopamine (6-OHDA) injections into the ventrolateral region of the caudate-putamen (CP) in rats, modeling preclinical stages of Parkinson's disease, induces a "depressive-like" behavior which is reversed by chronic treatment with pramipexole (PRA). The aim of the present study was to examine the influence of the above lesion and chronic PRA treatment on binding to the serotonin transporter (SERT) in different brain regions. As before, 6-OHDA (15 μg/2.5 μl) was administered bilaterally into the CP. PRA (1mg/kg) was injected subcutaneously twice a day for 2 weeks. Serotonergic and dopaminergic neurons of the dorsal raphe (DR) were immunostained for tryptophan hydroxylase and tyrosine hydroxylase, respectively, and were counted stereologically. Binding of [(3)H]GBR 12,935 to the dopamine transporter (DAT) and [(3)H]citalopram to SERT was analyzed autoradiographically. Intrastriatal 6-OHDA injections decreased the number of dopaminergic, but not serotonergic neurons in the DR. 6-OHDA reduced the DAT binding in the CP, and SERT binding in the nigrostriatal system (CP, substantia nigra (SN)), limbic system (ventral tegmental area (VTA), nucleus accumbens (NAC), amygdala, prefrontal cortex (PFCX), habenula, hippocampus) and DR. A significant positive correlation was found between DAT and SERT binding in the CP. Chronic PRA did not influence DAT binding but reduced SERT binding in the above structures, and deepened the lesion-induced losses in the core region of the NAC, SN, VTA and PFCX. The present study indicates that both the lesion of dopaminergic neurons and chronic PRA administration induce adaptive down-regulation of SERT binding. Moreover, although involvement of stimulation of dopaminergic transmission by chronic PRA in its "antidepressant" effect seems to be prevalent, additional contribution of SERT inhibition cannot be excluded. Copyright © 2015 IBRO. Published by Elsevier

  3. Attenuation of hyperalgesia responses via the modulation of 5-hydroxytryptamine signalings in the rostral ventromedial medulla and spinal cord in a 6-hydroxydopamine-induced rat model of Parkinson’s disease

    PubMed Central

    Wang, Chen-Tao; Mao, Cheng-Jie; Zhang, Xiao-Qi; Zhang, Cai-Yi; Lv, Dong-Jun; Yang, Ya-Ping; Xia, Kai-Lin; Liu, Jun-Yi; Wang, Fen; Hu, Li-Fang; Xu, Guang-Yin

    2017-01-01

    Background Although pain is one of the most distressing non-motor symptoms among patients with Parkinson’s disease, the underlying mechanisms of pain in Parkinson’s disease remain elusive. The aim of the present study was to investigate the role of serotonin (5-hydroxytryptamine) in the rostral ventromedial medulla (RVM) and spinal cord in pain sensory abnormalities in a 6-hydroxydopamine-treated rat model of Parkinson’s disease. Methods The rotarod test was used to evaluate motor function. The radiant heat test and von Frey test were conducted to evaluate thermal and mechanical pain thresholds, respectively. Immunofluorescence was used to examine 5-hydroxytryptamine neurons and fibers in the rostral ventromedial medulla and spinal cord. High-performance liquid chromatography was used to determine 5-hydroxytryptamine and 5-hydroxyindoleacetic acid levels. Results The duration of running time on the rotarod test was significantly reduced in 6-hydroxydopamine-treated rats. Nociceptive thresholds of both mechanical and heat pain were reduced compared to sham-treated rats. In addition to the degeneration of cell bodies and fibers in the substantia nigra pars compacta, the number of rostral ventromedial medulla 5-hydroxytryptamine neurons and 5-hydroxytryptamine fibers in the spinal dorsal horn was dramatically decreased. 5-Hydroxytryptamine concentrations in both the rostral ventromedial medulla and spinal cord were reduced. Furthermore, the administration of citalopram significantly attenuated pain hypersensitivity. Interestingly, Intra-rostral ventromedial medulla (intra-RVM) microinjection of 5,7-dihydroxytryptamine partially reversed pain hypersensitivity of 6-hydroxydopamine-treated rats. Conclusions These results suggest that the decreased 5-hydroxytryptamine contents in the rostral ventromedial medulla and spinal dorsal horn may be involved in hyperalgesia in the 6-hydroxydopamine-induced rat model of Parkinson’s disease. PMID:28326933

  4. Thiomers: Inhibition of cytochrome P450 activity.

    PubMed

    Iqbal, Javed; Sakloetsakun, Duangkamon; Bernkop-Schnürch, Andreas

    2011-08-01

    The aim of the present study was to investigate the potential of different thiolated polymers (thiomers) on the catalytic activity of CYP450s on one hand and to explore new inhibitors for CYP activity on the other hand. Several thiolated polymers including poly(acrylic acid)-cysteine (PAA-cysteine), chitosan-thioglycolic acid (chitosan-TGA), and thiolated PEG-g-PEI copolymer along with brij 35, myrj 52 and the well-established CYPP450 inhibitor verapamil were screened for their CYP3A4 and CYP2A6 inhibitory activity, and their IC(50) values were determined. Both enzyme inhibition assays were performed in 96-well microtiter plates. 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC) and 7-hydroxycoumarin (7-HC) were used as fluorescent substrates in order to determine CYP3A4 and CYP2A6 catalytic activity, respectively. All investigated compounds inhibited CYP3A4 as well as CYP2A6 activity. All tested (thiolated) polymers were found to be more potent inhibitors of CYP3A4 than of CYP2A6 catalytic activity. Apart from verapamil that is a known CYP3A4 inhibitor, brij 35 and myrj 52 were explored as potent inhibitors of CYP3A4 and CYP2A6 catalytic activity. Among the tested polymers, the rank order for CYP3A4 inhibition was PAA-cysteine (100 kDa)>brij 35>thiolated PEG-g-PEI copolymer (16 kDa)>myrj 52>PAA (100 kDa)>PAA-cysteine (450 kDa)>verapamil>PAA (450 kDa)>chitosan-TGA (150 kDa)>chitosan (150 kDa). On the other hand, the rank order of CYP2A6 inhibition was brij 35>PAA-cysteine (100kDa)>chitosan-TGA (150 kDa)>PAA (100 kDa)>thiolated PEG-g-PEI copolymer (16 kDa)>PAA-cysteine (450 kDa)>chitosan (150 kDa)>verapamil>PAA (450 kDa)>myrj 52. Thus, this study suggests that (thiolated) polymers display a promising potential to inhibit cytochrome P450s activity and might turn out to be potentially valuable tools for improving the oral bioavailability of actively secreted compounds by avoiding intestinal metabolism.

  5. The mechanism of cytochrome C oxidase inhibition by nitric oxide.

    PubMed

    Antunes, Fernando; Cadenas, Enrique

    2007-01-01

    The basic biochemistry of the inhibition of cytochrome oxidase by NO is reviewed. Three possible mechanisms that include the binding of NO to the fully reduced Fe(a3)-Cu(B) site, to the semi-reduced Fe(a3)-Cu(B) site, and to the fully oxidized Fe(a3)-Cu(B) site are confronted with the experimental data. Mathematical models are used to facilitate the analysis and to solve puzzling observations concerning the NO inhibition of cytochrome oxidase. It is concluded that the inhibition of cytochrome oxidase by NO is mixed, having both competitive and uncompetitive components, but under physiological electron flows the competitive component is largely predominant. The physiological and pathological relevance of this inhibition is briefly discussed.

  6. Dimerumic Acid and Deferricoprogen Activate Ak Mouse Strain Thymoma/Heme Oxygenase-1 Pathways and Prevent Apoptotic Cell Death in 6-Hydroxydopamine-Induced SH-SY5Y Cells.

    PubMed

    Tseng, Wei-Ting; Hsu, Ya-Wen; Pan, Tzu-Ming

    2016-08-03

    Parkinson's disease (PD) is a neurodegenerative disorder, which can be modeled using the neurotoxin 6-hydroxydopamine (6-OHDA) to generate oxidative stress. Here, we studied the effects of the antioxidants deferricoprogen (DFC) and dimerumic acid (DMA), produced by rice fermented with Monascus purpureus NTU 568, on 6-OHDA-induced apoptosis in SH-SY5Y cells and their potential protective mechanisms. DMA and DFC inhibited 6-OHDA-induced apoptosis and cellular reactive oxygen species (ROS) in SH-SY5Y human neuroblastoma cells. Molecular analysis demonstrated associated upregulation of the Ak mouse strain thymoma (Akt), heme oxygenase-1 (HO-1), and signal-regulated kinase (ERK) pathways along with inhibited phosphorylation of c-Jun N-terminal kinase (JNK) and p38 pathways and altered homodimeric glycoprotein, N-methyl-d-aspartate (NMDA) receptor, and immunoglobulin Fc receptor gene expression. These results suggested that the neuroprotection elicited by DMA and DFC against 6-OHDA-induced neurotoxicity was associated with the Akt, MAPK, and HO-1 pathways via regulating the gene expression of NMDA receptor, homodimeric glycoprotein, and immunoglobulin Fc receptor.

  7. RETRACTED: S-allyl cysteine protects against 6-hydroxydopamine-induced neurotoxicity in the rat striatum: involvement of Nrf2 transcription factor activation and modulation of signaling kinase cascades.

    PubMed

    Tobón-Velasco, Julio César; Vázquez-Victorio, Genaro; Macías-Silva, Marina; Cuevas, Elvis; Ali, Syed F; Maldonado, Perla D; González-Trujano, María Eva; Cuadrado, Antonio; Pedraza-Chaverrí, José; Santamaría, Abel

    2012-09-01

    Pharmacological activation at the basal ganglia of the transcription factor Nrf2, guardian of redox homeostasis, holds a strong promise for the slow progression of Parkinson's disease (PD). However, a potent Nrf2 activator in the brain still must be found. In this study, we have investigated the potential use of the antioxidant compound S-allyl cysteine (SAC) in the activation of Nrf2 in 6-hydoxydopamine (6-OHDA)-intoxicated rats. In the rat striatum, SAC by itself promoted the Nrf2 dissociation of Keap-1, its nuclear translocation, the subsequent association with small MafK protein, and further binding of the Nrf2/MafK complex to ARE sequence, as well as the up-regulation of Nrf2-dependent genes encoding the antioxidant enzymes HO-1, NQO-1, GR, and SOD-1. In vivo and in vitro experiments to identify signaling pathways activated by SAC pointed to Akt as the most likely kinase participating in Nrf2 activation by SAC. In PC12 cells, SAC stimulated the activation of Akt and ERK1/2 and inhibited JNK1/2/3 activation. In the rat striatum, the SAC-induced activation of Nrf2 is likely to contribute to inhibit the toxic effects of 6-OHDA evidenced by phase 2 antioxidant enzymes up-regulation, glutathione recovery, and attenuation of reactive oxygen species (ROS), nitric oxide (NO), and lipid peroxides formation. These early protective effects correlated with the long-term preservation of the cellular redox status, the striatal dopamine (DA) and tyrosine hydroxylase (TH) levels, and the improvement of motor skills. Therefore, this study indicates that, in addition to direct scavenging actions, the activation of Nrf2 by SAC might confer neuroprotective responses through the modulation of kinase signaling pathways in rodent models of PD, and suggests that this antioxidant molecule may have a therapeutic value in this human pathology.

  8. Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells.

    PubMed

    Tseng, Wei-Ting; Hsu, Ya-Wen; Pan, Tzu-Ming

    2016-08-01

    Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson's disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC). Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated. Materials and methods DMA (0-60 μM) or DFC (0-10 μM) was co-treated with 6-OHDA (200 μM, 24 h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression. Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells. Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research.

  9. Cytochrome c Oxidase Inhibition by Anesthetics: Thermodynamic Analysis

    NASA Astrophysics Data System (ADS)

    Vanderkooi, Garret; Chazotte, Brad

    1982-06-01

    The thermodynamic parameters that characterize the inhibition of cytochrome c oxidase activity, in rat liver submitochondrial particles, by n-butanol, tetracaine, and dibucaine were obtained. Three equilibria were assumed in order to account for the data: for the interaction of inhibitor with the native state of the enzyme, for the interaction of inhibitor with the thermally (reversibly) denatured state, and for the change between the native and thermally denatured states. Inhibition results from interaction with both the native and denatured states but, because the interaction is stronger with the denatured than with the native state, the native/denatured equilibrium is shifted to the right by the anesthetics. The enthalpies of interaction are -2.3, -4.7, and 3.7 kcal/mol (1 cal = 4.18 J) for the native state and -10, -6, and -14 kcal/mol for the denatured state, for n-butanol, tetracaine, and dibucaine, respectively. These values are much smaller than the previous estimates obtained by using the assumption that anesthetics interact only with the thermally denatured state of enzymes (e.g., -81 kcal/mol for tetracaine inhibition of luciferase). Our results suggest that local anesthetics inhibit enzyme activity by causing a reversible perturbation of protein conformation. The magnitude of the perturbation is much smaller (in energetic terms) than that which accompanies thermal denaturation.

  10. Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells

    SciTech Connect

    Hwang, Yong Pil; Jeong, Hye Gwang

    2010-01-01

    Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a popular traditional herbal medicine. Ginsenoside Rb1 (Rb1), an active component commonly found in ginseng root, is a phytoestrogen that exerts estrogen-like activity. In this study, we demonstrate that the phytoestrogen Rb1 inhibits 6-hydroxydopamine (6-OHDA)-induced oxidative injury via an ER-dependent Gbeta1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of SH-SY5Y cells with Rb1 significantly reduced 6-OHDA-induced caspase-3 activation and subsequent cell death. Rb1 also up-regulated HO-1 expression, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, Rb1 induced both Nrf2 nuclear translocation, which is upstream of HO-1 expression and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Also, Rb1-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that Rb1 augments the cellular antioxidant defenses through ER-dependent HO-1 induction via the Gbeta1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Thus our study indicates that Rb1 has a partial cytoprotective role in dopaminergic cell culture systems.

  11. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

    PubMed

    Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2015-10-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation.

  12. β-asarone and levodopa co-administration protects against 6-hydroxydopamine-induced damage in parkinsonian rat mesencephalon by regulating autophagy: down-expression Beclin-1 and light chain 3B and up-expression P62.

    PubMed

    Huang, Li-Ping; Deng, Min-Zhen; He, Yu-Ping; Fang, Yong-Qi

    2015-03-01

    In this study, we investigated Beclin-1, light chain (LC)3B, and p62 expression in 6-hydroxydopamine (6-OHDA)-induced parkinsonian rats after β-asarone and levodopa (l-dopa) co-administration. Unilateral 6-OHDA injection into the medial forebrain bundle was used to create the models, except in sham-operated rats. Rats were divided into eight groups: sham-operated group; 6-OHDA model group; madopar group (75 mg/kg, per os (p.o.)); l-dopa group (60 mg/kg, p.o.); β-asarone group (15 mg/kg, p.o.); β-asarone + l-dopa co-administered group (15 mg/kg + 60 mg/kg, p.o.); 3-methyladenine group (500 nmol, intraperitoneal injection); and rapamycin group (1 mg/kg, intraperitoneal injection). Then, Beclin-1, LC3B, and p62 expression in the mesencephalon were detected. The mesencephalon was also observed by transmission electron microscope. The results showed that Beclin-1 and LC3B expression decreased and that p62 expression increased significantly in the madopar, l-dopa, β-asarone, and co-administered groups when compared with the 6-OHDA model. Beclin-1 and LC3B expression in the β-asarone and co-administered groups were less than in the madopar or l-dopa groups, whereas p62 expression in the β-asarone and co-administered groups was higher than in the madopar or l-dopa groups. In addition, a significant decrease in autophagosome was exhibited in the β-asarone and co-administered groups when compared with the 6-OHDA group. Our findings indicate that Beclin-1 and LC3B expression decreased, whereas p62 expression increased after co-administration treatment. In sum, all data suggest that the co-administration of β-asarone and l-dopa may contribute to the treatment of 6-OHDA-induced damage in rats by inhibiting autophagy activity.

  13. Dexmedetomidine Regulates 6-hydroxydopamine-Induced Microglial Polarization.

    PubMed

    Zhang, Pei; Li, Yu; Han, Xuechang; Xing, Qunzhi; Zhao, Lei

    2017-02-28

    Microglia have undergone extensive characterization and have been shown to present distinct phenotypes, such as the M1 or M2 phenotypes, depending on their stimuli. As a highly specific neurotoxin, 6-hydroxydopamine (6-OHDA) can be used to further our understanding of the immune response in Parkinson's disease (PD). Dexmedetomidine (DEX), a centrally selective α2-adrenoceptor agonist, performs very well as an anti-anxiety medication, sedative and analgesic. In the present study, we investigated the effects of DEX on 6-OHDA-induced microglial polarization. Our results indicate that treatment with 6-OHDA promotes microglial polarization toward the M1 state in BV2 microglia cells by increasing the release of interleukin (IL)-6, IL-1β, or tumor necrosis factor-α, which can be prevented by pretreatment with DEX. In addition, we found that 6-OHDA blocked IL-4-mediated microglial M2 polarization by suppressing expression of the microglial M2 markers arginase-1 (Arg-1), resistin-like α (Retnla/Fizz1), and chitinase 3-like 3 (Chi3l3/Ym1), which could be ameliorated by pretreatment with DEX. Notably, the inhibitory effects of 6-OHDA on IL-4-mediated induction of the anti-inflammatory marker genes IL-10, IL-13, and transforming growth factor-β2 could be significantly alleviated by pretreatment with DEX in a dose-dependent manner (P < 0.01). Mechanistically, alternations in the activation of signal transducer and activator of transcription 6 were involved in this process. These findings suggest that administration of DEX has the potential to interrupt the process of microgliosis in PD.

  14. High-throughput mass spectrometric cytochrome P450 inhibition screening.

    PubMed

    Lim, Kheng B; Ozbal, Can C; Kassel, Daniel B

    2013-01-01

    We describe here a high-throughput assay to support rapid evaluation of drug discovery compounds for possible drug-drug interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of eight concentrations and against a panel of six cytochrome P450 (CYP) enzymes: 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. The method utilizes automated liquid handling for sample preparation, and online solid-phase extraction/tandem mass spectrometry (SPE/MS/MS) for sample analyses. The system is capable of generating two 96-well assay plates in 30 min, and completes the data acquisition and analysis of both plates in about 30 min. Many laboratories that perform the CYP inhibition screening automate only part of the processes leaving a throughput bottleneck within the workflow. The protocols described in this chapter are aimed to streamline the entire process from assay to data acquisition and processing by incorporating automation and utilizing high-precision instrument to maximize throughput and minimize bottleneck.

  15. Inhibiting Cytochrome C Oxidase Leads to Alleviated Ischemia Reperfusion Injury

    PubMed Central

    Yang, Zhaoyun; Duan, Zhongxin; Yu, Tian; Xu, Junmei

    2017-01-01

    Background and Objectives The overall purpose of this study was to investigate the role of cytochrome C oxidase (CcO) in preventing ischemia reperfusion-induced cardiac injury through gaseous signaling molecule pathways. Materials and Methods We used CcO inhibitor, potassium cyanide (KCN) to mimic the pre-treatment of gaseous signaling molecules in a global ischemia/reperfusion (IR) injury model in rats. Intracellular reactive oxygen species (ROS) was determined by measuring mitochondrial H2O2 and mitochondrial complex activity. Results KCN pre-treatment led to decreased infarction area after IR injury and improved cardiac function. KCN pre-treated group challenged with IR injury was associated with reduced ROS production through inhibition of activity and not downregulation of CcO expression. In addition, KCN pre-treatment was associated with enhanced expression and activity of mitochondrial antioxidase, suggesting the role of CcO in regulating IR injury through oxidative stress. Conclusion KCN pre-treatment reduced the severity of IR injury. The potential mechanism could be increased endogenous anti-oxidase activity and consequently, the enhanced clearance of ROS. PMID:28382074

  16. Direct inhibitions of the activities of steroidogenic cytochrome P-450 mono-oxygenase systems by anticonvulsants.

    PubMed

    Ohnishi, T; Ichikawa, Y

    1997-01-01

    The effects of anticonvulsants on the activities of cytochromes P-450(17alpha,lyase) (CYP17), P-450arom (CYP19), P-450C21 (CYP21), P-450SCC (CYP11A1), and P-450(11beta) (CYP11B1) mono-oxygenase systems were studied using rat testicular microsomes, human placental microsomes, bovine adrenocortical microsomes, bovine adrenocortical mitochondria and purified cytochrome P-450(11beta). Phenytoin, clonazepam and carbamazepine inhibited the steroidogenesis catalysed by these cytochrome P-450 mono-oxygenase systems and the Ki values for each anticonvulsant were determined. Neither hydantoin nor sodium valproate inhibited the activities of steroidogenic cytochromes P-450. When the activities of cytochromes P-450arom and P-450C21 were measured in the presence of anticonvulsants, the Ki values (0.15 mM) for phenytoin were close to the plasma concentration of phenytoin under therapeutic conditions. Phenytoin, clonazepam and carbamazepine directly inhibited the monooxygenase activities of cytochromes P-450, because they did not affect the activities of NADPH-cytochrome P-450 reductase, NADPH-adrenoferredoxin reductase and adrenoferredoxin.

  17. Cyanide inhibition of cytochrome c oxidase. A rapid-freeze e.p.r. investigation.

    PubMed Central

    Jensen, P; Wilson, M T; Aasa, R; Malmström, B G

    1984-01-01

    The inhibition of cytochrome c oxidase by cyanide, starting either with the resting or the pulsed enzyme, was studied by rapid-freeze quenching followed by quantitative e.p.r. It is found that a partial reduction of cytochrome oxidase by transfer of 2 electron equivalents from ferrocytochrome c to cytochrome a and CuA will induce a transition from a closed to an open enzyme conformation, rendering the cytochrome a3-CuB site accessible for cyanide binding, possibly as a bridging ligand. A heterogeneity in the enzyme is observed in that an e.p.r. signal from the cytochrome a3 3+-HCN complex is only found in 20% of the molecules, whereas the remaining cyanide-bound a3-CuB sites are e.p.r.-silent. PMID:6098268

  18. Superoxide dismutase-inhibitible reduction of cytochrome c by the alloxan radical. Implications for alloxan cytotoxicity.

    PubMed Central

    Winterbourn, C C

    1982-01-01

    Cytochrome c was reduced when superoxide was generated from xanthine oxidase in the presence of alloxan, and by the reaction of alloxan and with reduced glutathione. In each case, most of the reduction was inhibited by superoxide dismutase, but considerably more enzyme was required than with superoxide alone. This indicates that the superoxide dismutase-inhibitible cytochrome c reduction was mainly due to a direct reaction with the alloxan radical, and implies that other reactions that are inhibited by superoxide dismutase could be due to either alloxan radicals or superoxide. PMID:6299273

  19. Marked inhibition of hepatic cytochrome P450 activity in cholesterol-induced atherosclerosis in rabbits.

    PubMed

    Irizar, A; Ioannides, C

    1998-04-03

    The objective of the present study was to investigate the expression of major xenobiotic-metabolising cytochrome P450 proteins, and of other enzyme systems, in hepatic and extrahepatic tissues of rabbits rendered atherosclerotic by the dietary administration of 1% cholesterol diets for 8 weeks. Individual cytochrome P450 proteins were monitored using diagnostic substrates and immunologically in Western blot analysis. The activity of all hepatic isoforms studied was depressed in the atherosclerotic animals; when, however, apoprotein levels were determined immunologically, no major differences were evident between the control and the atherosclerotic rabbits. In vitro studies indicated that neither cholesterol nor palm oil inhibited cytochrome P450 activity. The effects of cholesterol treatment leading to atherosclerosis on kidney, heart and lung cytochrome P450 activities were isoform- and tissue-specific; no change was evident in the heart activities, but in the lung and kidney cytochrome P450 activities were clearly modulated by the treatment with cholesterol. Apoprotein levels did not always parallel the changes in activities. Western blot analysis of aortic cytochromes P450 revealed that administration of cholesterol-rich diets enhanced CYP2B and CYP3A apoprotein levels. Cholesterol feeding to rabbits gave rise to a marked decrease in hepatic glutathione S-transferase activity but did not influence glutathione reductase or total glutathione levels. The same treatment had no effect on catalase, glutathione peroxidase and superoxide dismutase. It is concluded that treatment of rabbits with cholesterol-rich diets leading to atherosclerosis gives rise to profound changes in the expression of cytochrome P450 proteins in the liver and other tissues; possible mechanisms are discussed.

  20. Inhibition of rabbit nasal and hepatic cytochrome P-450-dependent hexamethylphosphoramide (HMPA) N-demethylase by methylenedioxyphenyl compounds.

    PubMed

    Dahl, A R; Brezinski, D A

    1985-03-01

    Eighteen methylenedioxyphenyl (MDP) compounds, including some commonly inhaled by people, were tested for the ability to inhibit rabbit nasal microsomal cytochrome P-450-dependent hexamethylphosphoramide (HMPA) N-demethylase. For comparison, liver microsomes were also used. Nasal cytochrome P-450 from rabbits metabolized MDP compounds to form cytochrome P-450-metabolite (P-450-MI) complexes as indicated by difference spectra in the Soret region. Several of the MDP compounds were potent inhibitors of nasal P-450-dependent N-demethylase. If inhibition of nasal P-450 also occurs in vivo after inhibiting MDP compounds are inhaled, the metabolism of concurrently or subsequently inhaled compounds may be altered.

  1. Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition

    SciTech Connect

    Ishihara, Yasuhiro; Shiba, Dai; Shimamoto, Norio . E-mail: n-shimamoto@kph.bunri-u.ac.jp

    2006-07-15

    Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as {alpha}-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.

  2. Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons

    NASA Technical Reports Server (NTRS)

    Chalmers, G. R.; Edgerton, V. R.

    1989-01-01

    The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.

  3. Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons

    NASA Technical Reports Server (NTRS)

    Chalmers, G. R.; Edgerton, V. R.

    1989-01-01

    The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.

  4. Protection against chemical-induced lung injury by inhibition of pulmonary cytochrome P-450

    SciTech Connect

    Verschoyle, R.D.; Dinsdale, D. )

    1990-04-01

    Protection afforded by trialkyl phosphorothionates against the lung injury caused by trialkyl phosphorothiolates probably results from the inhibition by the P{double bond}S moiety of the thionates, of one or more pulmonary cytochrome P-450 isozymes. The aromatic hydrocarbons p-xylene and pseudocumene also protect against this injury and inhibit some P-450 isozymes, but by a different mechanism. OOS-Trimethylphosphorothionate and p-xylene were compared as protective agents against the effect of OOS-trimethylphosphorothiolate and two other lung toxins ipomeanol and 1-nitronaphthalene that are known to be activated by cytochrome P-450. The effects of these protective compounds, in vivo, on pulmonary cytochrome P-450 activity were also determined. Both compounds inhibited pentoxyresorufin O-deethylase activity, but not ethoxyresorufin O-deethylase. The phosphorothionate was most effective against lung injury caused by the phosphorothiolates and 1-nitronaphthalene, whereas p-xylene was much more effective against ipomeanol. {beta}-Naphthoflavone, which induces pulmonary ethoxyresorufin O-deethylase activity, did not protect against phosphorothiolate or 1-nitronaphthalene injury, and it was only marginally effective in decreasing the toxicity or ipomeanol.

  5. Cytochrome P450 inhibition by three licorice species and fourteen licorice constituents.

    PubMed

    Li, Guannan; Simmler, Charlotte; Chen, Luying; Nikolic, Dejan; Chen, Shao-Nong; Pauli, Guido F; van Breemen, Richard B

    2017-07-31

    The potential of licorice dietary supplements to interact with drug metabolism was evaluated by testing extracts of three botanically identified licorice species (Glycyrrhiza glabra L., Glycyrrhiza uralensis Fish. ex DC. and Glycyrrhiza inflata Batalin) and 14 isolated licorice compounds for inhibition of 9 cytochrome P450 enzymes using a UHPLC-MS/MS cocktail assay. G. glabra showed moderate inhibitory effects against CYP2B6, CYP2C8, CYP2C9, and CYP2C19, and weak inhibition against CYP3A4 (testosterone). In contrast, G. uralensis strongly inhibited CYP2B6 and moderately inhibited CYP2C8, CYP2C9 and CYP2C19, and G. inflata strongly inhibited CYP2C enzymes and moderately inhibited CYP1A2, CYP2B6, CYP2D6, and CYP3A4 (midazolam). The licorice compounds isoliquiritigenin, licoricidin, licochalcone A, 18β-glycyrrhetinic acid, and glycycoumarin inhibited one or more members of the CYP2C family of enzymes. Glycycoumarin and licochalcone A inhibited CYP1A2, but only glycycoumarin inhibited CYP2B6. Isoliquiritigenin, glabridin and licoricidin competitively inhibited CYP3A4, while licochalcone A (specific to G. inflata roots) was a mechanism-based inhibitor. The three licorice species commonly used in botanical dietary supplements have varying potential for drug-botanical interactions as inhibitors of cytochrome P450 isoforms. Each species of licorice displays a unique profile of constituents with potential for drug interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Inhibition of rat nasal cytochrome P-450-dependent mono-oxygenase by the essence heliotropin (piperonal)

    SciTech Connect

    Not Available

    1982-01-01

    The purpose of this study was to determine if heliotropin can inhibit cytochrome P-450 mediated oxidations in both nasal and hepatic microsomes. Heliotropin was an effective inhibitor of the methylation of the common fragrance dimethyl anthranilate by nasal microsomes; little inhibition was observed in liver microsomes. If the strong inhibitory activity of heliotropin in the nose occurs in vivo, then the inhalation of heliotropin prior to or along with inhalation of other airborne substances might profoundly alter the biological fate of those substances. (RJC)

  7. Cytochrome c oxidase maintains mitochondrial respiration during partial inhibition by nitric oxide.

    PubMed

    Palacios-Callender, Miriam; Hollis, Veronica; Frakich, Nanci; Mateo, Jesús; Moncada, Salvador

    2007-01-01

    Nitric oxide (NO), generated endogenously in NO-synthase-transfected cells, increases the reduction of mitochondrial cytochrome c oxidase (CcO) at O2 concentrations ([O2]) above those at which it inhibits cell respiration. Thus, in cells respiring to anoxia, the addition of 2.5 microM L-arginine at 70 microM O2 resulted in reduction of CcO and inhibition of respiration at [O2] of 64.0+/-0.8 and 24.8+/-0.8 microM, respectively. This separation of the two effects of NO is related to electron turnover of the enzyme, because the addition of electron donors resulted in inhibition of respiration at progressively higher [O2], and to their eventual convergence. Our results indicate that partial inhibition of CcO by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO. Thus, respiration is maintained without compromising the bioenergetic status of the cell. We suggest that this is a physiological mechanism regulated by the flux of electrons in the mitochondria and by the changing ratio of O2:NO, either during hypoxia or, as a consequence of increases in NO, as a result of cell stress.

  8. Cytochrome P450 inhibition potential of new psychoactive substances of the tryptamine class.

    PubMed

    Dinger, Julia; Woods, Campbell; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

    2016-01-22

    New psychoactive substances (NPS) are not tested for their cytochrome P450 (CYP) inhibition potential before consumption. Therefore, this potential was explored for tryptamine-derived NPS (TDNPS) including alpha-methyl tryptamines (AMTs), dimethyl tryptamines (DMTs), diallyl tryptamines (DALTs), and diisopropyl tryptamines (DiPTs) using test substrates preferred by the Food and Drug Administration in a cocktail assay. All tested TDNPS with the exception of DMT inhibited CYP2D6 activity with IC50 values below 100μM. DALTs inhibited CYP2D6 activity similar to paroxetine and quinidine and CYP1A2 activity comparable to fluvoxamine. 5-Methoxy-N,N-diallyltryptamine reduced in vivo the caffeine metabolism in rats consistent with in vitro results. Five of the AMTs also inhibited CYP1A2 activity comparable to amiodarone. AMT and 6-F-AMT inhibited CYP2A6 activity in the range of the test inhibitor tranylcypromine. CYP2B6 activity was inhibited by 19 tryptamines, but weakly compared to efavirenz. CYP2C8 activity was inhibited by five of the tested TDNPS and three showed values comparable to trimethoprim and gemfibrozil. Six tryptamines inhibited CYP2C9 and seven CYP2C19 activities comparable to fluconazole and chloramphenicol, respectively. Nineteen compounds showed inhibition of CYP2E1 and 18 of CYP3A activity, respectively. These results showed that the CYP inhibition by TDNPS might be clinically relevant, but clinical studies are needed to explore this further.

  9. Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

    SciTech Connect

    Reed, James R.; Cawley, George F.; Ardoin, Taylor G.; Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W.; Backes, Wayne L.

    2014-06-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

  10. Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes.

    PubMed

    Reed, James R; Cawley, George F; Ardoin, Taylor G; Dellinger, Barry; Lomnicki, Slawomir M; Hasan, Farhana; Kiruri, Lucy W; Backes, Wayne L

    2014-06-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230°C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Proteasome inhibition compromises direct retention of cytochrome P450 2C2 in the endoplasmic reticulum.

    PubMed

    Szczesna-Skorupa, Elzbieta; Kemper, Byron

    2008-10-15

    To determine whether protein degradation plays a role in the endoplasmic reticulum (ER) retention of cytochromes P450, the effects of proteasomal inhibitors on the expression and distribution of green fluorescent protein chimeras of CYP2C2 and related proteins was examined. In transfected cells, expression levels of chimeras of full-length CYP2C2 and its cytosolic domain, but not its N-terminal transmembrane sequence, were increased by proteasomal inhibition. Redistribution of all three chimeras from the reticular ER into a perinuclear compartment and, in a subset of cells, also to the cell surface was observed after proteasomal inhibition. Redistribution was blocked by the microtubular inhibitor, nocodazole, suggesting that redistribution to the cell surface followed the conventional vesicular transport pathway. Similar redistributions were detected for BAP31, a CYP2C2 binding chaperone; CYP2E1 and CYP3A4, which are also degraded by the proteasomal pathway; and for cytochrome P450 reductase, which does not undergo proteasomal degradation; but not for the ER membrane proteins, sec61 and calnexin. Redistribution does not result from saturation of an ER retention "receptor" since in some cases protein levels were unaffected. Proteasomal inhibition may, therefore, alter ER retention by affecting a protein critical for ER retention, either directly, or indirectly by affecting the composition of the ER membranes.

  12. On the mechanism and biology of cytochrome oxidase inhibition by nitric oxide.

    PubMed

    Antunes, Fernando; Boveris, Alberto; Cadenas, Enrique

    2004-11-30

    The detailed molecular mechanism for the reversible inhibition of mitochondrial respiration by NO has puzzled investigators: The rate constants for the binding of NO and O2 to the reduced binuclear center CuB/a3 of cytochrome oxidase (COX) are similar, and NO is able to dissociate slowly from this center whereas O2 is kinetically trapped, which altogether seems to favor the complex of COX with O2 over the complex of COX with NO. Paradoxically, the inhibition of COX by NO is observed at high ratios of O2 to NO (in the 40-500 range) and is very fast (seconds or faster). In this work, we used simple mathematical models to investigate this paradox and other important biological questions concerning the inhibition of COX by NO. The results showed that all known features of the inhibition of COX by NO can be accounted for by a direct competition between NO and O2 for the reduced binuclear center CuB/a3 of COX. Besides conciliating apparently contradictory data, this work provided an explanation for the so-called excess capacity of COX by showing that the COX activity found in tissues actually is optimized to avoid an excessive inhibition of mitochondrial respiration by NO, allowing a moderate, but not excessive, overlap between the roles of NO in COX inhibition and in cellular signaling. In pathological situations such as COX-deficiency diseases and chronic inflammation, an excessive inhibition of the mitochondrial respiration is predicted.

  13. On the mechanism and biology of cytochrome oxidase inhibition by nitric oxide

    PubMed Central

    Antunes, Fernando; Boveris, Alberto; Cadenas, Enrique

    2004-01-01

    The detailed molecular mechanism for the reversible inhibition of mitochondrial respiration by NO has puzzled investigators: The rate constants for the binding of NO and O2 to the reduced binuclear center CuB/a3 of cytochrome oxidase (COX) are similar, and NO is able to dissociate slowly from this center whereas O2 is kinetically trapped, which altogether seems to favor the complex of COX with O2 over the complex of COX with NO. Paradoxically, the inhibition of COX by NO is observed at high ratios of O2 to NO (in the 40–500 range) and is very fast (seconds or faster). In this work, we used simple mathematical models to investigate this paradox and other important biological questions concerning the inhibition of COX by NO. The results showed that all known features of the inhibition of COX by NO can be accounted for by a direct competition between NO and O2 for the reduced binuclear center CuB/a3 of COX. Besides conciliating apparently contradictory data, this work provided an explanation for the so-called excess capacity of COX by showing that the COX activity found in tissues actually is optimized to avoid an excessive inhibition of mitochondrial respiration by NO, allowing a moderate, but not excessive, overlap between the roles of NO in COX inhibition and in cellular signaling. In pathological situations such as COX-deficiency diseases and chronic inflammation, an excessive inhibition of the mitochondrial respiration is predicted. PMID:15546991

  14. Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly

    PubMed Central

    Graumann, Kristin; Schaumburg, Frieder; Reubold, Thomas F.; Hippe, Diana; Eschenburg, Susanne; Lüder, Carsten G. K.

    2015-01-01

    Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome c in vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway. PMID:28357287

  15. Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

    PubMed

    Hidaka, Muneaki; Fujita, Ken-ichi; Ogikubo, Tetsuya; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Arimori, Kazuhiko

    2004-06-01

    There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro.

  16. Molecular Modeling Analysis of the Inhibition of Mitochondrial Cytochrome BC1 Complex Activity by Tocol Derivatives

    NASA Astrophysics Data System (ADS)

    Singh, Awantika; Hauer-Jensen, Martin; Compadre, Cesar M.; Kumar, K. Sree

    2011-06-01

    The biological functions of vitamin E related compounds have been of interest in biomedical research for several decades. Among those compounds, α-, β-, δ-, and γ-tocopherols and their oxidation products, α-, β-, δ-, γ-tocopherylquinone and their analogs α-TQo, γ-TQo, TMC20 and TMC40 were recently shown to inhibit the mitochondrial cytochrome bc1 complex. In this investigation the effects of the structural variation on the inhibition of the mitochondrial cytochrome bc1 complex were analyzed using Comparative Molecular Field Analysis (CoMFA). CoMFA performed using steric and electrostatic molecular fields produced a very good correlation. The best CoMFA models were obtained using the manual alignment of 12 compounds with 5 components (q2 = 0.589, SPRESS = 0.515, r2 = 0.992, s = 0.068 and F value = 156.520). The resulting contour maps produced by the best CoMFA model were helpful in identifying the structural features required for the biological activity of compounds under study. These results would be helpful for predicting the activity of new compounds, and they could be used for guiding the design, synthesis and development of new and more effective agents.

  17. Inhibition of human cytochrome P450 enzymes by hops (Humulus lupulus) and hop prenylphenols

    PubMed Central

    Nikolić, Dejan; Chen, Shao-Nong; Huang, Ke; Li, Guannan; Pauli, Guido F.; van Breemen, Richard B.

    2014-01-01

    As hops (Humulus lupulus L.) are used in the brewing of beer and by menopausal women as estrogenic dietary supplements, the potential for hop extracts and hop constituents to cause drug-botanical interactions by inhibiting human cytochrome P450 enzymes was investigated. Inhibition of major human cytochrome P450 enzymes by a standardized hop extract and isolated hop prenylated phenols was evaluated using a fast and efficient assay based on ultrahigh pressure liquid chromatography-tandem mass spectrometry. The hop extract at 5 μg/mL inhibited CYP2C8 (93%), CYP2C9 (88%), CYP2C19 (70%), and CYP1A2 (27%) with IC50 values of 0.8, 0.9, 3.3, and 9.4 μg/mL, respectively, but time-dependent inactivation was observed only for CYP1A2. Isoxanthohumol from hops was the most potent inhibitor of CYP2C8 with an IC50 of 0.2 μM, whereas 8-prenylnaringenin was the most potent inhibitor of CYP1A2, CYP2C9 and CYP2C19 with IC50 values of 1.1 μM, 1.1 μM and 0.4 μM, respectively. Extracts of hops contain prenylated compounds such as the flavanones isoxanthohumol and 8-prenylnaringenin and the chalcone xanthohumol that can inhibit CYP450s, especially the CYP2C family, which may affect the efficacy and safety of some CYP2C substrate drugs when co-administered. PMID:24342125

  18. Inhibition of human cytochrome P450 enzymes by hops (Humulus lupulus) and hop prenylphenols.

    PubMed

    Yuan, Yang; Qiu, Xi; Nikolić, Dejan; Chen, Shao-Nong; Huang, Ke; Li, Guannan; Pauli, Guido F; van Breemen, Richard B

    2014-03-12

    As hops (Humulus lupulus L.) are used in the brewing of beer and by menopausal women as estrogenic dietary supplements, the potential for hop extracts and hop constituents to cause drug-botanical interactions by inhibiting human cytochrome P450 enzymes was investigated. Inhibition of major human cytochrome P450 enzymes by a standardized hop extract and isolated hop prenylated phenols was evaluated using a fast and efficient assay based on ultrahigh pressure liquid chromatography-tandem mass spectrometry. The hop extract at 5 μg/mL inhibited CYP2C8 (93%), CYP2C9 (88%), CYP2C19 (70%), and CYP1A2 (27%) with IC50 values of 0.8, 0.9, 3.3, and 9.4 μg/mL, respectively, but time-dependent inactivation was observed only for CYP1A2. Isoxanthohumol from hops was the most potent inhibitor of CYP2C8 with an IC50 of 0.2 μM, whereas 8-prenylnaringenin was the most potent inhibitor of CYP1A2, CYP2C9 and CYP2C19 with IC50 values of 1.1 μM, 1.1 μM and 0.4 μM, respectively. Extracts of hops contain prenylated compounds such as the flavanones isoxanthohumol and 8-prenylnaringenin and the chalcone xanthohumol that can inhibit CYP450s, especially the CYP2C family, which may affect the efficacy and safety of some CYP2C substrate drugs when co-administered.

  19. Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities.

    PubMed

    Kopečná-Zapletalová, Michaela; Krasulová, Kristýna; Anzenbacher, Pavel; Hodek, Petr; Anzenbacherová, Eva

    2017-04-01

    1. The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically. 2. The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with Ki of 35.95 ± 6.96 and 60.56 ± 3.53 μmol/l and CYP3A4 (inhibited by genistein with Ki of 23.25 ± 5.85 μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (Ki of 57.69 ± 2.36 μmol/l) and equol (Ki of 38.47 ± 2.32 μmol/l). 3. Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.

  20. Use of Human Plasma Samples to Identify Circulating Drug Metabolites that Inhibit Cytochrome P450 Enzymes.

    PubMed

    Eng, Heather; Obach, R Scott

    2016-08-01

    Drug interactions elicited through inhibition of cytochrome P450 (P450) enzymes are important in pharmacotherapy. Recently, greater attention has been focused on not only parent drugs inhibiting P450 enzymes but also on possible inhibition of these enzymes by circulating metabolites. In this report, an ex vivo method whereby the potential for circulating metabolites to be inhibitors of P450 enzymes is described. To test this method, seven drugs and their known plasma metabolites were added to control human plasma at concentrations previously reported to occur in humans after administration of the parent drug. A volume of plasma for each drug based on the known inhibitory potency and time-averaged concentration of the parent drug was extracted and fractionated by high-pressure liquid chromatography-mass spectrometry, and the fractions were tested for inhibition of six human P450 enzyme activities (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Observation of inhibition in fractions that correspond to the retention times of metabolites indicates that the metabolite has the potential to contribute to P450 inhibition in vivo. Using this approach, norfluoxetine, hydroxyitraconazole, desmethyldiltiazem, desacetyldiltiazem, desethylamiodarone, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion were identified as circulating metabolites that inhibit P450 activities at a similar or greater extent as the parent drug. A decision tree is presented outlining how this method can be used to determine when a deeper investigation of the P450 inhibition properties of a drug metabolite is warranted. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  1. An improved substrate cocktail for assessing direct inhibition and time-dependent inhibition of multiple cytochrome P450s

    PubMed Central

    Chen, Zhong-hua; Zhang, Su-xing; Long, Na; Lin, Li-shan; Chen, Tao; Zhang, Fei-peng; Lv, Xue-qin; Ye, Pei-zhen; Li, Ning; Zhang, Ke-zhi

    2016-01-01

    Aim: The substrate cocktail is frequently used to evaluate cytochrome P450 (CYP) enzyme-mediated drug interactions and potential interactions among the probe substrates. Here, we re-optimized the substrate cocktail method to increase the reliability and accuracy of screening for candidate compounds and expanded the method from a direct CYP inhibition assay to a time-dependent inhibition (TDI) assay. Methods: In the reaction mixtures containing human liver microsome (0.1 mg/mL), both the concentrations of a substrate cocktail (phenacetin for 1A2, coumarin for 2A6, bupropion for 2B6, diclofenac for 2C9, dextromethorphan for 2D6, and testosterone for 3A4) and the incubation time were optimized. Metabolites of the substrate probes were simultaneously analyzed by multiple-reaction monitoring (MRM) using a routine LC/MS/MS. Direct CYP inhibition was validated using 7 inhibitors (α-naphthoflavone, tranylcypromine, ticlopidine, fluconazole, quinidine, ketoconazole and 1-ABT). The time-dependent inhibition was partially validated with 5 inhibitors (ketoconazole, verapamil, quinidine, paroxetine and 1-ABT). Results: The inhibition curve profiles and IC50 values of 7 CYP inhibitors were approximate when a single substrate and the substrate cocktail were tested, and were consistent with the previously reported values. Similar results were obtained in the IC50 shifts of 5 inhibitors when a single substrate and the substrate cocktail were tested in the TDI assay. Conclusion: The 6-in-1 substrate cocktail (for 1A2, 2A6, 2B6, 2C9, 2D6 and 3A) is reliable for assessing CYP inhibition and time-dependent inhibition of drug candidates. PMID:27063220

  2. Selective inhibition of the cytochrome P450 isoform by hyperoside and its potent inhibition of CYP2D6.

    PubMed

    Song, Min; Hong, Miri; Lee, Min Young; Jee, Jun-Goo; Lee, You Mie; Bae, Jong-Sup; Jeong, Tae Cheon; Lee, Sangkyu

    2013-09-01

    Hyperoside, quercetin-3-O-galactoside, is a flavonoid isolated from Oenanthe javanica. In the present study, we investigated potential herb-drug inhibitory effects of hyperoside on nine cytochrome P450 (CYP) isoforms in pooled human liver microsomes (HLMs) and human recombinant cDNA expressed CYP using a cocktail probe assay. Hyperoside strongly inhibited CYP2D6-catalyzed dextromethorphan O-demethylation, with IC₅₀ values of 1.2 and 0.81 μM after 0 and 15 min of preincubation, and a Ki value of 2.01 μM in HLMs, respectively. Hyperoside strongly decreased CYP2D6 activity dose-, but not time-, dependently in HLMs. In addition, the Lineweaver-Burk and Secondary plots for the inhibition of CYP2D6 in HLMs fitted a competitive inhibition mode. Furthermore, hyperoside decreased CYP2D6-catalyzed dextromethorphan O-demethylation activity of human recombinant cDNA-expressed CYP2D6, with an IC₅₀ value of 3.87 μM. However, other CYPs were not inhibited significantly by hyperoside. In conclusion, our data demonstrate that hyperoside is a potent selective CYP2D6 inhibitor in HLMs, and suggest that hyperoside might cause herb-drug interactions when co-administrated with CYP2D substrates.

  3. Development of an in vitro cytochrome P450 cocktail inhibition assay for assessing the inhibition risk of drugs of abuse.

    PubMed

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2014-10-01

    Drugs of abuse are not tested for cytochrome P450 (CYP) inhibition potential before distribution. Therefore, a cocktail assay should be developed for testing the inhibition potential for all relevant CYPs. The following CYP test substrates and selective inhibitors were incubated in pooled human liver microsomes: phenacetin (alpha-naphthoflavone for CYP1A2), coumarin (tranylcypromine, CYP2A6), bupropion (sertraline, CYP2B6), amodiaquine (trimethoprim, CYP2C8), diclofenac (sulfaphenazole, CYP2C9), omeprazole (fluconazole, CYP2C19), dextromethorphan (quinidine, CYP2D6), chlorzoxazone (clomethiazole, CYP2E1), testosterone (verapamil, CYP3A). Samples were analyzed after protein precipitation using a Thermo Fisher Q-Exactive LC-high-resolution-MS/MS. The IC50 values were calculated by plotting the concentration of the formed metabolite, relative to the control sample, over the logarithm of the inhibitor concentration. They were determined either for single substrate or the cocktail incubation. Unfortunately, the cocktail assay had to be split because of interferences during incubation caused by substrates or metabolites, but the mixture of both incubates could be analyzed in one analytical run. The IC50 values determined in the single substrate or both cocktail incubations were comparable among themselves and with published data. In conclusion, the new inhibition cocktail assay was reproducible and applicable for testing the inhibition potential of drugs of abuse as exemplified for 2,5-dimethoxy-4-iodo-amfetamine (DOI). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. The curious case of benzbromarone: insight into super-inhibition of cytochrome P450.

    PubMed

    Parashar, Abhinav; Gade, Sudeep Kumar; Potnuru, Mahesh; Madhavan, Nandita; Manoj, Kelath Murali

    2014-01-01

    Cytochrome P450 (CYP) family of redox enzymes metabolize drugs and xenobiotics in liver microsomes. Isozyme CYP2C9 is reported to be inhibited by benzbromarone (BzBr) and this phenomenon was hitherto explained by classical active-site binding. Theoretically, it was impossible to envisage the experimentally derived sub-nM Ki for an inhibitor, when supra-nM enzyme and 10X KM substrate concentrations were employed. We set out to find a more plausible explanation for this highly intriguing "super-inhibition" phenomenon. In silico docking of various BzBr analogs with known crystal structure of CYP2C9 did not provide any evidence in support of active-site based inhibition hypothesis. Experiments tested the effects of BzBr and nine analogs on CYPs in reconstituted systems of lab-purified proteins, complex baculosomes & crude microsomal preparations. In certain setups, BzBr and its analogs could even enhance reactions, which cannot be explained by an active site hypothesis. Generally, it was seen that Ki became smaller by orders of magnitude, upon increasing the dilution order of BzBr analogs. Also, it was seen that BzBr could also inhibit other CYP isozymes like CYP3A4, CYP2D6 and CYP2E1. Further, amphipathic derivatives of vitamins C & E (scavengers of diffusible reactive oxygen species or DROS) effectively inhibited CYP2C9 reactions in different reaction setups. Therefore, the inhibition of CYP activity by BzBr analogs (which are also surface-active redox agents) is attributed to catalytic scavenging of DROS at phospholipid interface. The current work expands the scope of interpretations of inhibitions in redox enzymes and ushers in a new cellular biochemistry paradigm that small amounts of DROS may be obligatorily required in routine redox metabolism for constructive catalytic roles.

  5. Assessment of cytochrome p450 enzyme inhibition and inactivation in drug discovery and development.

    PubMed

    Nettleton, David O; Einolf, Heidi J

    2011-01-01

    Evaluation of the potential of a drug candidate to inhibit or inactivate cytochrome P450 (CYP) enzymes remains an important part of pharmaceutical drug Discovery and Development programs. CYP enzymes are considered to be one of the most important enzyme families involved in the metabolic clearance of the vast majority of prescribed drugs. Clinical drug-drug interactions (DDI) involving inhibition or time-dependent inactivation of these enzymes can result in dangerous side effects resulting from reduced clearance/increased exposure of the drug being affected (the 'victim' drug). In this regard, pharmaceutical companies have become quite vigilant in mitigating CYP inhibition/inactivation liabilities of drug candidates early in Discovery including continued risk assessment throughout Development. In this review, common strategies and decision making processes for the assessment of DDI risk in the different stages of pharmaceutical development are discussed. In addition, in vitro study designs, analysis, and interpretation of CYP inhibition and inactivation data are described in stage appropriate context. The in vitro tools and knowledge available now enable the Discovery Chemist to place the potential CYP DDI liability of a drug candidate into perspective and to aid in the optimization of chemical drug design to further mitigate this risk.

  6. Inhibition of human cytochrome P450 enzymes by the natural hepatotoxin safrole.

    PubMed

    Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw

    2005-05-01

    The hepatotoxin, safrole is a methylenedioxy phenyl compound, found in sassafras oil and certain other essential oils. Recombinant cytochrome P450 (CYP, P450) and human liver microsomes were studied to investigate the selective inhibitory effects of safrole on human P450 enzymes and the mechanisms of action. Using Escherichia coli-expressed human P450, our results demonstrated that safrole was a non-selective inhibitor of CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 in the IC(50) order CYP2E1 < CYP1A2 < CYP2A6 < CYP3A4 < CYP2D6. Safrole strongly inhibited CYP1A2, CYP2A6, and CYP2E1 activities with IC(50) values less than 20 microM. Safrole caused competitive, non-competitive, and non-competitive inhibition of CYP1A2, CYP2A6 and CYP2E1 activities, respectively. The inhibitor constants were in the order CYP1A2 < CYP2E1 < CYP2A6. In human liver microsomes, 50 microM safrole strongly inhibited 7-ethoxyresorufin O-deethylation, coumarin hydroxylation, and chlorzoxazone hydroxylation activities. These results revealed that safrole was a potent inhibitor of human CYP1A2, CYP2A6, and CYP2E1. With relatively less potency, CYP2D6 and CYP3A4 were also inhibited.

  7. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4.

    PubMed

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S; Zhou, Ruhong; Fadeel, Bengt

    2016-02-22

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

  8. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    PubMed Central

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-01-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743

  9. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    NASA Astrophysics Data System (ADS)

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-02-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

  10. Cyanide inhibition and pyruvate-induced recovery of cytochrome c oxidase.

    PubMed

    Nůsková, Hana; Vrbacký, Marek; Drahota, Zdeněk; Houštěk, Josef

    2010-10-01

    The mechanism of cyanide's inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ(m)) and the enzyme affinity to oxygen (p₅₀ value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60-80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects.

  11. Inhibition of acetylcholinesterase and cytochrome oxidase activity in Fasciola gigantica cercaria by phytoconstituents.

    PubMed

    Sunita, Kumari; Habib, Maria; Kumar, P; Singh, Vinay Kumar; Husain, Syed Akhtar; Singh, D K

    2016-02-01

    Fasciolosis is an important cattle and human disease caused by Fasciola hepatica and Fasciola gigantica. One of the possible methods to control this problem is to interrupt the life cycle of Fasciola by killing its larva (redia and cercaria) in host snail. Molecular identification of cercaria larva of F. gigantica was done by comparing the nucleotide sequencing with adult F. gigantica. It was noted that nucleotide sequencing of cercaria larva and adult F. gigantica were 99% same. Every month during the year 2011-2012, in vivo treatment with 60% of 4 h LC50 of phyto cercaricides citral, ferulic acid, umbelliferone, azadirachtin and allicin caused significant inhibition of acetylcholinesterase (AChE) and cytochrome oxidase activity in the treated cercaria larva of F. gigantica. Whereas, activity of both enzymes were not significantly altered in the nervous tissues of vector snail Lymnaea acuminata exposed to same treatments. Maximum reduction in AChE (1.35% of control in month of June) and cytochrome oxidase (3.71% of control in the month of July) activity were noted in the cercaria exposed to 60% of 4 h LC50 of azadirachtin and allicin, respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo'' to inhibition by the carbon monoxide-releasing molecule, CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration.

    PubMed

    Jesse, Helen E; Nye, Tacita L; McLean, Samantha; Green, Jeffrey; Mann, Brian E; Poole, Robert K

    2013-09-01

    CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO - a critical gasotransmitter - in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli - cytochrome bd-I, cytochrome bd-II and cytochrome bo', to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (Km 0.24μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo'. Cytochromes bo' and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. Rational design

  13. Inhibition of Peroxidase Activity of Cytochrome c: De Novo Compound Discovery and Validation.

    PubMed

    Bakan, Ahmet; Kapralov, Alexandr A; Bayir, Hulya; Hu, Feizhou; Kagan, Valerian E; Bahar, Ivet

    2015-09-01

    Cytochrome c (cyt c) release from mitochondria is accepted to be the point of no return for eliciting a cascade of interactions that lead to apoptosis. A strategy for containing sustained apoptosis is to reduce the mitochondrial permeability pore opening. Pore opening is enhanced by peroxidase activity of cyt c gained upon its complexation with cardiolipin in the presence of reactive oxygen species. Blocking access to the heme group has been proposed as an effective intervention method for reducing, if not eliminating, the peroxidase activity of cyt c. In the present study, using a combination of druggability simulations, pharmacophore modeling, virtual screening, and in vitro fluorescence measurements to probe peroxidase activity, we identified three repurposable drugs and seven compounds that are validated to effectively inhibit the peroxidase activity of cyt c. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  14. Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation

    PubMed Central

    Acin-Perez, Rebeca; Gatti, Domenico L.; Bai, Yidong; Manfredi, Giovanni

    2011-01-01

    Summary Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intra-mitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes, and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage. PMID:21641552

  15. In vitro inhibition and induction of human liver cytochrome p450 enzymes by milnacipran.

    PubMed

    Paris, Brandy L; Ogilvie, Brian W; Scheinkoenig, Julie A; Ndikum-Moffor, Florence; Gibson, Remi; Parkinson, Andrew

    2009-10-01

    Milnacipran (Savella) inhibits both norepinephrine and serotonin reuptake and is distinguished by a nearly 3-fold greater potency in inhibiting norepinephrine reuptake in vitro compared with serotonin. We evaluated the ability of milnacipran to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, milnacipran did not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19, or 2D6 (IC(50) >or= 100 microM); whereas, a comparator with dual reuptake properties [duloxetine (Cymbalta)] inhibited CYP2D6 (IC(50) = 7 microM) and CYP2B6 (IC(50) = 15 microM) with a relatively high potency. Milnacipran inhibited CYP3A4/5 in a substrate-dependent manner (i.e., midazolam 1'-hydroxylation IC(50) approximately 30 microM; testosterone 6beta-hydroxylation IC(50) approximately 100 microM); whereas, duloxetine inhibited both CYP3A4/5 activities with equal potency (IC(50) = 37 and 38 microM, respectively). Milnacipran produced no time-dependent inhibition (<10%) of P450 activity, whereas duloxetine produced time-dependent inhibition of CYP1A2, 2B6, 2C19, and 3A4/5. To evaluate P450 induction, freshly isolated human hepatocytes (n = 3) were cultured and treated once daily for 3 days with milnacipran (3, 10, and 30 microM), after which microsomal P450 activities were measured. Whereas positive controls (omeprazole, phenobarbital, and rifampin) caused anticipated P450 induction, milnacipran had minimal effect on CYP1A2, 2C8, 2C9, or 2C19 activity. The highest concentration of milnacipran (30 microM; >10 times plasma C(max)) produced 2.6- and 2.2-fold increases in CYP2B6 and CYP3A4/5 activity (making it 26 and 34% as effective as phenobarbital and rifampin, respectively). Given these results, milnacipran is not expected to cause clinically significant P450 inhibition or induction.

  16. Increased inhibition of cytochrome P450 3A4 with the tablet formulation of posaconazole.

    PubMed

    Petitcollin, A; Crochette, R; Tron, C; Verdier, M-C; Boglione-Kerrien, C; Vigneau, C; Bellissant, E; Lemaitre, F

    2016-10-01

    Being a substrate of the cytochrome P450 3A4 (CYP3A4) isoenzyme, sirolimus metabolism is decreased when posaconazole is administered concomitantly. However, because of the poor bioavailability of the oral suspension of posaconazole with which low plasma concentrations are obtained, CYP3A4 inhibition is weak and a 50-75% dose reduction of sirolimus is sufficient to avoid sirolimus overdosage. The new tablet formulation allows reaching posaconazole concentrations 3-4 fold higher than those obtained with the oral suspension. Based on a case of sirolimus overdosage following posaconazole tablets administration, we modelled the inhibition of sirolimus clearance by posaconazole, and then simulated several dosage regimens of sirolimus taken together with posaconazole tablets. We were able to describe well the interaction, and found a value of IC50 of posaconazole towards sirolimus clearance of 0.68 μg/mL. The simulations showed that even a 80% decrease of the daily dose of sirolimus is unsuitable in many cases with trough concentrations of posaconazole of 2 μg/mL. A decrease of 40% of the dose with spacing administrations of 3 days may be considered. The clinicians and pharmacologists must be warned that the use of posaconazole tablets may result in an inhibition of CYP3A4 of greater magnitude than with the oral suspension.

  17. Clinical outcomes and management of mechanism-based inhibition of cytochrome P450 3A4

    PubMed Central

    Zhou, Shufeng; Chan, Eli; Li, Xiaotian; Huang, Min

    2005-01-01

    Mechanism-based inhibition of cytochrome P450 (CYP) 3A4 is characterized by NADPH-, time-, and concentration-dependent enzyme inactivation, occurring when some drugs are converted by CYPs to reactive metabolites. Such inhibition of CYP3A4 can be due to the chemical modification of the heme, the protein, or both as a result of covalent binding of modified heme to the protein. The inactivation of CYP3A4 by drugs has important clinical significance as it metabolizes approximately 60% of therapeutic drugs, and its inhibition frequently causes unfavorable drug–drug interactions and toxicity. The clinical outcomes due to CYP3A4 inactivation depend on many factors associated with the enzyme, drugs, and patients. Clinical professionals should adopt proper approaches when using drugs that are mechanism-based CYP3A4 inhibitors. These include early identification of drugs behaving as CYP3A4 inactivators, rational use of such drugs (eg, safe drug combination regimen, dose adjustment, or discontinuation of therapy when toxic drug interactions occur), therapeutic drug monitoring, and predicting the risks for potential drug–drug interactions. A good understanding of CYP3A4 inactivation and proper clinical management are needed by clinical professionals when these drugs are used. PMID:18360537

  18. Inhibition of Cytochrome P450 by Propolis in Human Liver Microsomes

    PubMed Central

    Ryu, Chang Seon; Oh, Soo Jin; Oh, Jung Min; Lee, Ji-Yoon; Lee, Sang Yoon; Chae, Jung-woo; Kwon, Kwang-il; Kim, Sang Kyum

    2016-01-01

    Although propolis is one of the most popular functional foods for human health, there have been no comprehensive studies of herb-drug interactions through cytochrome P450 (CYP) inhibition. The purpose of this study was to determine the inhibitory effects of propolis on the activities of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 using pooled human liver microsomes (HLMs). Propolis inhibited CYP1A2, CYP2E1 and CYP2C19 with an IC50 value of 6.9, 16.8, and 43.1 μg/mL, respectively, whereas CYP2A6, 2B6, 2C9, 2D6, and 3A4 were unaffected. Based on half-maximal inhibitory concentration shifts between microsomes incubated with and without nicotinamide adenine dinucleotide phosphate, propolis-induced CYP1A2, CYP2C19, and CYP2E1 inhibition was metabolism-independent. To evaluate the interaction potential between propolis and therapeutic drugs, the effects of propolis on metabolism of duloxetine, a serotonin-norepinephrine reuptake inhibitor, were determined in HLMs. CYP1A2 and CYP2D6 are involved in hydroxylation of duloxetine to 4-hydroxy duloxetine, the major metabolite, which was decreased following propolis addition in HLMs. These results raise the possibility of interactions between propolis and therapeutic drugs metabolized by CYP1A2. PMID:27437087

  19. Cytochrome P450/ABC transporter inhibition simultaneously enhances ivermectin pharmacokinetics in the mammal host and pharmacodynamics in Anopheles gambiae.

    PubMed

    Chaccour, Carlos J; Hammann, Felix; Alustiza, Marta; Castejon, Sandra; Tarimo, Brian B; Abizanda, Gloria; Irigoyen Barrio, Ángel; Martí Soler, Helena; Moncada, Rafael; Bilbao, José Ignacio; Aldaz, Azucena; Maia, Marta; Del Pozo, José Luis

    2017-08-17

    Mass administration of endectocides, drugs that kill blood-feeding arthropods, has been proposed as a complementary strategy to reduce malaria transmission. Ivermectin is one of the leading candidates given its excellent safety profile. Here we provide proof that the effect of ivermectin can be boosted at two different levels by drugs inhibiting the cytochrome or ABC transporter in the mammal host and the target mosquitoes. Using a mini-pig model, we show that drug-mediated cytochrome P450/ABC transporter inhibition results in a 3-fold increase in the time ivermectin remains above mosquito-killing concentrations. In contrast, P450/ABC transporter induction with rifampicin markedly impaired ivermectin absorption. The same ketoconazole-mediated cytochrome/ABC transporter inhibition also occurs outside the mammal host and enhances the mortality of Anopheles gambiae. This was proven by using the samples from the mini-pig experiments to conduct an ex-vivo synergistic bioassay by membrane-feeding Anopheles mosquitoes. Inhibiting the same cytochrome/xenobiotic pump complex in two different organisms to simultaneously boost the pharmacokinetic and pharmacodynamic activity of a drug is a novel concept that could be applied to other systems. Although the lack of a dose-response effect in the synergistic bioassay warrants further exploration, our study may have broad implications for the control of parasitic and vector-borne diseases.

  20. QSAR modeling of in vitro inhibition of cytochrome P450 3A4.

    PubMed

    Mao, Boryeu; Gozalbes, Rafael; Barbosa, Frédérique; Migeon, Jacques; Merrick, Sandra; Kamm, Kelly; Wong, Eric; Costales, Chester; Shi, Wei; Wu, Cheryl; Froloff, Nicolas

    2006-01-01

    We report the QSAR modeling of cytochrome P450 3A4 (CYP3A4) enzyme inhibition using four large data sets of in vitro data. These data sets consist of marketed drugs and drug-like compounds all tested in four assays measuring the inhibition of the metabolism of four different substrates by the CYP3A4 enzyme. The four probe substrates are benzyloxycoumarin, testosterone, benzyloxyresorufin, and midazolam. We first show that using state-of-the-art QSAR modeling approaches applied to only one of these four data sets does not lead to predictive models that would be useful for in silico filtering of chemical libraries. We then present the development and the testing of a multiple pharmacophore hypothesis (MPH) that is formulated as a conceptual extension of the traditional QSAR approach to modeling the promiscuous binding of a large variety of drugs to CYP3A4. In the simplest form, the MPH approach takes advantage of the multiple substrate data sets and identifies the binding of test compounds as either proximal or distal relative to that of a given substrate. Application of the approach to the in silico filtering of test compounds for potential inhibitors of CYP3A4 is also presented. In addition to an improvement in the QSAR modeling for the inhibition of CYP3A4, the results from this modeling approach provide structural insights into the drug-enzyme interactions. The existence of multiple inhibition data sets in the BioPrint database motivates the original development of the concept of a multiple pharmacophore hypothesis and provides a unique opportunity for formulating alternative strategies of QSAR modeling of the inhibition of the in vitro metabolism of CYP3A4.

  1. Inhibition of human cytochrome P450 enzymes by Bacopa monnieri standardized extract and constituents.

    PubMed

    Ramasamy, Seetha; Kiew, Lik Voon; Chung, Lip Yong

    2014-02-24

    Bacopa monnieri and the constituents of this plant, especially bacosides, possess various neuropharmacological properties. Like drugs, some herbal extracts and the constituents of their extracts alter cytochrome P450 (CYP) enzymes, causing potential herb-drug interactions. The effects of Bacopa monnieri standardized extract and the bacosides from the extract on five major CYP isoforms in vitro were analyzed using a luminescent CYP recombinant human enzyme assay. B. monnieri extract exhibited non-competitive inhibition of CYP2C19 (IC50/Ki = 23.67/9.5 µg/mL), CYP2C9 (36.49/12.5 µg/mL), CYP1A2 (52.20/25.1 µg/mL); competitive inhibition of CYP3A4 (83.95/14.5 µg/mL) and weak inhibition of CYP2D6 (IC50 = 2061.50 µg/mL). However, the bacosides showed negligible inhibition of the same isoforms. B. monnieri, which is orally administered, has a higher concentration in the gut than the liver; therefore, this herb could exhibit stronger inhibition of intestinal CYPs than hepatic CYPs. At an estimated gut concentration of 600 µg/mL (based on a daily dosage of 300 mg/day), B. monnieri reduced the catalytic activities of CYP3A4, CYP2C9 and CYP2C19 to less than 10% compared to the total activity (without inhibitor = 100%). These findings suggest that B. monnieri extract could contribute to herb-drug interactions when orally co-administered with drugs metabolized by CYP1A2, CYP3A4, CYP2C9 and CYP2C19.

  2. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    SciTech Connect

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and {beta}-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from {beta}-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  3. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    PubMed Central

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2015-01-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450’s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and β-naphthoflavone treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from β-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury. PMID:20561902

  4. Gallium-Protoporphyrin IX Inhibits Pseudomonas aeruginosa Growth by Targeting Cytochromes

    PubMed Central

    Hijazi, Sarah; Visca, Paolo; Frangipani, Emanuela

    2017-01-01

    Pseudomonas aeruginosa is a challenging pathogen due to both innate and acquired resistance to antibiotics. It is capable of causing a variety of infections, including chronic lung infection in cystic fibrosis (CF) patients. Given the importance of iron in bacterial physiology and pathogenicity, iron-uptake and metabolism have become attractive targets for the development of new antibacterial compounds. P. aeruginosa can acquire iron from a variety of sources to fulfill its nutritional requirements both in the environment and in the infected host. The adaptation of P. aeruginosa to heme iron acquisition in the CF lung makes heme utilization pathways a promising target for the development of new anti-Pseudomonas drugs. Gallium [Ga(III)] is an iron mimetic metal which inhibits P. aeruginosa growth by interfering with iron-dependent metabolism. The Ga(III) complex of the heme precursor protoporphyrin IX (GaPPIX) showed enhanced antibacterial activity against several bacterial species, although no inhibitory effect has been reported on P. aeruginosa. Here, we demonstrate that GaPPIX is indeed capable of inhibiting the growth of clinical P. aeruginosa strains under iron-deplete conditions, as those encountered by bacteria during infection, and that GaPPIX inhibition is reversed by iron. Using P. aeruginosa PAO1 as model organism, we show that GaPPIX enters cells through both the heme-uptake systems has and phu, primarily via the PhuR receptor which plays a crucial role in P. aeruginosa adaptation to the CF lung. We also demonstrate that intracellular GaPPIX inhibits the aerobic growth of P. aeruginosa by targeting cytochromes, thus interfering with cellular respiration. PMID:28184354

  5. Fenofibrate Inhibits Cytochrome P450 Epoxygenase 2C Activity to Suppress Pathological Ocular Angiogenesis.

    PubMed

    Gong, Yan; Shao, Zhuo; Fu, Zhongjie; Edin, Matthew L; Sun, Ye; Liegl, Raffael G; Wang, Zhongxiao; Liu, Chi-Hsiu; Burnim, Samuel B; Meng, Steven S; Lih, Fred B; SanGiovanni, John Paul; Zeldin, Darryl C; Hellström, Ann; Smith, Lois E H

    2016-11-01

    Neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related-macular-degeneration are major causes of blindness. Fenofibrate treatment in type 2 diabetes patients reduces progression of diabetic retinopathy independent of its peroxisome proliferator-activated receptor (PPAR)α agonist lipid lowering effect. The mechanism is unknown. Fenofibrate binds to and inhibits cytochrome P450 epoxygenase (CYP)2C with higher affinity than to PPARα. CYP2C metabolizes ω-3 long-chain polyunsaturated fatty acids (LCPUFAs). While ω-3 LCPUFA products from other metabolizing pathways decrease retinal and choroidal neovascularization, CYP2C products of both ω-3 and ω-6 LCPUFAs promote angiogenesis. We hypothesized that fenofibrate inhibits retinopathy by reducing CYP2C ω-3 LCPUFA (and ω-6 LCPUFA) pro-angiogenic metabolites. Fenofibrate reduced retinal and choroidal neovascularization in PPARα-/-mice and augmented ω-3 LCPUFA protection via CYP2C inhibition. Fenofibrate suppressed retinal and choroidal neovascularization in mice overexpressing human CYP2C8 in endothelial cells and reduced plasma levels of the pro-angiogenic ω-3 LCPUFA CYP2C8 product, 19,20-epoxydocosapentaenoic acid. 19,20-epoxydocosapentaenoic acid reversed fenofibrate-induced suppression of angiogenesis ex vivo and suppression of endothelial cell functions in vitro. In summary fenofibrate suppressed retinal and choroidal neovascularization via CYP2C inhibition as well as by acting as an agonist of PPARα. Fenofibrate augmented the overall protective effects of ω-3 LCPUFAs on neovascular eye diseases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Gallium-Protoporphyrin IX Inhibits Pseudomonas aeruginosa Growth by Targeting Cytochromes.

    PubMed

    Hijazi, Sarah; Visca, Paolo; Frangipani, Emanuela

    2017-01-01

    Pseudomonas aeruginosa is a challenging pathogen due to both innate and acquired resistance to antibiotics. It is capable of causing a variety of infections, including chronic lung infection in cystic fibrosis (CF) patients. Given the importance of iron in bacterial physiology and pathogenicity, iron-uptake and metabolism have become attractive targets for the development of new antibacterial compounds. P. aeruginosa can acquire iron from a variety of sources to fulfill its nutritional requirements both in the environment and in the infected host. The adaptation of P. aeruginosa to heme iron acquisition in the CF lung makes heme utilization pathways a promising target for the development of new anti-Pseudomonas drugs. Gallium [Ga(III)] is an iron mimetic metal which inhibits P. aeruginosa growth by interfering with iron-dependent metabolism. The Ga(III) complex of the heme precursor protoporphyrin IX (GaPPIX) showed enhanced antibacterial activity against several bacterial species, although no inhibitory effect has been reported on P. aeruginosa. Here, we demonstrate that GaPPIX is indeed capable of inhibiting the growth of clinical P. aeruginosa strains under iron-deplete conditions, as those encountered by bacteria during infection, and that GaPPIX inhibition is reversed by iron. Using P. aeruginosa PAO1 as model organism, we show that GaPPIX enters cells through both the heme-uptake systems has and phu, primarily via the PhuR receptor which plays a crucial role in P. aeruginosa adaptation to the CF lung. We also demonstrate that intracellular GaPPIX inhibits the aerobic growth of P. aeruginosa by targeting cytochromes, thus interfering with cellular respiration.

  7. Interaction potential of Trigonella foenum graceum through cytochrome P450 mediated inhibition

    PubMed Central

    Ahmmed, Sk Milan; Mukherjee, Pulok K.; Bahadur, Shiv; Kar, Amit; Mukherjee, Kakali; Karmakar, Sanmoy; Bandyopadhyay, Arun

    2015-01-01

    Objective: The seeds of Trigonella foenum-graecum (TFG) (family: Leguminosae) are widely consumed both as a spice in food and Traditional Medicine in India. The present study was undertaken to evaluate the inhibitory effect of standardized extract of TFG and its major constituent trigonelline (TG) on rat liver microsome (RLM) and cytochrome P450 (CYP450) drug metabolizing isozymes (CYP3A4 and CYP2D6), which may indicate the possibility of a probable unwanted interaction. Materials and Methods: Reverse phase-high performance liquid chromatography method was developed to standardize the hydroalcoholic seed extract with standard TG. The inhibitory potential of the extract and TG was evaluated on RLM and CYP isozymes using CYP450-carbon monoxide (CYP450-CO) complex assay and fluorescence assay, respectively. Results: The content of TG in TFG was found to be 3.38% (w/w). The CYP-CO complex assay showed 23.32% inhibition on RLM. Fluorescence study revealed that the extract and the biomarker had some inhibition on CYP450 isozymes e.g. CYP3A4 and CYP2D6 (IC50 values of the extract: 102.65 ± 2.63–142.23 ± 2.61 µg/ml and TG: 168.73 ± 4.03–180.90 ± 2.49 µg/ml) which was very less compared to positive controls ketoconazole and quinidine. Inhibition potential of TFG was little higher than TG but very less compared to positive controls. Conclusions: From the present study, we may conclude that the TFG or TG has very less potential to inhibit the CYP isozymes (CYP3A4, CYP2D6), so administration of this plant extract or its biomarker TG may be safe. PMID:26600643

  8. Reversible inhibition of three important human liver cytochrome p450 enzymes by tiliroside.

    PubMed

    Sun, Dong-Xue; Lu, Jin-Cai; Fang, Zhong-Ze; Zhang, Yan-Yan; Cao, Yun-Feng; Mao, Yu-Xi; Zhu, Liang-Liang; Yin, Jun; Yang, Ling

    2010-11-01

    Tiliroside, an active flavonoid extensively found in many medicinal plants including Helichrysum italicum, Geranium mexicanum and Helianthemum glomeratum, has been demonstrated to exert multiple biological effects including antiinflammatory, antimicrobial, antioxidant and antitumor activities. Cytochrome P450 (CYP) enzymes play an important role in the Phase I oxidation metabolism of a wide range of xenobiotics and inhibition of CYP isoforms might influence the elimination of drugs and induce serious adverse drug response. The inhibition of seven CYP isoforms (CYP3A4, CYP1A2, CYP2A6, CYP2D6, CYP2C9, CYP2C8 and CYP2E1) by tiliroside was investigated using in vitro human liver microsomal incubation assays. The results showed that tiliroside strongly inhibited the activity of CYP3A4 (IC(50) = 9.0 ± 1.7 μm), CYP2C8 (IC(50) = 12.1 ± 0.9 μm) and CYP2C9 (IC(50) = 10.2 ± 0.9 μm) with other CYP isoforms negligibly influenced. Further kinetic analysis showed that inhibition of these three CYP isoforms by tiliroside is best fit to a competitive way. The K(i) value was calculated to be 5.5 μm, 3.3 μm, 9.4 μm for CYP3A4, CYP2C9 and CYP2C8, respectively. The relatively low K(i) values suggested that tiliroside might induce drug-drug interactions with many clinically used drugs which are mainly metabolized by these three CYP isoforms. Therefore, attention should be given to the probable drug-drug interaction between tiliroside-containing herbs and substrates of CYP3A4, CYP2C9 and CYP2C8. Copyright © 2010 John Wiley & Sons, Ltd.

  9. Endotoxin administration to humans inhibits hepatic cytochrome P450-mediated drug metabolism.

    PubMed Central

    Shedlofsky, S I; Israel, B C; McClain, C J; Hill, D B; Blouin, R A

    1994-01-01

    In experimental animals, injection of gram-negative endotoxin (LPS) decreases hepatic cytochrome P450-mediated drug metabolism. To evaluate this phenomenon in a human model of gram-negative sepsis, LPS was administered on two consecutive days to healthy male volunteers during which time a cocktail of antipyrine (AP-250 mg), hexobarbital (HB-500 mg), and theophylline (TH-150 mg) was ingested and the apparent oral clearance of each drug determined. Each subject had a control drug clearance study with saline injections. In the first experiment, six subjects received the drug cocktail 0.5 h after the first dose of LPS. In the second experiment, another six subjects received the drug cocktail 0.5 h after the second dose of LPS. In both experiments, LPS caused the expected physiologic responses of inflammation including fever with increases in serum concentrations of TNF alpha, IL-1 beta, IL-6, and acute phase reactants. In the first experiment, only minor decreases in clearances of the probe drugs were observed (7-12%). However in the second experiment, marked decreases in the clearances of AP (35, 95% CI 18-48%), HB (27, 95% CI 14-34%), and TH (22, 95% CI 12-32%) were seen. The decreases in AP clearance correlated with initial peak values of TNF alpha (r = 0.82) and IL-6 (r = 0.86). These data show that in humans the inflammatory response to even a very low dose of LPS significantly decreases hepatic cytochrome P450-mediated drug metabolism and this effect evolves over a 24-h period. It is likely that septic patients with much higher exposures to LPS have more profound inhibition of drug metabolism. PMID:7989576

  10. Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration.

    PubMed

    Yu, Hong; Lee, Icksoo; Salomon, Arthur R; Yu, Kebing; Hüttemann, Maik

    2008-01-01

    Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc(1) complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s(-1) whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s(-1). Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.

  11. Cytochrome P450 1 enzyme inhibition and anticancer potential of chromene amides from Amyris plumieri.

    PubMed

    Badal, S; Williams, S A; Huang, G; Francis, S; Vedantam, P; Vendantam, P; Dunbar, O; Jacobs, H; Tzeng, T J; Gangemi, J; Delgoda, R

    2011-03-01

    Cytochrome P450 (CYP) enzyme inhibitory properties of six chromenylated amide compounds (CAs) from Amyris plumieri are described. Inhibition of CYP microsomes (CYP1A1, CYP1A2, CYP1B1, CYP2D6, CYP3A4 and CYP2C19) was monitored using a fluorescent assay. Potent inhibition was found against CYP1A1 with IC(50) and K(i) for CA1 (acetamide), being the lowest at 1.547 ± 1.0 μM and 0.37 μM respectively, displaying non-competitive kinetics. The selectivity for CYP1A1 was increased in CA3 (butanamide), which also exhibited cytotoxicity against breast cancer cells, MCF7 with an IC(50) of 47.46 ± 1.62 μM. Structure-activity relationship studies provide insight at a molecular level for CAs with implications in chemoprevention and chemotherapy. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Food Polyphenol Apigenin Inhibits the Cytochrome P450 Monoxygenase Branch of the Arachidonic Acid Cascade.

    PubMed

    Steuck, Maryvonne; Hellhake, Stefan; Schebb, Nils Helge

    2016-11-30

    The product of cytochrome P450 monooxygenase (P450) ω-hydroxylation of arachidonic acid (AA), 20- hydroxyeicosatetraenoic acid (HETE), is a potent vasoconstrictor. Utilizing microsomes as well as individual CYP4 isoforms we demonstrate here that flavonoids can block 20-HETE formation. Apigenin inhibits CYP4F2 with an IC50 value of 4.6 μM and 20-HETE formation in human liver and kidney microsomes at 2.4-9.8 μM. Interestingly, the structurally similar naringenin shows no relevant effect on the formation of 20-HETE. Based on these in vitro data, it is impossible to evaluate if a relevant blockade of 20-HETE formation can result in humans from intake of polyphenols with the diet. However, the potency of apigenin is comparable to those of P450 inhibitors such as ketoconazole. Moreover, an IC50 value in the micromolar range is also described for the inhibition of CYP-mediated drug metabolism leading to food-drug interactions. The modulation of the arachidonic acid cascade by food polyphenols therefore warrants further investigation.

  13. Induction and inhibition of cytochrome P450 and drug-metabolizing enzymes by climbazole.

    PubMed

    Kobayashi, Yasuna; Suzuki, Michiya; Ohshiro, Naomi; Sunagawa, Takashi; Sasaki, Tadanori; Oguro, Takiko; Tokuyama, Shogo; Yamamoto, Toshinori; Yoshida, Takemi

    2002-01-01

    To determine the effect of climbazole on hepatic microsomal cytochrome P450 (P450) and drug-metabolizing enzymes, four different P450 isoforms (CYP2B1, 3A2, 2E1, and 2C12) were examined in female Long-Evans rats. Treatment of rats with climbazole resulted in the induction of P450 content. Climbazole both induced and inhibited aminopyrine N-demethylase activity, but not erythromycin N-demethylase activity. Uridine 5'-phosphate (UDP)-glucuronosyl transferase and glutathione S-transferase activities were also increased with climbazole treatment. Immunoblot analyses revealed that climbazole induces CYP2B1 and CYP3A2 at the lower dose examined, but it failed to increase CYP2B1 at the higher dose. Northern blot analysis revealed that climbazole markedly increases P450 2B1 mRNA. These results indicate that climbazole induces and inhibits P450-dependent drug-metabolizing enzymes in vivo and may have the dose-differential effect on CYP2B1 in rat liver.

  14. Inhibition of cytochrome P450 3A: relevant drug interactions in gastroenterology.

    PubMed

    Sagir, A; Schmitt, M; Dilger, K; Häussinger, D

    2003-01-01

    Cytochrome P450 3A (CYP3A) is involved in biotransformation of more than half of all drugs currently available. Drug interactions by inhibition of CYP3A are of major interest in patients receiving combinations of drugs. Some interactions with CYP3A inhibitors also involve inhibition of the multidrug export pump, P-glycoprotein. An increasing number of adverse drug reactions might be avoided on the basis of knowledge about CYP3A substrates and inhibitors. This article summarizes some examples of such interactions relevant to gastroenterologists. Serious cases by coadministration of CYP3A inhibitors resulting in acute hepatitis, hypotension, rhabdomyolyis, torsade de pointes, sedation, or ergotism are presented: interactions with azole antifungals (ketoconazole, itraconazole, fluconazole), HIV protease inhibitors (ritonavir, indinavir, saquinavir, nelfinavir), macrolide antibiotics (clarithromycin, erythromycin), and grapefruit juice. In addition, 1 case is reported who presented the highest trough levels of the CYP3A substrate budesonide in serum ever measured. Practitioners have to be aware of the high potential of metabolic drug interactions when they prescribe a CYP3A inhibitor. It is wise to check carefully comedication in patients complaining of side effects with substrates of CYP3A.

  15. Arachidonic Acid Inhibits Epithelial Na Channel Via Cytochrome P450 (CYP) Epoxygenase-dependent Metabolic Pathways

    PubMed Central

    Wei, Yuan; Lin, Dao-Hong; Kemp, Rowena; Yaddanapudi, Ganesh S.S.; Nasjletti, Alberto; Falck, John R.; Wang, Wen-Hui

    2004-01-01

    We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 μM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose–response curve of the AA effect on ENaC shows that 2 μM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) ω-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase–dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase–dependent metabolite, 11,12-EET. PMID:15545402

  16. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.

    PubMed

    González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A

    2015-08-11

    Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ's histone chaperone activity.

  17. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c

    PubMed Central

    González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A.

    2015-01-01

    Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ’s histone chaperone activity. PMID:26216969

  18. Activity, Inhibition, and Induction of Cytochrome P450 2J2 in Adult Human Primary Cardiomyocytes

    PubMed Central

    Evangelista, Eric A.; Kaspera, Rüdiger; Mokadam, Nahush A.; Jones, J. P.

    2013-01-01

    Cytochrome P450 2J2 plays a significant role in the epoxidation of arachidonic acid to signaling molecules important in cardiovascular events. CYP2J2 also contributes to drug metabolism and is responsible for the intestinal clearance of ebastine. However, the interaction between arachidonic acid metabolism and drug metabolism in cardiac tissue, the main expression site of CYP2J2, has not been examined. Here we investigate an adult-derived human primary cardiac cell line as a suitable model to study metabolic drug interactions (inhibition and induction) of CYP2J2 in cardiac tissue. The primary human cardiomyocyte cell line demonstrated similar mRNA-expression profiles of P450 enzymes to adult human ventricular tissue. CYP2J2 was the dominant isozyme with minor contributions from CYP2D6 and CYP2E1. Both terfenadine and astemizole oxidation were observed in this cell line, whereas midazolam was not metabolized suggesting lack of CYP3A activity. Compared with recombinant CYP2J2, terfenadine was hydroxylated in cardiomyocytes at a similar Km value of 1.5 μM. The Vmax of terfenadine hydroxylation in recombinant enzyme was found to be 29.4 pmol/pmol P450 per minute and in the cells 6.0 pmol/pmol P450 per minute. CYP2J2 activity in the cell line was inhibited by danazol, astemizole, and ketoconazole in submicromolar range, but also by xenobiotics known to cause cardiac adverse effects. Of the 14 compounds tested for CYP2J2 induction, only rosiglitazone increased mRNA expression, by 1.8-fold. This cell model can be a useful in vitro model to investigate the role of CYP2J2-mediated drug metabolism, arachidonic acid metabolism, and their association to drug induced cardiotoxicity. PMID:24021950

  19. Inhibition of cytochrome P450omega-hydroxylase: a novel endogenous cardioprotective pathway.

    PubMed

    Nithipatikom, Kasem; Gross, Eric R; Endsley, Michael P; Moore, Jeannine M; Isbell, Marilyn A; Falck, John R; Campbell, William B; Gross, Garrett J

    2004-10-15

    Cytochrome P450s (CYP) and their arachidonic acid (AA) metabolites have important roles in regulating vascular tone, but their function and specific pathways involved in modulating myocardial ischemia-reperfusion injury have not been clearly established. Thus, we characterized the effects of several selective CYPomega-hydroxylase inhibitors and a CYPomega-hydroxylase metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), on the extent of ischemia-reperfusion injury in canine hearts. During 60 minutes of ischemia and particularly after 3 hours of reperfusion, 20-HETE was produced at high concentrations. A nonspecific CYP inhibitor, miconazole, and 2 specific CYPomega-hydroxylase inhibitors, 17-octadecanoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly inhibited 20-HETE production during ischemia-reperfusion and produced a profound reduction in myocardial infarct size (expressed as a percent of the area at risk) (19.6+/-1.7% [control], 8.4+/-2.5% [0.96 mg/kg miconazole], 5.9+/-2.2% [0.28 mg/kg 17-ODYA], and 10.8+/-1.8% [0.40 mg/kg DDMS], P<0.05, respectively). Conversely, exogenous 20-HETE administration significantly increased infarct size (26.9+/-1.9%, P<0.05). Several CYPomega-hydroxylase isoforms, which are known to produce 20-HETE such as CYP4A1, CYP4A2, and CYP4F, were demonstrated to be present in canine heart tissue and their activity was markedly inhibited by incubation with 17-ODYA. These results indicate an important endogenous role for CYPomega-hydroxylases and in particular their product, 20-HETE, in exacerbating myocardial injury in canine myocardium. The full text of this article is available online at http://circres.ahajournals.org.

  20. Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity.

    PubMed Central

    Bennett, M C; Mlady, G W; Fleshner, M; Rose, G M

    1996-01-01

    Previously, we developed a rat model of persistent mitochondrial dysfunction based upon the chronic partial inhibition of the mitochondrial enzyme cytochrome oxidase (EC 1.9.3.1). Continuous systemic infusion of sodium azide at approximately 1 mg/kg per hr inhibited cytochrome oxidase activity and produced a spatial learning deficit. In other laboratories, glucocorticoids have been reported to exacerbate neuronal damage from various acute metabolic insults. Therefore, we tested the hypothesis that corticosterone, the primary glucocorticoid in the rat, would potentiate the sodium azide-induced learning deficit. To this end, we first identified nonimpairing doses of sodium azide (approximately 0.75 mg/kg per hr) and corticosterone (100-mg pellet, 3-week sustained-release). We now report that chronic co-administration of these individually nonimpairing treatments produced a severe learning deficit. Moreover, the low dose of corticosterone, which did not elevate serum corticosterone, acted synergistically with sodium azide to inhibit cytochrome oxidase activity. The latter result represents a previously unidentified effect of glucocorticoids that provides a candidate mechanism for glucocorticoid potentiation of neurotoxicity induced by metabolic insult. These results may have the clinical implication of expanding the definition of hypercortisolism in patient populations with compromised oxidative metabolism. Furthermore, they suggest that glucocorticoid treatment may contribute to pathology in disease or trauma conditions that involve metabolic insult. PMID:8577764

  1. Salidroside Protects Against 6-Hydroxydopamine-Induced Cytotoxicity by Attenuating ER Stress.

    PubMed

    Tao, Kai; Wang, Bao; Feng, Dayun; Zhang, Wei; Lu, Fangfang; Lai, Juan; Huang, Lu; Nie, Tiejian; Yang, Qian

    2016-02-01

    Parkinson's disease (PD) is a neurodegenerative disease characterized by a persistent decline of dopaminergic (DA) neurons in the substantia nigra pars compacta. Despite its frequency, effective therapeutic strategies that halt the neurodegenerative processes are lacking, reinforcing the need to better understand the molecular drivers of this disease. Importantly, increasing evidence suggests that the endoplasmic reticulum (ER) stress-induced unfolded protein response is likely involved in DA neuronal death. Salidroside, a major compound isolated from Rhodiola rosea L., possesses potent anti-oxidative stress properties and protects against DA neuronal death. However, the underlying mechanisms are not well understood. In the present study, we demonstrate that salidroside prevents 6-hydroxydopamine (6-OHDA)-induced cytotoxicity by attenuating ER stress. Furthermore, treatment of a DA neuronal cell line (SN4741) and primary cortical neurons with salidroside significantly reduced neurotoxin-induced increases in cytoplasmic reactive oxygen species and calcium, both of which cause ER stress, and cleaved caspase-12, which is responsible for ER stress-induced cell death. Together, these results suggest that salidroside protects SN4741 cells and primary cortical neurons from 6-OHDA-induced neurotoxicity by attenuating ER stress. This provides a rationale for the investigation of salidroside as a potential therapeutic agent in animal models of PD.

  2. Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain.

    PubMed

    Nishimura, Kaneyasu; Inoue, Takeshi; Yoshimoto, Kanji; Taniguchi, Takashi; Kitamura, Yoshihisa; Agata, Kiyokazu

    2011-12-01

    Planarians have robust regenerative ability dependent on X-ray-sensitive pluripotent stem cells, called neoblasts. Here, we report that planarians can regenerate dopaminergic neurons after selective degeneration of these neurons caused by treatment with a dopaminergic neurotoxin (6-hydroxydopamine; 6-OHDA). This suggests that planarians have a system to sense the degeneration of dopaminergic neurons and to recruit stem cells to produce dopaminergic neurons to recover brain morphology and function. We confirmed that X-ray-irradiated planarians do not regenerate brain dopaminergic neurons after 6-OHDA-induced lesioning, suggesting that newly generated dopaminergic neurons are indeed derived from pluripotent stem cells. However, we found that the majority of regenerated dopaminergic neurons were 5-bromo-2'-deoxyuridine-negative cells. Therefore, we carefully analyzed when proliferating stem cells became committed to become dopaminergic neurons during regeneration by a combination of 5-bromo-2'-deoxyuridine pulse-chase experiments, immunostaining/in situ hybridization, and 5-fluorouracil treatment. The results strongly suggested that G(2) -phase stem cells become committed to dopaminergic neurons in the mesenchymal space around the brain, after migration from the trunk region following S-phase. These new findings obtained from planarian regeneration provide hints about how to conduct cell-transplantation therapy for future regenerative medicine. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  3. Astragalus Polysaccharide Suppresses 6-Hydroxydopamine-Induced Neurotoxicity in Caenorhabditis elegans.

    PubMed

    Li, Haifeng; Shi, Ruona; Ding, Fei; Wang, Hongyu; Han, Wenjing; Ma, Fangli; Hu, Minghua; Ma, Chung Wah; Huang, Zebo

    2016-01-01

    Astragalus membranaceus is a medicinal plant traditionally used in China for a variety of conditions, including inflammatory and neural diseases. Astragalus polysaccharides are shown to reduce the adverse effect of levodopa which is used to treat Parkinson's disease (PD). However, the neuroprotective effect of Astragalus polysaccharides per se in PD is lacking. Using Caenorhabditis elegans models, we investigated the protective effect of astragalan, an acidic polysaccharide isolated from A. membranaceus, against the neurotoxicity of 6-hydroxydopamine (6-OHDA), a neurotoxin that can induce parkinsonism. We show that 6-OHDA is able to degenerate dopaminergic neurons and lead to the deficiency of food-sensing behavior and a shorter lifespan in C. elegans. Interestingly, these degenerative symptoms can be attenuated by astragalan treatment. Astragalan is also shown to alleviate oxidative stress through reducing reactive oxygen species level and malondialdehyde content and increasing superoxide dismutase and glutathione peroxidase activities and reduce the expression of proapoptotic gene egl-1 in 6-OHDA-intoxicated nematodes. Further studies reveal that astragalan is capable of elevating the decreased acetylcholinesterase activity induced by 6-OHDA. Together, our results demonstrate that the protective effect of astragalan against 6-OHDA neurotoxicity is likely due to the alleviation of oxidative stress and regulation of apoptosis pathway and cholinergic system and thus provide an important insight into the therapeutic potential of Astragalus polysaccharide in neurodegeneration.

  4. Neuroprotective effect of hydroxysafflor yellow A on 6-hydroxydopamine-induced Parkinson's disease in rats.

    PubMed

    Han, Bing; Hu, Jia; Shen, Jingyu; Gao, Yonglin; Lu, Yan; Wang, Tian

    2013-08-15

    Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting predominantly the dopaminergic mesotelencephalic system. Enormous progress has been made in the treatment of PD. Our previous study has shown that hydroxysafflor yellow A (HSYA) could attenuate the neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. In the present work, we examined whether HSYA had the neuroprotective effect on dopaminergic neurons of substantia nigra in a rat model of PD. Adult Sprague-Dawley rats were unilaterally injected with 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. The PD rats were treated with HSYA (2 or 8 mg/kg) via caudal vein injection daily for 4 weeks. Rotational tests showed that HSYA significantly attenuated apomorphine-induced turns in 6-OHDA-induced PD rats. HSYA treatment resulted in a significant protection against the loss of tyrosine hydroxylase-positive cells. Our data showed that HSYA also increased the levels of dopamine and its metabolites, glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor in striatum of PD rats. In conclusion, these results supported a role for HSYA in preserving dopamine neuron integrity and motor function in a rodent model of PD, and implied a potential neuroprotective role for HSYA in this disease.

  5. Protective effect of planarian DJ-1 against 6-hydroxydopamine-induced neurotoxicity.

    PubMed

    Tsushima, Jun; Nishimura, Kaneyasu; Tashiro, Natsuka; Takata, Kazuyuki; Ashihara, Eishi; Yoshimoto, Kanji; Ariga, Hiroyoshi; Agata, Kiyokazu; Kitamura, Yoshihisa

    2012-12-01

    DJ-1/PARK7 has multiple functions as an antioxidant, an oncogene, and a molecular chaperone in vertebrates, and loss-of-function mutations in DJ-1 cause early onset of Parkinson's disease. However, the function of invertebrate DJ-1 remains unknown. In order to investigate the function of planarian DJ-1, we isolated the planarian DJ-1 gene Dugesia japonica DJ-1 (DjDJ-1) and analyzed its expression and function. In situ hybridization analysis revealed that DjDJ-1 mRNA was expressed throughout the body, including the central nervous system, cells surrounding the pharynx, and stem cells. Planarian DjDJ-1 protein exhibited antioxidant function, similar to human DJ-1, as evidenced by the fact that recombinant DjDJ-1 protein reduced reactive oxygen species and protected human neuroblastoma SH-SY5Y cells from cell death. In addition, dopaminergic neurons in DjDJ-1(RNAi) planarians became susceptible to 6-hydroxydopamine, a dopaminergic neurotoxin. These results suggest that planarians have a DJ-1 ortholog, which has conserved antioxidant and neuroprotective functions.

  6. Astragalus Polysaccharide Suppresses 6-Hydroxydopamine-Induced Neurotoxicity in Caenorhabditis elegans

    PubMed Central

    Li, Haifeng; Ding, Fei; Wang, Hongyu; Han, Wenjing; Ma, Fangli; Hu, Minghua; Ma, Chung Wah

    2016-01-01

    Astragalus membranaceus is a medicinal plant traditionally used in China for a variety of conditions, including inflammatory and neural diseases. Astragalus polysaccharides are shown to reduce the adverse effect of levodopa which is used to treat Parkinson's disease (PD). However, the neuroprotective effect of Astragalus polysaccharides per se in PD is lacking. Using Caenorhabditis elegans models, we investigated the protective effect of astragalan, an acidic polysaccharide isolated from A. membranaceus, against the neurotoxicity of 6-hydroxydopamine (6-OHDA), a neurotoxin that can induce parkinsonism. We show that 6-OHDA is able to degenerate dopaminergic neurons and lead to the deficiency of food-sensing behavior and a shorter lifespan in C. elegans. Interestingly, these degenerative symptoms can be attenuated by astragalan treatment. Astragalan is also shown to alleviate oxidative stress through reducing reactive oxygen species level and malondialdehyde content and increasing superoxide dismutase and glutathione peroxidase activities and reduce the expression of proapoptotic gene egl-1 in 6-OHDA-intoxicated nematodes. Further studies reveal that astragalan is capable of elevating the decreased acetylcholinesterase activity induced by 6-OHDA. Together, our results demonstrate that the protective effect of astragalan against 6-OHDA neurotoxicity is likely due to the alleviation of oxidative stress and regulation of apoptosis pathway and cholinergic system and thus provide an important insight into the therapeutic potential of Astragalus polysaccharide in neurodegeneration. PMID:27885333

  7. Cat retinal ganglion cell receptive-field alterations after 6-hydroxydopamine induced dopaminergic amacrine cell lesions

    SciTech Connect

    Maguire, G.W.; Smith, E.L. III

    1985-06-01

    Optic tract single-unit recordings were used to study ganglion cell response functions of the intact cat eye after 6-hydroxydopamine (6-OHDA) lesioning of the dopaminergic amacrine cell (AC) population of the inner retina. The impairment of the dopaminergic AC was verified by high pressure-liquid chromatography with electrochemical detection of endogenous dopamine content and by (/sup 3/H)dopamine high-affinity uptake; the dopaminergic ACs of the treated eyes demonstrated reduced endogenous dopamine content and reduced (/sup 3/H)dopamine uptake compared with that of their matched controls. Normal appearing (/sup 3/H)GABA and (/sup 3/H)-glycine uptake in the treated retinas suggests the absence of any nonspecific action of the 6-OHDA on the neural retina. The impairment of the dopaminergic AC population was found to alter a number of response properties in off-center ganglion cells, but this impairment had only a modest effect on the on-center cells. An abnormally high proportion of the off-center ganglion cells in the 6-OHDA treated eyes possessed nonlinear, Y-type receptive fields. These cells also possessed shift-responses of greater than normal amplitude, altered intensity-response functions, reduced maintained activities, and more transient center responses. Of the on-center type cells, only the Y-type on-center cells were affected by 6-OHDA, possessing higher than normal maintained activities and altered intensity-response functions. The on-center X-cells were unaffected by 6-OHDA treatment. The dopaminergic AC of the photopically adapted cat retina therefore modulates a number of ganglion cell response properties and within the limits of this study is most prominent in off-center ganglion cell circuitry.

  8. Curcumin Prevents Aflatoxin B1 Hepatoxicity by Inhibition of Cytochrome P450 Isozymes in Chick Liver

    PubMed Central

    Zhang, Ni-Ya; Qi, Ming; Zhao, Ling; Zhu, Ming-Kun; Guo, Jiao; Liu, Jie; Gu, Chang-Qin; Rajput, Shahid Ali; Krumm, Christopher Steven; Qi, De-Sheng; Sun, Lv-Hui

    2016-01-01

    This study was designed to establish if Curcumin (CM) alleviates Aflatoxin B1 (AFB1)-induced hepatotoxic effects and to determine whether alteration of the expression of cytochrome P450 (CYP450) isozymes is involved in the regulation of these effects in chick liver. One-day-old male broilers (n = 120) were divided into four groups and used in a two by two factorial trial in which the main factors included supplementing AFB1 (< 5 vs. 100 μg/kg) and CM (0 vs. 150 mg/kg) in a corn/soybean-based diet. Administration of AFB1 induced liver injury, significantly decreasing albumin and total protein concentrations and increasing alanine aminotransferase and aspartate aminotransferase activities in serum, and induced hepatic histological lesions at week 2. AFB1 also significantly decreased hepatic glutathione peroxidase, catalase, and glutathione levels, while increasing malondialdehyde, 8-hydroxydeoxyguanosine, and exo-AFB1-8,9-epoxide (AFBO)-DNA concentrations. In addition, the mRNA and/or activity of enzymes responsible for the bioactivation of AFB1 into AFBO—including CYP1A1, CYP1A2, CYP2A6, and CYP3A4—were significantly induced in liver microsomes after 2-week exposure to AFB1. These alterations induced by AFB1 were prevented by CM supplementation. Conclusively, dietary CM protected chicks from AFB1-induced liver injury, potentially through the synergistic actions of increased antioxidant capacities and inhibition of the pivotal CYP450 isozyme-mediated activation of AFB1 to toxic AFBO. PMID:27834912

  9. Inhibition of cytochrome P450 activity enhances the systemic availability of triclabendazole metabolites in sheep.

    PubMed

    Virkel, G; Lifschitz, A; Sallovitz, J; Ballent, M; Scarcella, S; Lanusse, C

    2009-02-01

    Understanding the disposition kinetics and the pattern of metabolism is critical to optimise the flukicidal activity of triclabendazole (TCBZ) in ruminants. TCBZ is metabolised by both flavin-monooxygenase (FMO) and cytochrome P450 (P450) in the liver. Interference with these metabolic pathways may be useful to increase the systemic availabilities of TCBZ metabolites, which may improve the efficacy against Fasciola hepatica. The plasma disposition of TCBZ metabolites was evaluated following TCBZ co-administration with FMO [methimazole (MTZ)] and P450 [piperonyl butoxyde (PB) and ketoconazole (KTZ)] inhibitors in sheep. Twenty (20) healthy Corriedale x Merino weaned female lambs were randomly allocated into four experimental groups. Animals of each group were treated as follow: Group A, TCBZ alone (5 mg/kg, IV route); Group B, TCBZ (5 mg/kg, IV) + MTZ (3 mg/kg, IV); Group C, TCBZ (5 mg/kg, IV) + PB (30 mg/kg, IV) and Group D, TCBZ (5 mg/kg, IV) + KTZ (10 mg/kg, orally). Blood samples were taken over 240 h post-treatment and analysed by HPLC. TCBZ sulphoxide and sulphone were the main metabolites recovered in plasma. MTZ did not affect TCBZ disposition kinetics. TCBZ sulphoxide Cmax values were significantly increased (P < 0.05) after the TCBZ + PB (62%) and TCBZ + KTZ (37%) treatments compared to those measured in the TCBZ alone treatment. TCBZ sulphoxide plasma AUCs were higher (P < 0.05) in the presence of both PB (99%) and KTZ (41%). Inhibition of TCBZ P450-mediated oxidation in the liver accounted for the increased systemic availability of its active metabolite TCBZ sulphoxide. This work contributes to the search of different strategies to improve the use of this flukicidal drug in ruminants.

  10. Reduced expression of cytochrome oxidases largely explains cAMP inhibition of aerobic growth in Shewanella oneidensis

    PubMed Central

    Yin, Jianhua; Meng, Qiu; Fu, Huihui; Gao, Haichun

    2016-01-01

    Inhibition of bacterial growth under aerobic conditions by elevated levels of cyclic adenosine 3′,5′-monophosphate (cAMP), first revealed more than 50 years ago, was attributed to accumulation of toxic methylglyoxal (MG). Here, we report a Crp-dependent mechanism rather than MG accumulation that accounts for the phenotype in Shewanella oneidensis, an emerging research model for the bacterial physiology. We show that a similar phenotype can be obtained by removing CpdA, a cAMP phosphodiesterase that appears more effective than its Escherichia coli counterpart. Although production of heme c and cytochromes c is correlated well with cAMP levels, neither is sufficient for the retarded growth. Quantities of overall cytochromes c increased substantially in the presence of elevated cAMP, a phenomenon resembling cells respiring on non-oxygen electron acceptors. In contrast, transcription of Crp-dependent genes encoding both cytochromes bd and cbb3 oxidases is substantially repressed under the same condition. Overall, our results suggest that cAMP of elevated levels drives cells into a low-energetic status, under which aerobic respiration is inhibited. PMID:27076065

  11. Inhibition selectivity of grapefruit juice components on human cytochromes P450.

    PubMed

    Tassaneeyakul, W; Guo, L Q; Fukuda, K; Ohta, T; Yamazoe, Y

    2000-06-15

    Five compounds including furanocoumarin monomers (bergamottin, 6', 7'-dihydroxybergamottin (DHB)), furanocoumarin dimers (4-¿¿6-hydroxy-71-¿(1-hydroxy-1-methyl)ethyl-4-methyl-6-(7-oxo-7H- furo¿3,2-g1benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl- 2-octenyl]oxy]-7H-furo[3,2-g]¿1benzopyran-7-one (GF-I-1) and 4-¿¿6-hydroxy-7¿¿4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo¿3, 2-g1benzopyran-4-yl)-4-hexenylŏxy-3, 7-dimethyl-2-octenylŏxy-7H-furo¿3,2-g1benzopyran-7-one (GF-I-4)), and a sesquiterpene nootkatone have been isolated from grapefruit juice and screened for their inhibitory effects toward human cytochrome P450 (P450) forms using selective substrate probes. Addition of ethyl acetate extract of grapefruit juice into an incubation mixture resulted in decreased activities of CYP3A4, CYP1A2, CYP2C9, and CYP2D6. All four furanocoumarins clearly inhibited CYP3A4-catalyzed nifedipine oxidation in concentration- and time-dependent manners, suggesting that these compounds are mechanism-based inhibitors of CYP3A4. Of the furanocoumarins investigated, furanocoumarin dimers, GF-I-1 and GF-I-4, were the most potent inhibitors of CYP3A4. Inhibitor concentration required for half-maximal rate of inactivation (K(I)) values for bergamottin, DHB, GF-I-1, and GF-I-4 were calculated, respectively, as 40.00, 5. 56, 0.31, and 0.13 microM, whereas similar values were observed on their inactivation rate constant at infinite concentration of inhibitor (k(inact), 0.05-0.08 min(-1)). Apparent selectivity toward CYP3A4 does occur with the furanocoumarin dimers. In contrast, bergamottin showed rather stronger inhibitory effect on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on CYP3A4. DHB inhibited CYP3A4 and CYP1A2 activities at nearly equivalent potencies. Among P450 forms investigated, CYP2E1 was the least sensitive to the inhibitory effect of furanocoumarin components. A sesquiterpene nootkatone has no significant effect on P450 activities investigated except for CYP2A6 and CYP2C19

  12. Inhibition of cytochrome p450 enzymes by enrofloxacin in the sea bass (Dicentrarchus labrax).

    PubMed

    Vaccaro, E; Giorgi, M; Longo, V; Mengozzi, G; Gervasi, P G

    2003-01-10

    Currently, there are no reports on the effects of enrofloxacin (EF), a fluoroquinolone antibiotic, on the cytochrome p450 enzymes in fish, although its use as antimicrobial agent in aquaculture has been put forward. Therefore, the in vivo and in vitro effects of EF on hepatic p450 enzymes of sea bass, a widespread food-producing fish, have been evaluated. Sea bass pretreated with a single dose of EF (3 mg/kg i.p.) or with three daily doses of EF (1 mg/kg i.p.) markedly depressed the microsomal N-demethylation of aminopyrine, erythromycin, the O-deethylation of 7-ethoxycoumarin, ethoxyresorufin and the 6beta-testosterone hydroxylase. In vitro experiments showed that EF at 10 microM inhibited the above-mentioned activities and, in particular, the erythromycin N-demethylase (ERND) and 6beta-testosterone-hydroxylase, likely dependant on a p450 3A isoform. When the nature of ERND inhibition by EF was specifically studied with sea bass liver microsomes, it was found that EF is a potent mechanism-based inhibitor, with K(i) of 3.7 microM and a K(inact) of 0.045 min(-1). An immunoblot analysis with anti p450 3A27 of trout showed that the p450 3A isoform, constitutively expressed in sea bass, is particularly susceptible to inactivation by EF. In vitro experiments with sea bass microsomes have also demonstrated that EF is oxidative deethylated by the p450 system to ciprofloxacin (CF) and that this compound maintains the ability to inactivate the p450 enzymes. The mechanism by which EF or CF inactivate the p450 enzymes has not been studied but an attack of p450 on the cyclopropan ring, present, both in EF and CF structure, with the formation of electrophilic intermediates (i.e. radicals) has been postulated. In conclusion, the EF seems to be a powerful inhibitor of p450s in the sea bass. Therefore, the clinical use of this antibiotic in aquaculture has to be considered with caution.

  13. Inhibition of Cytochromes P450 and the Hydroxylation of 4-Monochlorobiphenyl in Whole Poplar

    PubMed Central

    Zhai, Guangshu; Lehmler, Hans-Joachim; Schnoor, Jerald L.

    2013-01-01

    Cytochromes P450 (CYPs) are potential enzymes responsible for hydroxylation of many xenobiotics and endogenous chemicals in living organisms. It has been found that 4-monochlorobiphenyl (PCB3), mainly an airborne pollutant, can be metabolized to hydroxylated transformation products (OH-PCB3s) in whole poplars. However, the enzymes involved in the hydroxylation of PCB3 in whole poplars have not been identified. Therefore, two CYP suicide inhibitors, 1-aminobenzotriazole (ABT) and 17-octadecynoic acid (ODYA), were selected to probe the hydroxylation reaction of PCB3 in whole poplars in this work. Poplars (Populus deltoides × nigra, DN34) were exposed to PCB3 with or without inhibitor for 11 days. Results showed both ABT and ODYA can decrease the concentrations and yields of five OH-PCB3s in different poplar parts via the inhibition of CYPs. Furthermore, both ABT and ODYA demonstrated a dose-dependent relationship to the formation of OH-PCB3s in whole poplars. The higher the inhibitor concentrations, the lower the total yields of OH-PCB3s. For ABT spiked-additions, the total mass yield of five OH-PCB3s was inhibited by a factor of 1.6 times at an ABT concentration of 2.5 mg L−1, 4.0 times at 12.5 mg L−1, and 7.0 times at 25 mg L−1. For the inhibitor ODYA, the total mass of five OH-PCB3s was reduced by 2.1 times compared to the control at an ODYA concentration of 2.5 mg L−1. All results pointed to the conclusion that CYP enzymes were the agents which metabolized PCB3 to OH-PCB3s in whole poplars because suicide CYP inhibitors ABT and ODYA both led to sharp decreases of OH-PCB3s formation in whole poplars. A dose-response curve for each of the suicide inhibitors was developed. PMID:23320482

  14. tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance

    PubMed Central

    Zhang, Haoming; Lin, Hsia-lien; Walker, Vyvyca J.; Hamdane, Djemel

    2009-01-01

    We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a KI of 0.44 μM and kinact of 0.12 min−1. The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 Å away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ∼20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4. PMID:19720728

  15. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2.

    PubMed

    Reed, James R; dela Cruz, Albert Leo N; Lomnicki, Slawo M; Backes, Wayne L

    2015-12-01

    Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductase and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2-CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Inhibition of cytochrome P450 2B4 by environmentally persistent free radical-containing particulate matter.

    PubMed

    Reed, James R; dela Cruz, Albert Leo N; Lomnicki, Slawo M; Backes, Wayne L

    2015-05-15

    Combustion processes generate particulate matter (PM) that can affect human health. The presence of redox-active metals and aromatic hydrocarbons in the post-combustion regions results in the formation of air-stable, environmentally persistent free radicals (EPFRs) on entrained particles. Exposure to EPFRs has been shown to negatively influence pulmonary and cardiovascular functions. Cytochromes P450 (P450/CYP) are endoplasmic reticulum resident proteins that are responsible for the metabolism of foreign compounds. Previously, it was shown that model EPFRs, generated by exposure of silica containing 5% copper oxide (CuO-Si) to either dicholorobenzene (DCB230) or 2-monochlorophenol (MCP230) at ≥ 230 °C, inhibited six forms of P450 in rat liver microsomes (Toxicol. Appl. Pharmacol. (2014) 277:200-209). In this study, the inhibition of P450 by MCP230 was examined in more detail by measuring its effect on the rate of metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) and 7-benzyloxyresorufin (7BRF) by the purified, reconstituted CYP2B4 system. MCP230 inhibited the CYP2B4-mediated metabolism of 7EFC at least 10-fold more potently than non-EPFR controls (CuO-Si, silica, and silica generated from heating silica and MCP at 50 °C, so that EPFRs were not formed (MCP50)). The inhibition by EPFRs was specific for the P450 and did not affect the ability of the redox partner, P450 reductase (CPR) from reducing cytochrome c. All of the PM inhibited CYP2B4-mediated metabolism noncompetitively with respect to substrate. When CYP2B4-mediated metabolism of 7EFC was measured as a function of the CPR concentration, the mechanism of inhibition was competitive. EPFRs likely inhibit CYP2B4-mediated substrate metabolism by physically disrupting the CPR·P450 complex. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2

    PubMed Central

    Reed, James R.; dela Cruz, Albert Leo N.; Lomnicki, Slawo M.; Backes, Wayne L.

    2015-01-01

    Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductase and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2-CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2. PMID:26423927

  18. Reversal of cyanide inhibition of cytochrome c oxidase by the auxiliary substrate nitric oxide: an endogenous antidote to cyanide poisoning?

    PubMed

    Pearce, Linda L; Bominaar, Emile L; Hill, Bruce C; Peterson, Jim

    2003-12-26

    Nitric oxide (NO) is shown to overcome the cyanide inhibition of cytochrome c oxidase in the presence of excess ferrocytochrome c and oxygen. Addition of NO to the partially reduced cyanide-inhibited form of the bovine enzyme is shown by electron paramagnetic resonance spectroscopy to result in substitution of cyanide at ferriheme a3 by NO with reduction of the heme. The resulting nitrosylferroheme a3 is a 5-coordinate structure, the proximal bond to histidine having been broken. NO does not simply act as a reversibly bound competitive inhibitor but is an auxiliary substrate consumed in a catalytic cycle along with ferrocytochrome c and oxygen. The implications of this observation with regard to estimates of steady-state NO levels in vivo is discussed. Given the multiple sources of NO available to mitochondria, the present results appear to explain in part some of the curious biomedical observations reported by other laboratories; for example, the kidneys of cyanide poisoning victims surprisingly exhibit no significant irreversible damage, and lethal doses of potassium cyanide are able to inhibit cytochrome c oxidase activity by only approximately 50% in brain mitochondria.

  19. Traditional Preparations and Methanol Extracts of Medicinal Plants from Papua New Guinea Exhibit Similar Cytochrome P450 Inhibition

    PubMed Central

    Rai, Prem P.; Matainaho, Teatulohi K.; Piskaut, Pius; Franklin, Michael R.

    2016-01-01

    The hypothesis underlying this current work is that fresh juice expressed from Papua New Guinea (PNG) medicinal plants (succus) will inhibit human Cytochrome P450s (CYPs). The CYP inhibitory activity identified in fresh material was compared with inhibition in methanol extracts of dried material. Succus is the most common method of traditional medicine (TM) preparation for consumption in PNG. There is increasing concern that TMs might antagonize or complicate drug therapy. We have previously shown that methanol extracts of commonly consumed PNG medicinal plants are able to induce and/or inhibit human CYPs in vitro. In this current work plant succus was prepared from fresh plant leaves. Inhibition of three major CYPs was determined using human liver microsomes and enzyme-selective model substrates. Of 15 species tested, succus from 6/15 was found to inhibit CYP1A2, 7/15 inhibited CYP3A4, and 4/15 inhibited CYP2D6. Chi-squared tests determined differences in inhibitory activity between succus and methanol preparations. Over 80% agreement was found. Thus, fresh juice from PNG medicinal plants does exhibit the potential to complicate drug therapy in at risk populations. Further, the general reproducibility of these findings suggests that methanol extraction of dried material is a reasonable surrogate preparation method for fresh plant samples. PMID:27642356

  20. Traditional Preparations and Methanol Extracts of Medicinal Plants from Papua New Guinea Exhibit Similar Cytochrome P450 Inhibition.

    PubMed

    Larson, Erica C; Pond, Christopher D; Rai, Prem P; Matainaho, Teatulohi K; Piskaut, Pius; Franklin, Michael R; Barrows, Louis R

    2016-01-01

    The hypothesis underlying this current work is that fresh juice expressed from Papua New Guinea (PNG) medicinal plants (succus) will inhibit human Cytochrome P450s (CYPs). The CYP inhibitory activity identified in fresh material was compared with inhibition in methanol extracts of dried material. Succus is the most common method of traditional medicine (TM) preparation for consumption in PNG. There is increasing concern that TMs might antagonize or complicate drug therapy. We have previously shown that methanol extracts of commonly consumed PNG medicinal plants are able to induce and/or inhibit human CYPs in vitro. In this current work plant succus was prepared from fresh plant leaves. Inhibition of three major CYPs was determined using human liver microsomes and enzyme-selective model substrates. Of 15 species tested, succus from 6/15 was found to inhibit CYP1A2, 7/15 inhibited CYP3A4, and 4/15 inhibited CYP2D6. Chi-squared tests determined differences in inhibitory activity between succus and methanol preparations. Over 80% agreement was found. Thus, fresh juice from PNG medicinal plants does exhibit the potential to complicate drug therapy in at risk populations. Further, the general reproducibility of these findings suggests that methanol extraction of dried material is a reasonable surrogate preparation method for fresh plant samples.

  1. Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo′' to inhibition by the carbon monoxide-releasing molecule, CORM-3☆☆☆

    PubMed Central

    Jesse, Helen E.; Nye, Tacita L.; McLean, Samantha; Green, Jeffrey; Mann, Brian E.; Poole, Robert K.

    2013-01-01

    Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (Km 0.24 μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a

  2. Automated high throughput ADME assays for metabolic stability and cytochrome P450 inhibition profiling of combinatorial libraries.

    PubMed

    Jenkins, Kelly M; Angeles, Reginald; Quintos, Marianne T; Xu, Rongda; Kassel, Daniel B; Rourick, Robyn A

    2004-03-10

    Early determinations of pharmaceutical properties can serve as predictors of a compound's likely development success. Our laboratory has implemented high throughput in vitro absorption, distribution, metabolism and excretion (ADME) assays which address absorption, metabolism, and physico-chemical properties in an effort to identify potential development liabilities early, thereby minimizing discovery to market attrition. In response to the throughput demands of parallel synthesis, we have incorporated a SAGIAN core robotics system for the determination of both metabolic stability in human liver microsomes (HLMs) and cytochrome P450 (CYP450) inhibition. This automated solution has led to an increase in capacity, throughput and reliability for both in vitro assays. The SAGIAN core robotics system integrates devices such as liquid handlers, plate hotels and incubators through the use of an ORCA robotic arm. The HLM stability assay utilizes a Multimek 96-channel pipettor for liquid handling. The incubation plates are transferred off-line for final semi-quantitative analysis using high throughput parallel LC/MS. The CYP inhibition method combines both liquid handlers and an integrated fluorescence plate reader to perform single concentration percent inhibition assays for 88 compounds. Cytochrome P450 inhibition is measured for both CYP3A4 and CYP2D6 isozymes. This system represents a fully integrated approach to high throughput ADME evaluation in support of drug discovery. The core system concept creates a plug-and-play approach, which combines a series of modular stations to build a robotic platform, which is flexible, upgradable, and easily reconfigured when assays change or are newly developed. The application of these strategies as a means of assessing metabolic stability and CYP inhibition of synthetic libraries is discussed.

  3. Inhibition of cathepsin B activity reduces apoptosis by preventing cytochrome c release from mitochondria in porcine parthenotes

    PubMed Central

    KIM, Seon-Hyang; ZHAO, Ming-Hui; LIANG, Shuang; CUI, Xiang-Shun; KIM, Nam-Hyung

    2015-01-01

    Cathepsin B, a lysosomal cysteine protease of the papain family, has recently been implicated in the quality and developmental competence of bovine preimplantation embryos. In this study, to determine whether inhibition of cathepsin B activity can improve porcine oocyte maturation and early embryo developmental competence, we supplemented in vitro maturation or embryo culture media with E-64, a cathepsin B inhibitor. Cathepsin B activity was high in poor quality germinal vesicle stage oocytes, but no differences in mRNA expression or protein localization were observed between good and poor quality oocytes, which were categorized based on morphology. Following treatment with 1 μM E-64, cathepsin B activity sharply decreased in 4-cell and blastocyst stage embryos. E-64 had no effect on cell number but significantly (P < 0.05) increased blastocyst formation and decreased the number of apoptotic cells in blastocysts. It also significantly (P < 0.05) enhanced mitochondrial membrane potential in blastocysts, reducing the release of cytochrome c and resulting in decreased expression of Caspase-3 and Caspase-9. In conclusion, inhibition of cathepsin B activity in porcine parthenotes using 1 μM E-64 resulted in attenuation of apoptosis via a reduction in the release of cytochrome c from mitochondria. PMID:25903788

  4. Inhibition of cathepsin B activity reduces apoptosis by preventing cytochrome c release from mitochondria in porcine parthenotes.

    PubMed

    Kim, Seon-Hyang; Zhao, Ming-Hui; Liang, Shuang; Cui, Xiang-Shun; Kim, Nam-Hyung

    2015-01-01

    Cathepsin B, a lysosomal cysteine protease of the papain family, has recently been implicated in the quality and developmental competence of bovine preimplantation embryos. In this study, to determine whether inhibition of cathepsin B activity can improve porcine oocyte maturation and early embryo developmental competence, we supplemented in vitro maturation or embryo culture media with E-64, a cathepsin B inhibitor. Cathepsin B activity was high in poor quality germinal vesicle stage oocytes, but no differences in mRNA expression or protein localization were observed between good and poor quality oocytes, which were categorized based on morphology. Following treatment with 1 μM E-64, cathepsin B activity sharply decreased in 4-cell and blastocyst stage embryos. E-64 had no effect on cell number but significantly (P < 0.05) increased blastocyst formation and decreased the number of apoptotic cells in blastocysts. It also significantly (P < 0.05) enhanced mitochondrial membrane potential in blastocysts, reducing the release of cytochrome c and resulting in decreased expression of Caspase-3 and Caspase-9. In conclusion, inhibition of cathepsin B activity in porcine parthenotes using 1 μM E-64 resulted in attenuation of apoptosis via a reduction in the release of cytochrome c from mitochondria.

  5. Spiroethers of German chamomile inhibit production of aflatoxin G and trichothecene mycotoxin by inhibiting cytochrome P450 monooxygenases involved in their biosynthesis.

    PubMed

    Yoshinari, Tomoya; Yaguchi, Atsushi; Takahashi-Ando, Naoko; Kimura, Makoto; Takahashi, Haruo; Nakajima, Takashi; Sugita-Konishi, Yoshiko; Nagasawa, Hiromichi; Sakuda, Shohei

    2008-07-01

    The essential oil of German chamomile showed specific inhibition toward aflatoxin G(1) (AFG(1)) production, and (E)- and (Z)-spiroethers were isolated as the active compounds from the oil. The (E)- and (Z)-spiroethers inhibited AFG(1) production of Aspergillus parasiticus with inhibitory concentration 50% (IC(50)) values of 2.8 and 20.8 microM, respectively, without inhibiting fungal growth. Results of an O-methylsterigmatocystin (OMST) conversion study indicated that the spiroethers specifically inhibited the OMST to AFG(1) pathway. A cytochrome P450 monooxygenase, CYPA, is known as an essential enzyme for this pathway. Because CYPA has homology with TRI4, a key enzyme catalyzing early steps in the biosynthesis of trichothecenes, the inhibitory actions of the two spiroethers against TRI4 reactions and 3-acetyldeoxynivalenol (3-ADON) production were tested. (E)- and (Z)-spiroethers inhibited the enzymatic activity of TRI4 dose-dependently and interfered with 3-ADON production by Fusarium graminearum, with IC(50) values of 27.1 and 103 microM, respectively. Our results suggest that the spiroethers inhibited AFG(1) and 3-ADON production by inhibiting CYPA and TRI4, respectively.

  6. Study Liver Cytochrome P450 3A4 Inhibition and Hepatotoxicity Using DMSO-Differentiated HuH-7 Cells.

    PubMed

    Liu, Yitong

    2016-01-01

    Metabolically competent, inexpensive, and robust in vitro cell models are needed for studying liver drug-metabolizing enzymes and hepatotoxicity. Human hepatoma HuH-7 cells develop into a differentiated in vitro model resembling primary human hepatocytes after a 2-week dimethyl sulfoxide (DMSO) treatment. DMSO-treated HuH-7 cells express elevated cytochrome P450 3A4 (CYP3A4) enzyme gene expression and activity compared to untreated HuH-7 cells. This cell model could be used to study CYP3A4 inhibition by reversible and time-dependent inhibitors, including drugs, food-related substances, and environmental chemicals. The DMSO-treated HuH-7 model is also a suitable tool for investigating hepatotoxicity. This chapter describes a detailed methodology for developing DMSO-treated HuH-7 cells, which are subsequently used for CYP3A4 inhibition and hepatotoxicity studies.

  7. Photodynamic therapy-induced apoptosis in lymphoma cells: translocation of cytochrome c causes inhibition of respiration as well as caspase activation.

    PubMed

    Varnes, M E; Chiu, S M; Xue, L Y; Oleinick, N L

    1999-02-24

    L5178Y-R mouse lymphoma (LY-R) cells undergo rapid apoptosis when treated with photodynamic therapy (PDT) sensitized with the silicon phthalocyanine Pc 4. In this study we show that cytochrome c is released into the cytosol within 10 min of an LD99.9 dose of PDT. Cellular respiration is inhibited by 42% at 15 min, and 60% at 30 min after PDT treatment, and caspase 3-like protease activity is elevated by 15 min post-PDT. In digitonin-permeabilized cells addition of cytochrome c to the respiration buffer reverses PDT-induced inhibition of state 3 respiration via Complex I by 40-60%, and via Complex III by 50-90%. In contrast, extramitochondrial cytochrome c does not stimulate respiration in permeabilized control cells, and catalyzes only a low rate of oxygen consumption via electron transfer to cytochrome b5 on the outer mitochondrial membrane. These results demonstrate that PDT-induced inhibition of respiration is primarily due to leakage of cytochrome c into the cytosol rather than to damage to the major enzyme complexes of the electron transport chain. Whether or not inhibition of respiration influences the time course or extent of Pc 4-PDT-induced apoptosis in LY-R cells is not clear at the present time.

  8. In vitro inhibition of methadone and oxycodone cytochrome P450-dependent metabolism: reversible inhibition by H2-receptor agonists and proton-pump inhibitors.

    PubMed

    Moody, David E; Liu, Fenyun; Fang, Wenfang B

    2013-10-01

    In vitro inhibition of oxycodone metabolism to noroxycodone and oxymorphone and R- and S-methadone metabolism to R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was measured for four H2-receptor antagonists and five proton-pump inhibitors (PPIs) using human liver microsomes (HLM) and cDNA-expressed human cytochrome P450s (rCYPs). Inhibitors were first incubated with HLM at three concentrations with and without preincubation of inhibitor, enzyme source and reducing equivalents to also screen for time-dependent inhibition (TDI). Cimetidine and famotidine (10-1,000 µM) inhibited all the four pathways >50%. Nizatidine and ranitidine did not. All the five PPIs (1-200 µM) inhibited one or more pathways >50%. Half maximal inhibitory concentrations (IC50s) were then determined using rCYPs. Cimetidine and famotidine both inhibited CYP3A4-mediated formation of noroxycodone and CYP2D6-mediated formation of oxymorphone, and famotidine inhibited CYP3A4-mediated formation of R- and S-EDDP, but IC50s were so high that only >10× therapeutic concentrations may have potential for reversible in vivo inhibition. The PPIs were more potent inhibitors; many have the potential for reversible in vivo inhibition at therapeutic concentrations. Omeprazole, esomeprazole and pantoprazole had greater effects on CYP3A4-mediated reactions, whereas lansoprazole was selective for CYP2D6-mediated formation of oxymorphone. Preincubation enhanced cimetidine inhibition of noroxycodone formation and rabeprazole inhibition of all pathways. Future studies will explore irreversible TDI.

  9. In vitro cytochrome P450 inhibition potential of methylenedioxy-derived designer drugs studied with a two-cocktail approach.

    PubMed

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2016-02-01

    In vitro cytochrome P450 (CYP) inhibition assays are common approaches for testing the inhibition potential of drugs for predicting potential interactions. In contrast to marketed medicaments, drugs of abuse, particularly the so-called novel psychoactive substances, were not tested before distribution and consumption. Therefore, the inhibition potential of methylenedioxy-derived designer drugs (MDD) of different drug classes such as aminoindanes, amphetamines, benzofurans, cathinones, piperazines, pyrrolidinophenones, and tryptamines should be elucidated. The FDA-preferred test substrates, split in two cocktails, were incubated with pooled human liver microsomes and analysed after protein precipitation using LC-high-resolution-MS/MS. IC50 values were determined of MDD showing more than 50 % inhibition in the prescreening. Values were calculated by plotting the relative metabolite concentration formed over the logarithm of the inhibitor concentration. All MDD showed inhibition against CYP2D6 activity and most of them in the range of the clinically relevant CYP2D6 inhibitors quinidine and fluoxetine. In addition, the beta-keto compounds showed inhibition of the activity of CYP2B6, 5,6-MD-DALT of CYP1A2 and CYP3A, and MDAI of CYP2A6, all in the range of clinically relevant inhibitors. In summary, all MDD showed inhibition of the activity of CYP2D6, six of CYP1A2, three of CYP2A6, 13 of CYP2B6, two of CYP2C9, six of CYP2C19, one of CYP2E1, and six of CYP3A. These results showed that the CYP inhibition by MDD might be clinically relevant, but further studies are needed for final conclusions.

  10. Mechanism of inhibition of purified leaping mullet (Liza saliens) NADPH-cytochrome P450 reductase by toxic metals: aluminum and thallium.

    PubMed

    Bozcaarmutlu, Azra

    2007-01-01

    Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 microM and 3 microM, respectively. The Lineweaver-Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The K(i) values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 microM, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase.

  11. Inhibition of rat respiratory-tract cytochrome P-450 isozymes following inhalation of m-Xylene: possible role of metabolites.

    PubMed

    Vaidyanathan, Anu; Foy, J W-D; Schatz, Robert

    2003-06-27

    Xylene is used as a solvent in paints, cleaning agents, and gasoline. Exposure occurs primarily by inhalation. The volatility and lipophilicity of the xylenes make the lung and nasal mucosa the primary target organs. m-Xylene (m-XYL) has been shown to alter cytochrome P-450 (CYP) activity in an organ- and isozyme-specific manner. The purpose of this work was to determine if the metabolism of m-XYL to the inhibitory metabolite m-tolualdehyde (m-ALD) is the cause of inhibition of CYP isozymes following in vivo inhalation exposure to m-XYL (100, 300 ppm), 3-methylbenzyl alcohol (3-MBA) (50, 100 ppm), or m-ALD (50, 100 ppm). A single 6-h inhalation exposure of rats to m-XYL inhibited pulmonary CYPs 2B1, 2E1, and 4B1 in a dose-dependent manner. Inhalation of 3-MBA inhibited pulmonary CYPs 2B1 and 4B1 in a dose-dependent manner. m-ALD inhibited pulmonary CYPs 2B1 and 2E1 in a dose-dependent manner, while 4B1 activity was increased dose dependently. Nasal mucosa CYP 2B1 and 2E1 activity was inhibited following exposure to m-XYL dose dependently, 3-MBA inhibited nasal mucosa CYPs 2E1 and 4B1 dose dependently. CYPs 2B1, 2E1, and 4B1 were inhibited in a dose-dependent fashion following inhalation of m-ALD. Following high-performance liquid chromatography (HPLC) analysis, m-ALD was detected after in vivo exposure to m-XYL, m-ALD, and 3-MBA in a dose-dependent manner, with highest m-ALD levels in the nasal mucosa and lung. Alteration of cytochrome P-450 activity by m-XYL could result in increased or decreased toxicity, changing the metabolic profiles of xenobiotics in coexposure scenarios in an organ-specific manner.

  12. In vitro hepatotoxicity and cytochrome P450 induction and inhibition characteristics of carnosic acid, a dietary supplement with antiadipogenic properties.

    PubMed

    Dickmann, Leslie J; VandenBrink, Brooke M; Lin, Yvonne S

    2012-07-01

    Carnosic acid is a phenolic diterpene isolated from rosemary (Rosmarinus officinalis), which may have anticancer, antiadipogenic, and anti-inflammatory properties. Recently, carnosic acid was shown to prevent weight gain and hepatic steatosis in a mouse model of obesity and type II diabetes. Based on these results, carnosic acid has been suggested as a potential treatment for obesity and nonalcoholic fatty liver disease; however, little is known about the safety of carnosic acid at doses needed to elicit a pharmacological effect. For this reason, hepatotoxicity and cytochrome P450 inhibition and induction studies were performed in primary human hepatocytes and microsomes. Measuring cellular ATP, carnosic acid showed a dose-dependent increase in hepatotoxicity with an EC(50) value of 94.8 ± 36.7 μM in three human hepatocyte donors without a concurrent increase in the apoptosis markers caspase-3/7. In human liver microsomes, carnosic acid did not exhibit significant time-dependent inhibition for any of the cytochrome P450 enzymes investigated, although it did inhibit CYP2C9- and CYP3A4-catalyzed reactions with K(i) values of 9.2 and 4.3 μM, respectively. Carnosic acid also induced CYP2B6 and CYP3A4 mRNA and enzyme activity in a dose-dependent manner. At 10 μM, carnosic acid increased CYP2B6 enzyme activity 61.6 and 49.3% in two donors compared with phenobarbital, and it increased CYP3A enzyme activity 82.6 and 142% compared with rifampicin. These results indicate the potential for drug interactions with carnosic acid and illustrate the need for an appropriate safety assessment before being used as a weight loss supplement.

  13. Structure–function relationships of inhibition of mosquito cytochrome P450 enzymes by flavonoids of Andrographis paniculata.

    PubMed

    Kotewong, Rattanawadee; Duangkaew, Panida; Srisook, Ekaruth; Sarapusit, Songklod; Rongnoparut, Pornpimol

    2014-09-01

    The cytochrome P450 monooxygenases are known to play a major role in pyrethroid resistance, by means of increased rate of insecticide detoxification as a result of their overexpression. Inhibition of detoxification enzymes may help disrupting insect detoxifying defense system. The Anopheles minimus CYP6AA3 and CYP6P7 have shown pyrethroid degradation activity and been implicated in pyrethroid resistance. In this study inhibition of the extracts and constituents of Andrographis paniculata Nees. leaves and roots was examined against benzyloxyresorufin O-debenzylation (BROD) of CYP6AA3 and CYP6P7. Four purified flavones (5,7,4′-trihydroxyflavone, 5-hydroxy-7,8-dimethoxyflavone, 5-hydroxy-7,8,2′,3′-tetramethoxyflavone, and 5,4′-dihydroxy-7,8,2′,3′-tetramethoxyflavone), one flavanone (5-hydroxy-7,8-dimethoxyflavanone) and a diterpenoid (14-deoxy-11,12-didehydroandrographolide) containing inhibitory effects toward both enzymes were isolated from A. paniculata. Structure–function relationships were observed for modes and kinetics of inhibition among flavones, while diterpenoid and flavanone were inferior to flavones. Docking of flavones onto enzyme homology models reinforced relationships on flavone structures and inhibition modes. Cell-based inhibition assays employing 3-(4,5-dimethylthiazol-2-y-l)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that these flavonoids efficiently increased susceptibility of CYP6AA3- and CYP6P7-expressing Spodoptera frugiperda (Sf9) cells to cypermethrin toxicity, due to inhibition effects on mosquito enzymes. Thus synergistic effects on cypermethrin toxicity of A. paniculata compounds as a result of enzyme inhibition could be useful for mosquito vector control and insecticide resistance management in the future.

  14. The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: chemical mechanism and physiological significance.

    PubMed

    Cooper, Chris E; Brown, Guy C

    2008-10-01

    The four gases, nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H(2)S) and hydrogen cyanide (HCN) all readily inhibit oxygen consumption by mitochondrial cytochrome oxidase. This inhibition is responsible for much of their toxicity when they are applied externally to the body. However, recently these gases have all been implicated, to greater or lesser extents, in normal cellular signalling events. In this review we analyse the chemistry of this inhibition, comparing and contrasting mechanism and discussing physiological consequences. The inhibition by NO and CO is dependent on oxygen concentration, but that of HCN and H(2)S is not. NO and H(2)S are readily metabolised by oxidative processes within cytochrome oxidase. In these cases the enzyme may act as a physiological detoxifier of these gases. CO oxidation is much slower and unlikely to be as physiologically important. The evidence for normal physiological levels of these gases interacting with cytochrome oxidase is equivocal, in part because there is little robust data about their steady state concentrations. A reasonable case can be made for NO, and perhaps CO and H(2)S, inhibiting cytochrome oxidase in vivo, but endogenous levels of HCN seem unlikely to be high enough.

  15. Inhibition of cytochrome P450 3A4 activity by schisandrol A and gomisin A isolated from Fructus Schisandrae chinensis.

    PubMed

    Wan, C-K; Tse, A K; Yu, Z-L; Zhu, G-Y; Wang, H; Fong, D W F

    2010-07-01

    We studied the effects of schisandrol A (SCH) and gomisin A (GOM), two of the main bioactive components of Fructus Schisandrae chinensis, on cytochrome P450-3A4 (CYP3A4) activity and cellular glutathione (GSH) level. In a cell-free system both SCH and GOM inhibited CYP3A4 activity with IC(50) values of 32.02 microM and 1.39 microM, respectively. SCH or GOM at concentrations up to 100 microM did not alter cellular GSH level in regular HepG2 cells and P-glycoprotein overexpressing HepG2-DR cells. Since SCH and GOM may reverse multidrug resistance (MDR) by impeding the activity of P-glycoprotein, a membrane xenobiotic exporter, SCH or GOM could affect cellular drug metabolism in addition to drug uptake.

  16. A dual inhibition: microRNA-552 suppresses both transcription and translation of cytochrome P450 2E1.

    PubMed

    Miao, Lingling; Yao, Hailan; Li, Chenggang; Pu, Mengfan; Yao, Xuan; Yang, Hui; Qi, Xinming; Ren, Jin; Wang, Yizheng

    2016-04-01

    MicroRNAs (miRNAs) can direct post-transcriptional or transcriptional gene silencing. Here, we report that miR-552 is in the nucleus and cytosol and inhibits human cytochrome P450 (CYP) 2E1 expression at both transcriptional and post-transcriptional levels. MiR-552 via its non-seed sequence forms hybrids with a loop hairpin of the cruciform structure in CYP2E1 promoter region to inhibit SMARCE1 and RNA polymerase II binding to the promoter and CYP2E1 transcription. Expressing SMARCE1 reverses the inhibitory effects of miR-552 on CYP2E1 mRNA expression. MiR-552 with mutations in non-seed region losses its transcriptional, but retains its post-transcriptional repression to CYP2E1. In contrast, mutation in miR-552 seed sequence suppresses its inhibitory effects on CYP2E1 expression at protein, but not at mRNA, levels. Our results suggest that miR-552 is a miRNA with a dual inhibitory ability at transcriptional and post-transcriptional levels leading to an effective inhibition.

  17. Zinc inhibition of bacterial cytochrome bc1 reveals the role of cytochrome b E295 in proton release at the Qo site†

    PubMed Central

    Lee, Dong-Woo; Khoury, Youssef El; Francia, Francesco; Zambelli, Barbara; Ciurli, Stefano; Venturoli, Giovanni; Hellwig, Petra; Daldal, Fevzi

    2011-01-01

    The cytochrome (cyt) bc1 complex (cyt bc1) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc1 is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn2+-induced inhibition of Rhodobacter capsulatus cyt bc1 using enzyme kinetics, isothermal titration calorimetry (ITC) and electrochemically-induced FTIR difference spectroscopy with the purpose to understand the Zn2+-binding mechanism and its inhibitory effect on cyt bc1 function. Analogous studies were carried out on a mutant of cyt b, E295, a residue previously proposed to bind Zn2+ on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 into valine, a non-coordinating residue, results in the reduction of Zn2+-binding affinity. The kinetic study showed that wild-type cyt bc1 and its E295V mutant have similar levels of apparent Km values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 μM and 3.1 ± 0.4 μM, respectively), whereas their KI values for Zn2+ are 8.3 μM and 38.5 μM, respectively. The calorimetry-based KD values for the high affinity site of cyt bc1 are of the same order of magnitude as the KI values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn2+, whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn2+. Our overall results indicate that the proton-active E295 residue near the Qo site of cyt bc1 can bind directly to Zn2+, resulting in a decrease of the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to

  18. Inhibition of testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/C-17,20-lyase) by estrogens.

    PubMed

    Onoda, M; Hall, P F

    1981-09-01

    Highly purified cytochrome P-450 from neonatal pig testicular microsomes is capable of catalyzing both 17 alpha-hydroxylation and C-17,20-lyase activity. Estradiol was found to inhibit both activities of the purified enzyme with delta 4 and with delta 5 substrates (progesterone, pregnenolone, and the corresponding 17 alpha-hydroxysteroids). For the delta 4 series, inhibition of lyase is competitive and that of 17 alpha-hydroxylase is noncompetitive; for the delta 5 series, inhibition was noncompetitive for both activities. Ki values for lyase activity were determined from enzyme kinetics (5.0 microM for the delta 4 substrate and 20 microM for the delta 5 substrate). Estradiol produces a typical type I spectral shift with the pure enzyme (Ks = 3.0 microM where Ks is the concentration of steroid required to give half maximal spectral shift), so that Ki values were also determined directly from binding studies by using substrate-induced difference spectroscopy. Fifty per cent inhibition of the maximal spectral shift induced by the 17 alpha-hydroxysubstrates (Ki) are 3.8 and 7.6 microM for the delta 4 and delta 5 substrates, respectively. Values for Ki are higher with the substrates of 17 alpha-hydroxylase (progesterone and pregnenolone), by either method, than the corresponding Ki values for the lyase substrates. The concentration of estradiol in Leydig cells of neonatal pig testis is approximately 1.5 nmol/g. It is proposed that estradiol may influence testicular steroidogensis under physiological conditions by competitive inhibition of lyase activity.

  19. Substrate specificity of human liver cytochrome P-450 debrisoquine 4-hydroxylase probed using immunochemical inhibition and chemical modeling.

    PubMed

    Wolff, T; Distlerath, L M; Worthington, M T; Groopman, J D; Hammons, G J; Kadlubar, F F; Prough, R A; Martin, M V; Guengerich, F P

    1985-05-01

    A significant population of humans (5 to 10%) are phenotypic poor metabolizers of debrisoquine. We have isolated the cytochrome P-450 isozyme from rat liver responsible for this activity and have shown that antibodies raised against the protein are able to inhibit this catalytic activity in human liver microsomes (Distlerath, L. M., and Guengerich, F. P., Proc. Natl. Acad. Sci. USA, 81: 7348-7352, 1984). These antibodies were utilized to determine which metabolic transformations are linked to debrisoquine 4-hydroxylation in human liver microsomes using techniques of immunochemical inhibition. The antibodies almost completely inhibited debrisoquine 4-hydroxylation and bufuralol 1'-hydroxylation in microsomes prepared from several different human livers. The oxidation of the pyrrolizidine alkaloids lasiocarpine and monocrotaline were inhibited by roughly one-third. The antibodies did not inhibit N,N-dimethylnitrosamine N-demethylation, oxidation of vinylidene chloride to 2,2-chloroacetaldehyde, oxidation of trichloroethylene to chloral, N-oxidation of azoprocarbazine, morphine N-demethylation, diazepam N-demethylation, oxidation of benzo(a)pyrene to alkali-soluble metabolites, oxidation of benzo(a)pyrene 7,8-dihydrodiol to products covalently bound to DNA, the N- and ring-oxidation of 1- and 2-naphthylamine and 2-aminofluorene, or the conversion of aflatoxin B1 to DNA adducts or aflatoxin Q1. Studies with space-filling models of the drugs the metabolism of which is associated with debrisoquine 4-hydroxylase in the literature indicated that all can be fitted to a general structure in which a basic nitrogen is about 5 A away from the site of carbon hydroxylation and a hydrophobic domain is near the site of hydroxylation. These results may be useful in predicting which chemicals may or may not be metabolized in an atypical manner by a segment of the human population.

  20. Integrated structure- and ligand-based in silico approach to predict inhibition of cytochrome P450 2D6.

    PubMed

    Martiny, Virginie Y; Carbonell, Pablo; Chevillard, Florent; Moroy, Gautier; Nicot, Arnaud B; Vayer, Philippe; Villoutreix, Bruno O; Miteva, Maria A

    2015-12-15

    Cytochrome P450 (CYP) is a superfamily of enzymes responsible for the metabolism of drugs, xenobiotics and endogenous compounds. CYP2D6 metabolizes about 30% of drugs and predicting potential CYP2D6 inhibition is important in early-stage drug discovery. We developed an original in silico approach for the prediction of CYP2D6 inhibition combining the knowledge of the protein structure and its dynamic behavior in response to the binding of various ligands and machine learning modeling. This approach includes structural information for CYP2D6 based on the available crystal structures and molecular dynamic simulations (MD) that we performed to take into account conformational changes of the binding site. We performed modeling using three learning algorithms--support vector machine, RandomForest and NaiveBayesian--and we constructed combined models based on topological information of known CYP2D6 inhibitors and predicted binding energies computed by docking on both X-ray and MD protein conformations. In addition, we identified three MD-derived structures that are capable all together to better discriminate inhibitors and non-inhibitors compared with individual CYP2D6 conformations, thus ensuring complementary ligand profiles. Inhibition models based on classical molecular descriptors and predicted binding energies were able to predict CYP2D6 inhibition with an accuracy of 78% on the training set and 75% on the external validation set. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5'-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

    PubMed

    Kim, Ju-Hyun; Kwon, Soon-Sang; Kong, Tae Yeon; Cheong, Jae Chul; Kim, Hee Seung; In, Moon Kyo; Lee, Hye Suk

    2017-03-10

    AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography-tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

  2. Bioactivation of dibrominated biphenyls by cytochrome P450 activity to metabolites with estrogenic activity and estrogen sulfotransferase inhibition capacity.

    PubMed

    van Lipzig, Marola M H; Commandeur, Jan N; de Kanter, Frans J J; Damsten, Micaela C; Vermeulen, Nico P E; Maat, Evelina; Groot, Ed J; Brouwer, Abraham; Kester, Monique H A; Visser, Theo J; Meerman, John H N

    2005-11-01

    Exposure of humans and wildlife to xenobiotics, such as halogenated biphenyls, that interfere with the endogenous estrogen balance may lead to endocrine disruption. Such compounds may either mimic or block estradiol's action by agonistic or antagonistic action, respectively. They may also affect endogenous estradiol concentrations by induction or inhibition of enzymes that metabolize estradiol. In the present study, we demonstrate that estrogenic metabolites of two brominated biphenyls, 2,2'-dibromobiphenyl (2,2'-DBB) and 4,4'-dibromobiphenyl (4,4'-DBB), are formed by rat liver microsomal cytochrome P450 (CYP) activity. Bioactivation of 2,2'-DBB and 4,4'-DBB yielded various mono- and dihydroxylated bromobiphenyl metabolites, which were collected by preparative HPLC and analyzed by LC/MS. Several of the metabolites bound to the estrogen receptor (ER) activated the ER and inhibited human estrogen sulfotransferase (hEST). Seven monohydroxylated metabolites were positively identified using synthetic monohydroxylated reference compounds. These synthetic monohydroxylated bromobiphenyls also bound to and activated the ER and inhibited hEST. The highest ER affinity was observed for 4-OH-2,2'-DBB, with an EC50 of 6.6 nM. The highest ER activation was observed for 4-OH-3,4'-DBB (EC50 of 74 nM) while 4-OH-4'-MBB and 4-OH-2,2'-DBB induced a supramaximal (as compared to estradiol) ER activation. The strongest hEST inhibition was found with 4-OH-3,4'-DBB (EC50 = 40 nM). In conclusion, we show that two dibrominated biphenyls are bioactivated by CYP activity into very potent estrogenic metabolites and inhibitors of hEST. These findings are of vital importance for accurate risk assessment of exposure to environmental contaminants, such as halogenated biphenyls. Neglecting bioactivation through biotransformation will lead to underestimation of health risks of this class of xenobiotics.

  3. Cytochrome P450 2D6 and 3A4 enzyme inhibition by amine stimulants in dietary supplements.

    PubMed

    Liu, Yitong; Santillo, Michael F

    2016-01-01

    A number of dietary supplements used for weight loss and athletic performance enhancement have been recently shown to contain a variety of stimulants, for which there is a lack of pharmacological and toxicological information. One concern for these emerging compounds is their potential to inhibit metabolic enzymes in the liver such as cytochromes P450 (CYP), which can lead to unexpected interactions among dietary supplements, drugs, and other xenobiotics. In this study, inhibition of human recombinant CYP2D6 and CYP3A4 by 27 amine stimulants associated with dietary supplements and their analogs was evaluated by luminescence assays. The strongest CYP2D6 inhibitors were coclaurine (IC50  = 0.14 ± 0.01 μM) and N-benzylphenethylamine (IC50  = 0.7 ± 0.2 μM), followed by several other relatively strong inhibitors (IC50 , 2-12 μM) including β-methylphenethylamine, N,β-dimethylphenethylamine (phenpromethamine), 1,3-dimethylamylamine (DMAA), N,α-diethylphenethylamine, higenamine (norcoclaurine) and N,N-diethylphenethylamine. Only nine compounds inhibited CYP3A4 by 20-55% at 100 μM. Results of this study illustrate that several amine stimulants associated with dietary supplements inhibit CYP2D6 and CYP3A4 in vitro, and these compounds may participate in adverse drug-dietary supplement interactions in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    PubMed

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

    2013-09-05

    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

  5. High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions

    PubMed Central

    Li, Guannan; Huang, Ke; Nikolic, Dejan

    2015-01-01

    Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry–based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography–tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example. PMID:26285764

  6. Studies on Inhibition of Respiratory Cytochrome bc1 Complex by the Fungicide Pyrimorph Suggest a Novel Inhibitory Mechanism

    PubMed Central

    Xiao, Yu-Mei; Esser, Lothar; Zhou, Fei; Li, Chang; Zhou, Yi-Hui; Yu, Chang-An; Qin, Zhao-Hai; Xia, Di

    2014-01-01

    The respiratory chain cytochrome bc1 complex (cyt bc1) is a major target of numerous antibiotics and fungicides. All cyt bc1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site. The primary cause of resistance to bc1 inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc1 to overcome resistance. Pyrimorph, a synthetic fungicide, inhibits the growth of a broad range of plant pathogenic fungi, though little is known concerning its mechanism of action. In this study, using isolated mitochondria from pathogenic fungus Phytophthora capsici, we show that pyrimorph blocks mitochondrial electron transport by affecting the function of cyt bc1. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc1 with IC50 values of 85.0 μM and 69.2 μM, respectively, indicating that it targets the essential subunits of cyt bc1 complexes. Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the QP site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol. In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site. PMID:24699450

  7. High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions.

    PubMed

    Li, Guannan; Huang, Ke; Nikolic, Dejan; van Breemen, Richard B

    2015-11-01

    Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry-based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography-tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  8. Selective inhibition by chloramphenicol of pregnenolone-16. cap alpha. -carbonitrile-inducible rat liver cytochrome P-450 isozymes

    SciTech Connect

    Graves, P.E.; Kaminsky, L.S.; Halpert, J.

    1986-03-01

    Pregnenolone-16 ..cap alpha..-carbonitrile (PCN) has been shown to induce, in male rats, cytochrome P-450 isozymes responsible for the formation of R-10-hydroxywarfarin and R-dehydrowarfarin. Antibodies to the major PCN-inducible isozyme (PB/PCN-E) inhibit both activities in microsomal preparations. Recently the authors have shown that PCN treatment of female rats also induces the formation of both R-warfarin metabolites. However, in both sexes chloramphenicol (CAP) treatment selectively inhibits only the rate of formation of the R-dehydrowarfarin. A decrease in microsomal P-450 content occurs after in vivo administration of CAP to PCN-treated rats of both sexes. This is in contrast to the lack of effect of CAP on P-450 levels in phenobarbital-treated rats. Covalent binding of /sup 14/C-CAP to microsomal protein in vitro was increased 3 to 4-fold following PCN treatment. Chromatographic evidences suggests the presence of at least two PCN-induced isozymes of similar molecular weights in both male and female rat liver microsomes. These data are consistent with the multiplicity of PCN-inducible P-450 in rat liver.

  9. Differential time course of cytochrome P450 2D6 enzyme inhibition by fluoxetine, sertraline, and paroxetine in healthy volunteers.

    PubMed

    Liston, Heidi L; DeVane, C Lindsay; Boulton, David W; Risch, Samuel C; Markowitz, John S; Goldman, Juliet

    2002-04-01

    The selective serotonin reuptake inhibitors (SSRIs) paroxetine, sertraline, and fluoxetine have varying degrees of potency in inhibiting the hepatic cytochrome P450 (CYP) 2D6 enzyme. However, the time course for maximum inhibition to occur or for inhibition to dissipate when dosing is discontinued, requires clarification. In an open label, parallel group study of 45 healthy volunteers, the time course of CYP2D6 inhibition of the above SSRIs was evaluated. Subjects were randomized to receive paroxetine at 20 mg/day for 10 days; sertraline at 50 mg/day for 3 days, followed by sertraline at 100 mg/day for 10 days; or fluoxetine at 20 mg/day for 28 days. CYP2D6 activity was assessed using the dextromethorphan metabolic ratio (DMR) on antidepressant days 5 and 10 for sertraline and paroxetine and at weekly intervals for fluoxetine. Following SSRI discontinuation, calculation of a CYP2D6 inhibition half-life (t(1/2)inh) revealed the time course of fluoxetine inhibition (t(1/2)inh = 7.0 +/- 1.5 days) to be significantly longer than either paroxetine (t(1/2)inh = 2.9 +/- 1.9) or sertraline (t(1/2)inh = 3.0 +/- 3.0) (p < 0.01), but the latter were not significantly different from each other (p > 0.05). Time for the extrapolated DMR versus time log-linear plots to return to baseline was significantly different between fluoxetine (63.2 +/- 5.6 days) and both paroxetine (20.3 +/- 6.4 days) and sertraline (25.0 +/- 11.0 days) (p < 0.01), making the rank order (from longest to shortest) of time for CYP2D6 inhibition to dissipate: fluoxetine > sertraline >or= paroxetine. Differences between mean baseline DMR values and measured values obtained after drug discontinuation for each drug group became nonsignificant on discontinuation day 5 for both paroxetine and sertraline and on discontinuation day 42 for fluoxetine. These data define the time course of a persistent effect that fluoxetine, sertraline, and paroxetine have on CYP2D6 following drug discontinuation and should be

  10. Inhibition of human drug-metabolising cytochrome P450 and UDP-glucuronosyltransferase enzyme activities in vitro by uremic toxins.

    PubMed

    Barnes, Kyra J; Rowland, Andrew; Polasek, Thomas M; Miners, John O

    2014-09-01

    To investigate the potential inhibitory effects of uremic toxins on the major human hepatic drug-metabolising cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes in vitro. Benzyl alcohol, p-cresol, indoxyl sulfate, hippuric acid and a combination of the four uremic toxins were co-incubated with human liver microsomes and selective probe substrates for the major human drug-metabolising CYP and UGT enzymes. The percentage of enzyme inhibition was calculated by measuring the rates of probe metabolite formation in the absence and presence of the uremic toxins. Kinetics studies were conducted to evaluate the K i values and mechanism(s) of the inhibition of CYP2E1, CYP3A4, UGT1A1 and UGT1A9 by p-cresol. The individual uremic toxins inhibited CYP and UGT enzymes to a variable extent. p-Cresol was the most potent individual inhibitor, producing >50% inhibition of CYP2E1, CYP3A4, UGT1A1, UGT1A9 and UGT2B7 at a concentration of 100 μM. The greatest inhibition was observed with UGT1A9. p-Cresol was shown to be an uncompetitive inhibitor of UGT1A9, with unbound K i values of 9.1 and 2.5 μM in the absence and presence of bovine serum albumin (BSA), respectively. K i values for p-cresol inhibition of human liver microsomal CYP2E1, CYP3A4 and UGT1A1 ranged from 43 to 89 μM. A combination of the four uremic toxins produced >50% decreases in the activities of CYP1A2, CYP2C9, CYP2E1, CYP3A4, UGT1A1, UGT1A9 and UGT2B7. Uremic toxins may contribute to decreases in drug hepatic clearance in individuals with kidney disease by inhibition of hepatic drug-metabolising enzymes.

  11. Atovaquone and ELQ-300 Combination Therapy as a Novel Dual-Site Cytochrome bc1 Inhibition Strategy for Malaria

    PubMed Central

    Smilkstein, Martin J.; Morrisey, Joanne M.; Li, Yuexin; Forquer, Isaac P.; Kelly, Jane X.; Pou, Sovitj; Winter, Rolf W.; Nilsen, Aaron; Vaidya, Akhil B.

    2016-01-01

    Antimalarial combination therapies play a crucial role in preventing the emergence of drug-resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc1 complex (cyt bc1) are among the few compounds that are effective for both acute antimalarial treatment and prophylaxis. There are two known sites for inhibition within cyt bc1: atovaquone (ATV) blocks the quinol oxidase (Qo) site of cyt bc1, while some members of the endochin-like quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinone reductase (Qi) site and retain full potency against ATV-resistant Plasmodium falciparum strains with Qo site mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and combination therapy consisting of ATV plus ELQ-300 (ATV:ELQ-300), using P. yoelii murine models of malaria. In our monotherapy assessments, we found that ATV functioned as a single-dose curative compound in suppressive tests whereas ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked recrudescence even in a high-parasitemia acute infection model. ATV:ELQ-300 therapy was highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg of body weight. Compared to the ATV:proguanil (Malarone) formulation, ATV:ELQ-300 was more efficacious in multiday, acute infection models and was equally effective at blocking the emergence of ATV-resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt bc1 is a valuable strategy for antimalarial combination therapy and that Qi site inhibitors such as ELQ-300 represent valuable partner drugs for the clinically successful Qo site inhibitor ATV. PMID:27270285

  12. Effect of cytochrome P450 2D1 inhibition on hydrocodone metabolism and its behavioral consequences in rats.

    PubMed

    Tomkins, D M; Otton, S V; Joharchi, N; Li, N Y; Balster, R F; Tyndale, R F; Sellers, E M

    1997-03-01

    Humans that lack cytochrome P450 2D6 (CYP2D6) activity may have an altered risk of drug dependence or abuse because this enzyme is important in the metabolism of some drugs of abuse, including hydrocodone. In rats, hydrocodone conversion to hydromorphone is catalyzed by CYP2D1, the rat homolog of the human CYP2D6. To determine the impact of impaired hydromorphone formation on the behavioral effects of the parent compound, hydrocodone-induced analgesia and hyperactivity, hydrocodone discrimination and self-administration were examined in male Wistar rats, with or without pretreatment with CYP2D1 inhibitors (quinine and budipine). In vivo, quinine (20 mg/kg) and budipine (10 mg/kg) produced a marked suppression in brain and plasma hydromorphone levels detected after the peripheral administration of hydrocodone, thus confirming that the doses used suppressed CYP2D1 activity. In contrast, CYP2D1 inhibition had no impact on the analgesic or discriminative stimulus effects of hydrocodone, nor did this type of manipulation alter hydrocodone self-administration. The effects of quinine on the locomotor activating effects of hydrocodone were subtle at best. Because inhibition of CYP2D1 in this rat strain is proposed to be a useful animal counterpart for studying the impact of CYP2D6 polymorphism in humans, these data suggest that differences in CYP2D6 phenotype will have limited influence on the drug response to hydrocodone after nonoral administration. This has recently been verified in a study showing that inhibition of hydrocodone biotransformation to hydromorphone does not affect measures of abuse liability. Therefore, hydrocodone's behavioral effects are most likely attributable to its own intrinsic effects at mu opioid receptors.

  13. Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy.

    PubMed

    Mast, Natalia; Lin, Joseph B; Pikuleva, Irina A

    2015-09-01

    Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6- and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification.

  14. Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy

    PubMed Central

    Mast, Natalia; Lin, Joseph B.

    2015-01-01

    Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6- and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification. PMID:26082378

  15. Atovaquone and ELQ-300 Combination Therapy as a Novel Dual-Site Cytochrome bc1 Inhibition Strategy for Malaria.

    PubMed

    Stickles, Allison M; Smilkstein, Martin J; Morrisey, Joanne M; Li, Yuexin; Forquer, Isaac P; Kelly, Jane X; Pou, Sovitj; Winter, Rolf W; Nilsen, Aaron; Vaidya, Akhil B; Riscoe, Michael K

    2016-08-01

    Antimalarial combination therapies play a crucial role in preventing the emergence of drug-resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc1 complex (cyt bc1) are among the few compounds that are effective for both acute antimalarial treatment and prophylaxis. There are two known sites for inhibition within cyt bc1: atovaquone (ATV) blocks the quinol oxidase (Qo) site of cyt bc1, while some members of the endochin-like quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinone reductase (Qi) site and retain full potency against ATV-resistant Plasmodium falciparum strains with Qo site mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and combination therapy consisting of ATV plus ELQ-300 (ATV:ELQ-300), using P. yoelii murine models of malaria. In our monotherapy assessments, we found that ATV functioned as a single-dose curative compound in suppressive tests whereas ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked recrudescence even in a high-parasitemia acute infection model. ATV:ELQ-300 therapy was highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg of body weight. Compared to the ATV:proguanil (Malarone) formulation, ATV:ELQ-300 was more efficacious in multiday, acute infection models and was equally effective at blocking the emergence of ATV-resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt bc1 is a valuable strategy for antimalarial combination therapy and that Qi site inhibitors such as ELQ-300 represent valuable partner drugs for the clinically successful Qo site inhibitor ATV.

  16. In vitro inhibition of cytochrome P450 3A4 by Aronia melanocarpa constituents.

    PubMed

    Bräunlich, Marie; Christensen, Hege; Johannesen, Siri; Slimestad, Rune; Wangensteen, Helle; Malterud, Karl E; Barsett, Hilde

    2013-01-01

    Extracts, subfractions, isolated anthocyanins and procyanidins, and two phenolic acids from aronia [Aronia melanocarpa] were investigated for their CYP3A4 inhibitory effects, using midazolam as the probe substrate and recombinant insect cell microsomes expressing CYP3A4 as the enzyme source. Procyanidin B5 was a considerably stronger CYP3A4 inhibitor in vitro than the isomeric procyanidin B2 and comparable to bergamottin, a known CYP3A4 inhibitor from grapefruit juice. The inhibitory activity of proanthocyanidin-containing fractions was correlated to the degree of polymerization. Among the anthocyanins, cyanidin 3-arabinoside showed stronger CYP3A4 inhibition than cyanidin 3-galactoside and cyanidin 3-glucoside. Thus, the ability to inhibit CYP3A4 in vitro seems to be influenced by the sugar unit linked to the anthocyanidin. Georg Thieme Verlag KG Stuttgart · New York.

  17. Metabolism profiling, and cytochrome P450 inhibition & induction in drug discovery.

    PubMed

    Yan, Z; Caldwell, G W

    2001-11-01

    To reduce the high attrition rates of NCEs in preclinical and clinical development uncovering pharmacokinetics, toxicokinetics, drug metabolism, and drug-drug interactions early in drug discovery would be highly valuable. There have been many in vitro screens developed for these areas that have higher sample throughput, which is consistent with the iterative cycle of a typical drug discovery research project. We have presented the present status and given detailed descriptions of biotransformation, metabolic stability assays, identification of drug metabolizing P450 enzymes, prediction of pharmacokinetic parameters from in vitro metabolism data, structure elucidation of metabolites, CYP450 inhibition assays and CYP450 induction assays from a drug discovery perspective. Strategies for the proper sequencing of primary and secondary assays employedfor drug metabolism and CYP450 inhibition & induction is discussed.

  18. Glycyrrhetinic acid might increase the nephrotoxicity of bakuchiol by inhibiting cytochrome P450 isoenzymes

    PubMed Central

    Zhao, Zijing; Yuan, Mei

    2016-01-01

    Background Licorice, a popular traditional Chinese medicine (TCM), is widely used to moderate the effects (detoxification) of other herbs in TCM and often combined with Fructus Psoraleae. However, the classical TCM book states that Fructus Psoraleae is incompatible with licorice; the mechanism underlying this incompatibility has not been identified. Glycyrrhetinic acid (GA), the active metabolite of licorice, may increase the toxicity of bakuchiol (BAK), the main chemical ingredient in Psoralea corylifolia, by inhibiting its detoxification enzymes CYP450s. Methods The effect of concomitant GA administration on BAK-induced nephrotoxicity was investigated, and the metabolic interaction between BAK and GA was further studied in vitro and in vivo. The cytotoxicity was assessed using an MTT assay in a co-culture model of HK-2 cell and human liver microsomes (HLMs). The effect of GA on the metabolism of BAK, and on the activities of CYP isoforms were investigated in HLMs. The toxicokinetics and tissue exposure of BAK as well as the renal and hepatic functional markers were measured after the administration of a single oral dose in rats. Results In vitro studies showed that the metabolic detoxification of BAK was significantly reduced by GA, and BAK was toxic to HK-2 cells, as indicated by 25∼40% decreases in viability when combined with GA. Further investigation revealed that GA significantly inhibited the metabolism of BAK in HLMs in a dose-dependent manner. GA strongly inhibits CYP3A4 and weakly inhibits CYP2C9 and CYP1A2; these CYP isoforms are involved in the metabolism of BAK. In vivo experiment found that a single oral dose of BAK combined with GA or in the presence of 1-aminobenzotriazole (ABT), altered the toxicokinetics of BAK in rats, increased the internal exposure, suppressed the elimination of BAK prototype, and therefore may have enhanced the renal toxicity. Conclusion The present study demonstrated that GA inhibits CYP isoforms and subsequently may

  19. Time-dependent inhibition of human drug metabolizing cytochromes P450 by tricyclic antidepressants

    PubMed Central

    Polasek, Thomas M; Miners, John O

    2008-01-01

    AIMS To investigate time-dependent inhibition (TDI) of human drug metabolizing CYP enzymes by tricyclic antidepressants (TCAs). METHODS CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A/CYP3A4 activities were investigated following co- and preincubation with TCAs using human liver microsomes (HLM) and human recombinant CYP proteins (expressed in Escherichia coli) as the enzyme sources. A two-step incubation method was employed to examine the in vitro mechanism-based inactivation (MBI) criteria. Potential metabolite–intermediate complex (MIC) formation was studied by spectral analysis. RESULTS TCAs generally exhibited significant TDI of recombinant CYP1A2, CYP2C19 and CYP2D6 (>10% positive inhibition differences between co- and preincubation conditions). TDI of recombinant CYP2C9 was minor (<10%), and was minor or absent in experiments utilizing recombinant CYP3A4 or HLM as the enzyme sources. Where observed, TDI of recombinant CYP occurred via alkylamine MIC formation, but evidence to support similar behaviour in HLM was limited. Indeed, only secondary amine TCAs reduced the apparent P450 content of HLM (3–6%) consistent with complexation. As a representative TCA, nortriptyline fulfilled the in vitro MBI criteria using recombinant CYP2C19 and CYP3A4 (KI and kinact values of 4 µm and 0.19 min−1, and 70 µm and 0.06 min−1), but not with the human liver microsomal enzymes. CONCLUSIONS TCAs appear to have minimal potential for MBI of human liver microsomal CYP enzymes involved in drug metabolism. HLM and recombinant CYP (expressed in E. coli) are not equivalent enzyme sources for evaluating the TDI associated with some drugs. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Much of the literature evidence for mechanism-based inactivation (MBI) of CYP by tricyclic antidepressants is limited to studies in rat liver microsomes. One report from this laboratory characterized MBI of human recombinant CYP2C8 by nortriptyline. WHAT THIS STUDY ADDS Tricyclic antidepressants form

  20. Inhibition of cytochrome P450 enzymes in the Australian brushtail possum, Trichosurus vulpecula: comparison with that of the rat, rabbit, sheep and chicken.

    PubMed

    Olkowski, A; Gooneratne, R; Eason, C

    1998-08-01

    A comparative study was conducted of the inhibition of liver microsomal cytochrome P450 phase I biotransformation enzyme activity of the Australian brushtail possum, rat, rabbit, sheep and chicken. The possum has caused considerable agricultural and ecological problems since its introduction to New Zealand. This work investigated species differences in cytochrome P450 inhibition by selected imidazole derivatives that may be exploited for designing a more species-specific method of toxicological control of the New Zealand possum population. The imidazole derivatives used were ketoconazole, clotrimazole, miconazole and cimetidine. The potency of these inhibitors varied, with clotrimazole and miconazole being most potent, followed by ketoconazole. Cimetidine was the least effective inhibitor. The inhibitory effect of imidazole derivatives on cytochrome p450 phase I biotransformation enzymes appeared more effective in the possum than in other species. All inhibitors used produced type II spectra upon interaction with cytochrome P450 preparations. Possum and chicken microsomal preparations showed absorbancy maxima at 428 nm, rabbit and rat and 429 nm, and sheep at 431 nm.

  1. Effects of Cytochrome P450 Inhibition and Induction on the Phenotyping Metrics of the Basel Cocktail: A Randomized Crossover Study.

    PubMed

    Derungs, Adrian; Donzelli, Massimiliano; Berger, Benjamin; Noppen, Christoph; Krähenbühl, Stephan; Haschke, Manuel

    2016-01-01

    Activity of human cytochrome P450 enzymes (CYPs) shows high inter-and intra-individual variability, which is determined by genetic and non-genetic factors. Using a combination of CYP-specific probe drugs, phenotyping cocktails allow simultaneous assessment of the activity of different CYP isoforms. The objective of this study was to characterize the phenotyping metrics of the Basel cocktail in healthy male subjects with induced and inhibited CYP activity. In a randomized crossover study, the probe drugs for simultaneous phenotyping of CYP1A2 (caffeine), CYP2B6 (efavirenz), CYP2C9 (losartan), 2C19 (omeprazole), CYP2D6 (metoprolol), and CYP3A4 (midazolam) were administered to 16 subjects without pretreatment (baseline), after pretreatment with a combination of CYP inhibitors (ciprofloxacin, ketoconazole, and paroxetine), and after CYP induction with rifampicin. All subjects were genotyped. Pharmacokinetic profiles of the probe drugs and their main metabolites and metabolic ratios 2, 4, 6, and 8 h after probe drug application were determined in plasma and compared with the corresponding area under the plasma concentration-time curve (AUC) ratios. The Basel phenotyping cocktail was well tolerated by all subjects independent of pretreatment. Good correlations of metabolic ratios with AUC ratios of the corresponding probe drugs and their metabolites for all three conditions (baseline, CYP inhibition, and CYP induction) were found at 2 h after probe drug administration for CYP3A4, at 4 h for CYP1A2 and CYP2C19, and at 6 h for CYP2B6 and CYP2D6. While CYP inhibition significantly changed AUC ratios and metabolic ratios at these time points for all six CYP isoforms, CYP induction did not significantly change AUC ratios for CYP2C9. For CYP3A4, total 1'-hydroxymidazolam concentrations after pretreatment of samples with β-glucuronidase were needed to obtain adequate reflection of CYP induction by the metabolic ratio. Inhibition of CYP activity can be detected with the

  2. Inhibition of Mitochondrial Cytochrome c Release and Suppression of Caspases by Gamma-Tocotrienol Prevent Apoptosis and Delay Aging in Stress-Induced Premature Senescence of Skin Fibroblasts

    PubMed Central

    Makpol, Suzana; Abdul Rahim, Norhazira; Kien Hui, Chua; Wan Ngah, Wan Zurinah

    2012-01-01

    In this study, we determined the molecular mechanism of γ-tocotrienol (GTT) in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs). Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal) and promoted G0/G1 cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (P < 0.05). GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (P < 0.05). Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (P < 0.05) in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins. PMID:22919441

  3. Inhibition of mitochondrial cytochrome c release and suppression of caspases by gamma-tocotrienol prevent apoptosis and delay aging in stress-induced premature senescence of skin fibroblasts.

    PubMed

    Makpol, Suzana; Abdul Rahim, Norhazira; Hui, Chua Kien; Ngah, Wan Zurinah Wan

    2012-01-01

    In this study, we determined the molecular mechanism of γ-tocotrienol (GTT) in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs). Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal) and promoted G(0)/G(1) cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (P < 0.05). GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (P < 0.05). Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (P < 0.05) in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins.

  4. Organophosphorothionate pesticides inhibit the bioactivation of imipramine by human hepatic cytochrome P450s

    SciTech Connect

    Di Consiglio, Emma; Meneguz, Annarita; Testai, Emanuela . E-mail: testai@iss.it

    2005-06-15

    The drug-toxicant interaction between the antidepressant imipramine (IMI) and three organophosphorothionate pesticides (OPTs), to which humans may be chronically and simultaneously exposed, has been investigated in vitro. Concentrations of IMI (2-400 {mu}M) and OPTs ({<=}10 {mu}M) representative of actual human exposure have been tested with recombinant human CYPs and human liver microsomes (HLM). The different CYPs involved in IMI demethylation to the pharmacologically active metabolite desipramine (DES) were CYP2C19 > CYP1A2 > CYP3A4. The OPTs significantly inhibited (up to >80%) IMI bioactivation catalyzed by the recombinant CYPs tested, except CYP2D6, and by HLM; the inhibition was dose-dependent and started at low pesticide concentrations (0.25-2.5 {mu}M). The OPTs, having lower K {sub m} values, efficiently competed with IMI for the enzyme active site, as in the case of CYP2C19. However, with CYP1A2 and CYP3A4, a time- and NADPH-dependent mechanism-based inactivation also occurred, consistently with irreversible inhibition due to the release of the sulfur atom, binding to the active CYP during OPT desulfuration. At low IMI and OPT concentrations, lower IC50 values have been obtained with recombinant CYP1A2 (0.7-1.1 {mu}M) or with HLM rich in 1A2-related activity (2-10.8 {mu}M). The K {sub i} values (2-14 {mu}M), independent on substrate concentrations, were quite low and similar for the three pesticides. Exposure to OPTs during IMI therapeutic treatments may lead to decreased DES formation, resulting in high plasma levels of the parent drug, eventual impairment of its pharmacological action and possible onset of adverse drug reactions (ADRs)

  5. Correlation between the potency of flavonoids for cytochrome c reduction and inhibition of cardiolipin-induced peroxidase activity.

    PubMed

    Lagoa, Ricardo; Samhan-Arias, Alejandro K; Gutierrez-Merino, Carlos

    2017-03-20

    There are large differences between flavonoids to protect against apoptosis, a process in which cytochrome c (Cyt c) plays a key role. In this work, we show that 7 of 13 flavonoids studied have a capacity to reduce Cyt c similar or higher than ascorbate, the flavonols quercetin, kaempferol and myricetin, flavanol epigallocatechin-gallate, anthocyanidins cyanidin and malvidin, and the flavone luteolin. In contrast, the kaempferol 3(O)- and 3,4'(O)-methylated forms, the flavanone naringenin, and also apigenin and chrysin, had a negligible reducing capacity. Equilibrium dialysis and quenching of 1,6-diphenyl-1,3,5-hexatriene fluorescence experiments showed that flavonoids did not interfere with Cyt c binding to cardiolipin (CL)/phosphatidylcholine (PC) vesicles. However, the CL-induced loss of Cyt c Soret band intensity was largely attenuated by flavonoids, pointing out a stabilizing action against Cyt c unfolding in the complex. Moreover, flavonoids that behave as Cyt c reductants also inhibited the pro-apoptotic CL-induced peroxidase activity of Cyt c, indicating that modulation of Cyt c signaling are probable mechanisms behind the protective biological activities of flavonoids. © 2017 BioFactors, 2017.

  6. Cerebrovascular Protective Effect of Boldine Against Neural Apoptosis via Inhibition of Mitochondrial Bax Translocation and Cytochrome C Release

    PubMed Central

    Qiu, Xiaozhong; Shi, Ling; Zhuang, Hanting; Zhang, Hongtao; Wang, Juan; Wang, Lijun; Sun, Peng; Yu, Lili; Liu, Longxi

    2017-01-01

    Background In the present study, we explored the protective effect and mechanism of action of boldine (BOL) against neural apoptosis, which is a mediator of TBI. Material/Methods The effect of BOL on mitochondrial and cytosol proteins of extracted from cerebral cortical tissue of mice was evaluated. The grip test was used to assess the neurological deficit and brain water content of the subjects after administration of BOL to assess its effect on SOD, GSH, and MDA activity in brain ischemic tissues. To further confirm the effect of the BOL, the histopathological analysis and morphology of neurons were studied by Nissl staining. The effect of BOL against TBI-induced neural apoptosis by immuno-histochemistry and Western blotting assay were also studied. Result BOL showed significant improvement against TBI in a dose-dependent manner. In the BOL-treated group, the apoptotic index was significantly reduced, but the level of caspase-3 was greatly diminished. Additionally, the level of the Bax in mitochondria (mit) and cytosol was elevated in the TBI-treated group as compared to the sham group. Further BOL at the test dose causes significant reduction in the level of mitochondrial MDA together with increase in SOD activity as compared to the TBI alone group. Conclusions BOL showed a cerebroprotective effect against TBI by attenuating the oxidative stress and the mitochondrial apoptotic pathway. It also inhibited mitochondrial Bax translocation and cytochrome c release. PMID:28841638

  7. Maintenance of Atlantic salmon (Salmo salar) at elevated temperature inhibits cytochrome P450 aromatase activity in isolated ovarian follicles.

    PubMed

    Watts, Marianne; Pankhurst, Ned W; King, Henry R

    2004-02-01

    Atlantic salmon (Salmo salar) broodstock were transferred from natural (12-16 degrees C) to controlled temperatures of 14, 18 or 22 degrees C for 3 months during vitellogenesis. Fertility and survival were significantly reduced in eggs from broodstock held at 22 degrees C relative to 14 or 18 degrees C. Endocrine mechanisms were disrupted after only one month at 22 degrees C, as evidenced by decreased plasma vitellogenin (Vtg) and increased plasma testosterone (T) levels and, at later stages, decreased levels of plasma 17beta-estradiol (E2). In vitro incubations of isolated ovarian follicles were carried out at monthly intervals, with follicles exposed to human chorionic gonadotropin, N-2-0-dibutyryladenosine 3,5-cyclic monophosphate, and the gonadal steroid precursors 17-hydroxyprogesterone, androstenedione, and T. After one month of exposure to controlled temperature, T synthesis was generally enhanced in response to all treatments at all temperatures, but E2 synthesis was inhibited at 22 degrees C, suggesting temperature impairment of cytochrome P450 aromatase (P450arom) synthesis or activity. The effect became less marked as follicles matured suggesting that temperature sensitivity is stage dependent. The results of this study suggest that the inhibitory effects of elevated temperature on E2 and Vtg synthesis, and subsequent egg development found in the present and earlier studies, arise at least partly, from temperature modulation of P450arom.

  8. Inhibition and induction of cytochrome P450 2B1 in rat liver by promazine and chlorpromazine.

    PubMed

    Murray, M

    1992-09-25

    Phenothiazine tranquilizers have been associated with pharmacokinetic drug interactions in man. In this study the in vivo and in vitro effects of the clinically important phenothiazines promazine (PZ) and chlorpromazine (CPZ) on drug oxidations catalysed by specific cytochrome P450 (P450) enzymes were investigated in the rat. In vitro, the two drugs were relatively ineffective inhibitors of constitutive P450 activities, but were inhibitory toward the principal phenobarbital-inducible P450 2B1 and, to a lesser extent, P450 1A1. Administration of PZ and CPZ to male rats did not markedly influence the total microsomal P450 content of the liver. However, the quantitatively important male-specific P450 2C11 was down-regulated by CPZ and concomitant induction of P450 2B1 and associated 7-pentylresorufin O-depentylase activity were noted. A small increase in the activity of microsomal 7-ethylresorufin O-deethylase was also observed following administration of both drugs to rats, suggesting induction of P450 1A1/2. Considered together, it is apparent that the two phenothiazines are preferential inhibitors and inducers of P450 2B1 in rat liver. Drug interactions in humans involving phenothiazines may reflect a combined effect of induction and inhibition processes as well as down-regulation of other P450s, such as that produced by CPZ on P450 2C11.

  9. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    PubMed Central

    Ye, Qinyuan; Lian, Fuzhi; Chavez, Pollyanna R.G.; Chung, Jayong; Ling, Wenhua; Qin, Hua; Seitz, Helmut K.

    2012-01-01

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethanol liquid diet or a non-ethanol liquid diet, with or without CMZ for one and ten months. A single intraperitoneal injection of diethylnitrosamine (DEN, 20 mg/kg) was given to initiate hepatic carcinogenesis. CYP2E1 expression, inflammatory proteins, cell proliferation, protein-bound 4-HNE, etheno-DNA adducts, 8-hydroxy-2'-deoxyguanosine (8-OHdG), retinoid concentrations, and hepatic carcinogenesis were examined. Ethanol feeding for 1 month with DEN resulted in significantly increased hepatic CYP2E1 levels and increased nuclear accumulation of NF-κB protein and TNF-α expression, which were associated with increased cyclin D1 expression and p-GST positive altered hepatic foci. All of these changes induced by ethanol feeding were significantly inhibited by the one month CMZ treatment. At 10-months of treatment, hepatocellular adenomas were detected in ethanol-fed rats only, but neither in control rats nor in animals receiving ethanol and CMZ. The 8-OHdG formation was found to be significantly increased in ethanol fed animals and normalized with CMZ treatment. In addition, alcohol-reduced hepatic retinol and retinoic acid concentrations were restored by CMZ treatment to normal levels in the rats at 10 months of treatment. These data demonstrate that the inhibition of ethanol-induced CYP2E1 as a key pathogenic factor can counteract the tumor-promoting action of ethanol by decreasing TNF-α expression, NF-κB activation, and oxidative DNA damage as well as restoring normal hepatic levels of retinoic acid in DEN-treated rats. PMID:23543859

  10. Induction or inhibition of cytochrome P450 2E1 modifies the acute toxicity of acrylonitrile in rats: biochemical evidence.

    PubMed

    Suhua, Wang; Rongzhu, Lu; Wenrong, Xu; Guangwei, Xing; Xiaowu, Zhao; Shizhong, Wang; Ye, Zhang; Fangan, Han; Aschner, Michael

    2010-06-01

    The present study was designed to examine the effects of the inhibition or induction of CYP2E1 activity on acute acrylonitrile (AN) toxicity in rats. Increased or decreased hepatic CYP2E1 activity was achieved by pretreatment with acetone or trans-1,2-dichloroethylene (DCE), respectively. AN (50 mg/kg) was administered by intraperitoneal injection. Onset of convulsions and death were observed in rats with increased CYP2E1 activity, whereas convulsions and death did not appear in rats within 1 h after treatment with AN alone. Convulsions occurred in all AN-treated animals with increased CYP2E1 activity at approximately 18 min. The levels of cyanide (CN(-)), a terminal metabolite of AN, were significantly increased in the brains and livers of the AN-treated rats with increased CYP2E1 activity, compared with the levels in rats treated with AN alone, DCE + AN or acetone + DCE + AN. The cytochrome c oxidase (CcOx) activities in the brains and livers of the rats treated with AN or AN + acetone were significantly lower than those in the normal control rats and the rats treated with DCE, whereas the CcOx activities in the brains and livers of rats with decreased CYP2E1 activity were significantly higher than those in AN-treated rats. Brain lipid peroxidation was enhanced, and the antioxidant capacity was significantly compromised in rats with decreased CYP2E1 activity compared with rats with normal or increased CYP2E1 activity. Therefore, inhibition of CYP2E1 and simultaneous antioxidant therapy should be considered as supplementary therapeutic interventions in acute AN intoxication cases with higher CYP2E1 activity, thus a longer window of opportunity would be got to offer further emergency medication.

  11. Metabolic interactions of magnolol with cytochrome P450 enzymes: uncompetitive inhibition of CYP1A and competitive inhibition of CYP2C.

    PubMed

    Kim, Sang-Bum; Kang, Hee Eun; Cho, Hyun-Jong; Kim, Yeong Shik; Chung, Suk-Jae; Yoon, In-Soo; Kim, Dae-Duk

    2016-01-01

    Magnolol (MAG; 5,5'-diallyl-2,2'-biphenyldiol) is a major bioactive component of Magnolia officinalis. We investigated the metabolic interactions of MAG with hepatic cytochrome P450 monooxygenase (CYP) through in vitro microsomal metabolism study using human (HLM) and rat liver microsomes (RLM). CYP2C and 3A subfamilies were significantly involved in the metabolism of MAG, while CYP1A subfamily was not in HLM and RLM. The relative contribution of phase I enzymes including CYP to the metabolism of MAG was comparable to that of uridine diphosphate glucuronosyltransferase (UGT) in RLM. Moreover, MAG potently inhibited the metabolic activity of CYP1A (IC50 of 1.62 μM) and 2C (IC50 of 5.56 μM), while weakly CYP3A (IC50 of 35.0 μM) in HLM and RLM. By the construction of Dixon plot, the inhibition type of MAG on CYP activity in RLM was determined as follows: uncompetitive inhibitor for CYP1A (Ki of 1.09-12.0 μM); competitive inhibitor for CYP2C (Ki of 10.0-15.2 μM) and 3A (Ki of 93.7-183 μM). Based on the comparison of the current IC50 and Ki values with a previously reported liver concentration (about 13 μM) of MAG after its seven times oral administration at a dose of 50 mg/kg in rats, it is suggested that MAG could show significant inhibition of CYP1A and 2C, but not CYP3A, in the in vivo rat system. These results could lead to further studies in clinically significant metabolism-mediated MAG-drug interactions.

  12. Let-7b Inhibits Human Cancer Phenotype by Targeting Cytochrome P450 Epoxygenase 2J2

    PubMed Central

    Yang, Shenglan; Gong, Wei; Wang, Yan; Cianflone, Katherine; Tang, Jiarong; Wang, Dao Wen

    2012-01-01

    Background MicroRNAs (miRNAs) are small, noncoding RNA molecules of 20 to 22 nucleotides that regulate gene expression by binding to their 3′ untranslated region (3′UTR). Increasing data implicate altered miRNA participation in the progress of cancer. We previously reported that CYP2J2 epoxygenase promotes human cancer phenotypes. But whether and how CYP2J2 is regulated by miRNA is not understood. Methods and Results Using bioinformatics analysis, we found potential target sites for miRNA let-7b in 3′UTR of human CYP2J2. Luciferase and western blot assays revealed that CYP2J2 was regulated by let-7b. In addition, let-7b decreased the enzymatic activity of endogenous CYP2J2. Furthermore, let-7b may diminish cell proliferation and promote cell apoptosis of tumor cells via posttranscriptional repression of CYP2J2. Tumor xenografts were induced in nude mice by subcutaneous injection of MDA-MB-435 cells. The let-7b expression vector, pSilencer-let-7b, was injected through tail vein every 3 weeks. Let-7b significantly inhibited the tumor phenotype by targeting CYP2J2. Moreover, quantitative real-time polymerase chain reaction and western blotting were used to determine the expression levels of let-7b and CYP2J2 protein from 18 matched lung squamous cell cancer and adjacent normal lung tissues; the expression level of CYP2J2 was inversely proportional to that of let-7b. Conclusions Our results demonstrated that the decreased expression of let-7b could lead to the high expression of CYP2J2 protein in cancerous tissues. These findings suggest that miRNA let-7b reduces CYP2J2 expression, which may contribute to inhibiting tumor phenotypes. PMID:22761738

  13. A Unified Proteochemometric Model for Prediction of Inhibition of Cytochrome P450 Isoforms

    PubMed Central

    Lapins, Maris; Worachartcheewan, Apilak; Spjuth, Ola; Georgiev, Valentin; Prachayasittikul, Virapong; Nantasenamat, Chanin; Wikberg, Jarl E. S.

    2013-01-01

    A unified proteochemometric (PCM) model for the prediction of the ability of drug-like chemicals to inhibit five major drug metabolizing CYP isoforms (i.e. CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was created and made publicly available under the Bioclipse Decision Support open source system at www.cyp450model.org. In regards to the proteochemometric modeling we represented the chemical compounds by molecular signature descriptors and the CYP-isoforms by alignment-independent description of composition and transition of amino acid properties of their protein primary sequences. The entire training dataset contained 63 391 interactions and the best PCM model was obtained using signature descriptors of height 1, 2 and 3 and inducing the model with a support vector machine. The model showed excellent predictive ability with internal AUC = 0.923 and an external AUC = 0.940, as evaluated on a large external dataset. The advantage of PCM models is their extensibility making it possible to extend our model for new CYP isoforms and polymorphic CYP forms. A key benefit of PCM is that all proteins are confined in one single model, which makes it generally more stable and predictive as compared with single target models. The inclusion of the model in Bioclipse Decision Support makes it possible to make virtual instantaneous predictions (∼100 ms per prediction) while interactively drawing or modifying chemical structures in the Bioclipse chemical structure editor. PMID:23799117

  14. Cytochrome P450 omega-hydroxylase inhibition reduces infarct size during reperfusion via the sarcolemmal KATP channel.

    PubMed

    Gross, Eric R; Nithipatikom, Kasem; Hsu, Anna K; Peart, Jason N; Falck, John R; Campbell, William B; Gross, Garrett J

    2004-12-01

    Inhibition of 20-hydroxyeicosatrienoic acid (20-HETE), by pretreatment with pharmacological inhibitors of cytochrome P450 (CYP) omega-hydroxylase, has been shown to reduce infarct size in canines when administered prior to ischemia. However, it is unknown whether these agents reduce infarct size when administered just prior to reperfusion and if the sarcolemmal and/or mitochondrial K(ATP) channels (sK(ATP) and mK(ATP)) contribute to cardioprotection. Therefore, we determined whether specific CYP inhibitors for epoxygenases and omega-hydroxylases are cardioprotective when given either prior to ischemia or prior to reperfusion and furthermore, if selective inhibition of the sK(ATP) by HMR-1098 or mK(ATP) by 5-hydroxydecanoic acid (5-HD) could abrogate this effect. Male Sprague-Dawley rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. Groups received either miconazole (MIC, non-selective CYP inhibitor, 3 mg/kg), 17-octadecynoic acid (17-ODYA, CYP omega-hydroxylase inhibitor, 0,3 or 3 mg/kg), N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS, CYP omega-hydroxylase inhibitor, 0,4 or 4 mg/kg), N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH, CYP epoxygenase inhibitor, 3 mg/kg), or vehicle either 10 minutes prior to ischemia or 5 minutes prior to reperfusion. Rats also received either HMR-1098 (6 mg/kg) or 5-HD (10 mg/kg) 10 minutes prior to reperfusion, with subsets of rats also receiving either MIC or 17-ODYA 5 minutes prior to reperfusion. DDMS and 17-ODYA dose dependently reduced infarct size. Rats treated with MIC, 17-ODYA and DDMS, but not MS-PPOH, produced comparable reductions in infarct size when administered prior to ischemia or reperfusion compared to vehicle. HMR-1098, but not 5-HD, also blocked the infarct size reduction afforded by MIC and 17-ODYA. These data suggest a novel cardioprotective pathway involving CYP omega-hydroxylase inhibition and subsequent activation of the sK(ATP) channel during reperfusion.

  15. Integrated in silico-in vitro strategy for addressing cytochrome P450 3A4 time-dependent inhibition.

    PubMed

    Zientek, Michael; Stoner, Chad; Ayscue, Robyn; Klug-McLeod, Jacquelyn; Jiang, Ying; West, Michael; Collins, Claire; Ekins, Sean

    2010-03-15

    Throughout the past decade, the expectations from the regulatory agencies for safety, drug-drug interactions (DDIs), pharmacokinetic, and disposition characterization of new chemical entities (NCEs) by pharmaceutical companies seeking registration have increased. DDIs are frequently assessed using in silico, in vitro, and in vivo methodologies. However, a key gap in this screening paradigm is a full structural understanding of time-dependent inhibition (TDI) on the cytochrome P450 systems, particularly P450 3A4. To address this, a number of high-throughput in vitro assays have been developed. This work describes an automated assay for TDI using two concentrations at two time points (2 + 2 assay). Data generated with this assay for over 2000 compounds from multiple therapeutic programs were used to generate in silico Bayesian classification models of P450 3A4-mediated TDI. These in silico models were validated using several external test sets and multiple random group testing (receiver operator curve value >0.847). We identified a number of substructures that were likely to elicit TDI, the majority containing indazole rings. These in vitro and in silico approaches have been implemented as a part of the Pfizer screening paradigm. The Bayesian models are available on the intranet to guide synthetic strategy, predict whether a NCE is likely to cause a TDI via P450 3A4, filter for in vitro testing, and identify substructures important for TDI as well as those that do not cause TDI. This represents an integrated in silico-in vitro strategy for addressing P450 3A4 TDI and improving the efficiency of screening.

  16. Picroside II Exerts a Neuroprotective Effect by Inhibiting the Mitochondria Cytochrome C Signal Pathway Following Ischemia Reperfusion Injury in Rats.

    PubMed

    Zhang, Hongyan; Zhai, Li; Wang, Tingting; Li, Shan; Guo, Yunliang

    2017-02-01

    Stroke is a common neurodegenerative disease in the wide world, and mitochondrial defects underlie the pathogenesis of ischemia, especially during reperfusion. Picroside II, the principal active component of Picrorhiza, is a traditional Chinese medicine. Our previous study demonstrated that the best therapeutic dose and time window were injection of picroside II at a dose of 10-20 mg/kg body weight following cerebral ischemia by 1.5-2.0 h. In this paper, the neuroprotective effect and the mechanism of picroside II were investigated, as well as its involvement in antioxidant and mitochondria cytochrome C (CytC) signal pathway following ischemia reperfusion (I/R) injury in rats. After 24 h of cerebral I/R, the neurobehavioral function was measured by modified neurological severity score test; the content of reactive oxygen species in brain tissue was measured by enzyme-linked immunosorbent assay; the cerebral infarction volume was detected by TTC staining; the morphology of brain tissue was observed by hematoxylin-eosin; the apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling assay; the ultrastructure of the cortical brain tissues was observation by transmission electron microscopy; the expressions of CytC and Caspase-3 were determined by immunohistochemical assay and Western blot. The results indicated that picroside II could scavenge ROS contents, decrease the cerebral infarction volume and apoptotic cells, protect the structure of mitochondria, down-regulate the expression of CytC and Caspase-3 in cerebral I/R rats. It can be concluded that picroside II exerts a neuroprotective effect by inhibiting the mitochondria CytC signal pathway following ischemia reperfusion injury in rats.

  17. Caspase-resistant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria.

    PubMed

    Nguyen, M; Breckenridge, D G; Ducret, A; Shore, G C

    2000-09-01

    BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two identical caspase recognition sites (AAVD.G) that are preferentially cleaved by initiator caspases, including caspase 8. Cleavage of BAP31 during apoptosis generates a p20 fragment that remains integrated in the membrane and, when expressed ectopically, is a potent inducer of cell death. To examine the consequences of maintaining the structural integrity of BAP31 during apoptosis, the caspase recognition aspartate residues were mutated to alanine residues, and Fas-mediated activation of caspase 8 and cell death were examined in human KB epithelial cells stably expressing the caspase-resistant mutant crBAP31. crBAP31 only modestly slowed the time course for activation of caspases, as assayed by the processing of procaspases 8 and 3 and the measurement of total DEVDase activity. As a result, cleavage of the caspase targets poly(ADP-ribosyl) polymerase and endogenous BAP31, as well as the redistribution of phosphatidylserine and fragmentation of DNA, was observed. In contrast, cytoplasmic membrane blebbing and fragmentation and apoptotic redistribution of actin were strongly inhibited, cell morphology was retained near normal, and the irreversible loss of cell growth potential following removal of the Fas stimulus was delayed. Of note, crBAP31-expressing cells also resisted Fas-mediated release of cytochrome c from mitochondria, and the mitochondrial electrochemical potential was only partly reduced. These results argue that BAP31 cleavage is important for manifesting cytoplasmic apoptotic events associated with membrane fragmentation and reveal an unexpected cross talk between mitochondria and the endoplasmic reticulum during Fas-mediated apoptosis in vivo.

  18. In vitro evaluation of valproic acid as an inhibitor of human cytochrome P450 isoforms: preferential inhibition of cytochrome P450 2C9 (CYP2C9)

    PubMed Central

    Wen, Xia; Wang, Jun-Sheng; Kivistö, Kari T; Neuvonen, Pertti J; Backman, Janne T

    2001-01-01

    Aims To evaluate the potency and specificity of valproic acid as an inhibitor of the activity of different human CYP isoforms in liver microsomes. Methods Using pooled human liver microsomes, the effects of valproic acid on seven CYP isoform specific marker reactions were measured: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), tolbutamide hydroxylase (CYP2C9), S-mephenytoin 4′-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1) and midazolam 1′-hydroxylase (CYP3A4). Results Valproic acid competitively inhibited CYP2C9 activity with a Ki value of 600 µm. In addition, valproic acid slightly inhibited CYP2C19 activity (Ki = 8553 µm, mixed inhibition) and CYP3A4 activity (Ki = 7975 µm, competitive inhibition). The inhibition of CYP2A6 activity by valproic acid was time-, concentration- and NADPH-dependent (KI = 9150 µm, Kinact=0.048 min−1), consistent with mechanism-based inhibition of CYP2A6. However, minimal inhibition of CYP1A2, CYP2D6 and CYP2E1 activities was observed. Conclusions Valproic acid inhibits the activity of CYP2C9 at clinically relevant concentrations in human liver microsomes. Inhibition of CYP2C9 can explain some of the effects of valproic acid on the pharmacokinetics of other drugs, such as phenytoin. Co-administration of high doses of valproic acid with drugs that are primarily metabolized by CYP2C9 may result in significant drug interactions. PMID:11736863

  19. Effects of selective inhibition of cytochrome P-450 omega-hydroxylases and ischemic preconditioning in myocardial protection.

    PubMed

    Nithipatikom, Kasem; Endsley, Michael P; Moore, Jeannine M; Isbell, Marilyn A; Falck, John R; Campbell, William B; Gross, Garrett J

    2006-02-01

    Cytochrome P-450 (CYP) omega-hydroxylases and their arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), produce a detrimental effect on ischemia-reperfusion injury in canine hearts, and the inhibition of CYP omega-hydroxylases markedly reduces myocardial infarct size expressed as a percentage of the area at risk (IS/AAR, %). In this study, we demonstrated that a specific CYP omega-hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly reduced 20-HETE production during ischemia-reperfusion and reduced myocardial infarct size compared with control [19.5 +/- 1.0% (control), 9.6 +/- 1.5% (0.40 mg/kg DDMS), 4.0 +/- 2.0% (0.81 mg/kg DDMS), P < 0.01]. In addition, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE, a putative 20-HETE antagonist) significantly reduced myocardial infarct size from control [10.3 +/- 1.3% (0.032 mg/kg 20-HEDE) and 5.9 +/- 1.9% (0.064 mg/kg 20-HEDE), P < 0.05]. We further demonstrated that one 5-min period of ischemic preconditioning (IPC) reduced infarct size to a similar extent as that observed with the high doses of DDMS and 20-HEDE, and the higher dose of DDMS given simultaneously with IPC augmented the infarct size reduction [9.9 +/- 2.8% (IPC) to 2.5 +/- 1.4% (0.81 mg/kg DDMS), P < 0.05] to a greater degree than that observed with either treatment alone. These results suggest an important negative role for endogenous CYP omega-hydroxylases and their product, 20-HETE, to exacerbate myocardial injury in canine myocardium. Furthermore, for the first time, this study demonstrates that the effect of IPC and the inhibition of CYP omega-hydroxylase synthesis (DDMS) or its actions (20-HEDE) may have additive effects in protecting the canine heart from ischemia-reperfusion injury.

  20. Regulation of p-nitroanisole O-demethylation in perfused rat liver. Adenine nucleotide inhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase.

    PubMed Central

    Kauffman, F C; Evans, R K; Reinke, L A; Thurman, R G

    1979-01-01

    Perfusion of rat livers with 10 mM-fructose or pretreatment of the rat with 6-aminonicotinamide (70 mg/kg) 6 h before perfusion decreased intracellular ATP concentrations and increased the rate of p-nitroanisole O-demethylation. This increase was accompanied by a decrease in the free [NADP+]/[NADPH] ratio calculated from concentrations of substrates assumed to be in near-equilibrium with isocitrate dehydrogenase. After pretreatment with 6-aminonicotinamide the [NADP+]/[NADPH] ratio also declined. Reduction of NADP+ during mixed-function oxidation may be explained by inhibition of of one or more NADPH-generating enzymes. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and "malic" enzyme, partially purified from livers of phenobarbital-treated rats, were inhibited by ATP and ADP. Inhibitor constants of ATP for the four dehydrogenases varied considerably, ranging from 9 micrometer for "malic" enzyme to 1.85 mM for glucose 6-phosphate dehydrogenase. NADPH-cytochrome c reductase was also inhibited by ATP (Ki 2.8 mM) and by ADP (Ki 0.9 mM), but not by AMP. Concentrations of ATP and ADP that inhibited glucose 6-phosphate dehydrogenase and the reductase were comparable with concentrations in the intact liver. Thus agents that lower intracellular ATP may accelerate rates of mixed-function oxidation by a concerted mechanism involving deinhibition of NADPH-cytochrome c reductase and one or more NADPH-generating enzymes. PMID:44195

  1. Cellular antioxidant adaptive survival response to 6-hydroxydopamine-induced nitrosative cell death in C6 glioma cells.

    PubMed

    Lee, Chan; Park, Gyu Hwan; Jang, Jung-Hee

    2011-05-10

    Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons in the substantia nigra. 6-Hydroxydopamine (6-OHDA) is a catecholaminergic neurotoxin widely used to produce experimental models of PD and has been reported to cause oxidative and/or nitrosative stress. In this study, we have investigated 6-OHDA-induced nitrosative cell death and its self-defense mechanism in C6 glioma cells. Treatment of C6 cells with 6-OHDA increased expression of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO). Furthermore 6-OHDA treatment led to peroxynitrite generation and nitrotyrosine formation. 6-OHDA-induced nitrosative stress ultimately caused apoptotic cell death as determined by decreased Bcl-2/Bax ratio, activation of c-Jun N-terminal kinase (JNK), and cleavage of caspase-3 and poly(ADP-ribose)polymerase (PARP), which were attenuated by peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron(III) (FeTPPS). In another experiment, exposure of C6 glioma cells to 6-OHDA resulted in an increased expression of heme oxygenase-1 (HO-1) and 6-OHDA-induced cytotoxicity was effectively suppressed by the HO-1 inducer SnCl(2) and aggravated by HO-1 inhibitor zinc protoporphyrin (ZnPP), supporting the cytoprotective role of HO-1. To elucidate the molecular mechanism underlying 6-OHDA-mediated HO-1 induction, we have examined the possible involvement of NF-E2-related factor 2 (Nrf2), which plays an important role in the transcriptional regulation of phase II detoxifying and antioxidant enzymes. 6-OHDA treatment increased nuclear translocation and transcriptional activity of Nrf2, which seemed to be partly mediated by activation of upstream kinases such as Akt/protein kinase B (PKB). Taken together these findings suggest that HO-1 up-regulation via Nrf2 activation may mediate the cellular adaptive survival response to 6-OHDA-induced nitrosative cell death in C6 glioma cells. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. Effects of discontinuing a high-fat diet on mitochondrial proteins and 6-hydroxydopamine-induced dopamine depletion in rats.

    PubMed

    Ma, Delin; Shuler, Jeffrey M; Raider, Kayla D; Rogers, Robert S; Wheatley, Joshua L; Geiger, Paige C; Stanford, John A

    2015-07-10

    Diet-induced obesity can increase the risk for developing age-related neurodegenerative diseases including Parkinson's disease (PD). Increasing evidence suggests that mitochondrial and proteasomal mechanisms are involved in both insulin resistance and PD. The goal of this study was to determine whether diet intervention could influence mitochondrial or proteasomal protein expression and vulnerability to 6-Hydroxydopamine (6-OHDA)-induced nigrostriatal dopamine (DA) depletion in rats' nigrostriatal system. After a 3 month high-fat diet regimen, we switched one group of rats to a low-fat diet for 3 months (HF-LF group), while the other half continued with the high-fat diet (HF group). A chow group was included as a control. Three weeks after unilateral 6-OHDA lesions, HF rats had higher fasting insulin levels and higher Homeostasis model assessment of insulin resistance (HOMA-IR), indicating insulin resistance. HOMA-IR was significantly lower in HF-LF rats than HF rats, indicating that insulin resistance was reversed by switching to a low-fat diet. Compared to the Chow group, the HF group exhibited significantly greater DA depletion in the substantia nigra but not in the striatum. DA depletion did not differ between the HF-LF and HF group. Proteins related to mitochondrial function (such as AMPK, PGC-1α), and to proteasomal function (such as TCF11/Nrf1) were influenced by diet intervention, or by 6-OHDA lesion. Our findings suggest that switching to a low-fat diet reverses the effects of a high-fat diet on systemic insulin resistance, and mitochondrial and proteasomal function in the striatum. Conversely, they suggest that the effects of the high-fat diet on nigrostriatal vulnerability to 6-OHDA-induced DA depletion persist.

  3. Regional distributions of manganese, iron, copper, and zinc in the brains of 6-hydroxydopamine-induced parkinsonian rats.

    PubMed

    Tarohda, Tohru; Ishida, Yasushi; Kawai, Keiichi; Yamamoto, Masayoshi; Amano, Ryohei

    2005-09-01

    Time courses of changes in manganese, iron, copper, and zinc concentrations were examined in regions of the brain of a 6-hydroxydopamine (6-OHDA)-induced rat model of Parkinson's disease using inductively coupled plasma mass spectrometry (ICP-MS). The concentrations were simultaneously determined in brain section at the level of the substantia nigra 1, 3, 7, 10, 14, and 21 days after the 6-OHDA treatment and compared with those of control rats. The distributions of these elements were obtained for 18 regions of the sagittal section (1-mm thick). The ICP-MS results indicated that Mn, Fe, Cu, and Zn levels of the 6-OHDA-induced parkinsonian brain were observed to increase in all regions that lay along the dopaminergic pathway. In the substantia nigra, the increase in Mn level occurred rapidly from 3 to 7 days and preceded those in the other elements, reaching a plateau in the 6-OHDA brain. Iron and Zn levels increased gradually until 7 days and then increased rapidly from 7 to 10 days. The increase in the copper level was slightly delayed. In other regions, such as the globus pallidus, putamen, and amygdala, the levels of Mn, Fe, Cu, and Zn increased with time after 6-OHDA treatment, although the time courses of their changes were region-specific. These findings contribute to our understanding of the roles of Mn and Fe in the induction of neurological symptoms and progressive loss of dopaminergic neurons in the development of Parkinson's disease. Manganese may hold the key to disturbing cellular Fe homeostasis and accelerating Fe levels, which play the most important role in the development of Parkinson's disease.

  4. Neuroprotective effect of D-psicose on 6-hydroxydopamine-induced apoptosis in rat pheochromocytoma (PC12) cells.

    PubMed

    Takata, Maki K; Yamaguchi, Fuminori; Nakanose, Koichi; Watanabe, Yasuo; Hatano, Naoya; Tsukamoto, Ikuko; Nagata, Mitsuhiro; Izumori, Ken; Tokuda, Masaaki

    2005-11-01

    We evaluated the neuroprotective effects of D-psicose, one of the rare sugars, on 6-hydroxydopamine (6-OHDA)-induced apoptosis in catecholaminergic PC12 cells, the in vitro model of Parkinson's disease (PD). Apoptotic characteristics of PC12 cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) assay. The results showed that D-psicose at a concentration of 50 mM, exerted significant protective effects against the 6-OHDA (200 muM)-induced PC12 cell apoptosis, while other sugars had little or no protective effects. We have observed a significant increase in the level of intracellular glutathione after 24 h in 6-OHDA (200 muM) treated cells, while a decrease in the level was observed at 3 h and 6 h. Also, a synergistic exposure to D-psicose and 6-OHDA for 24 h showed a significant increase in intracellular glutathione level. Therefore, these results suggest that D-psicose may play a potential role as a neuroprotective agent in the treatment of neurodegenerative diseases by inducing an up-regulation of intracellular glutathione.

  5. Treadmill exercise alleviates short-term memory impairment in 6-hydroxydopamine-induced Parkinson’s rats

    PubMed Central

    Cho, Han-Sam; Shin, Mal-Soon; Song, Wook; Jun, Tae-Won; Lim, Baek-Vin; Kim, Young-Pyo; Kim, Chang-Ju

    2013-01-01

    Progressive loss of dopaminergic neurons in substantia nigra is a key pathogenesis of Parkinson’s disease. In the present study, we investigated the effects of treadmill exercise on short-term memory, apoptotic dopaminergic neuronal cell death and fiber loss in the nigrostriatum, and cell proliferation in the hippocampal dentate gyrus of Parkinson’s rats. Parkinson’s rats were made by injection of 6-hydroxydopamine (6-OHDA) into the striatum using stereotaxic instrument. Four weeks after 6-OHDA injection, the rats in the 6-OHDA-injection group exhibited significant rotational asymmetry following apomorphine challenge. The rats in the exercise groups were put on the treadmill to run for 30 min once a day for 14 consecutive days starting 4 weeks after 6-OHDA injection. In the present results, extensive degeneration of the dopaminergic neurons in the substantia nigra with loss of dopaminergic fibers in the striatum were produced in the rats without treadmill running, which resulted in short-term memory impairment. However, the rats performing treadmill running for 2 weeks alleviated nigrostriatal dopaminergic cell loss and alleviated short-term memory impairment with increasing cell proliferation in the hippocampal dentate gyrus of Parkinson’s rats. The present results show that treadmill exercise may provide therapeutic value for the Parkinson’s disease. PMID:24278884

  6. Effects of electroacupuncture on metabolic changes in motor cortex and striatum of 6-hydroxydopamine-induced Parkinsonian rats.

    PubMed

    Li, Min; Wang, Ke; Su, Wen-Ting; Jia, Jun; Wang, Xiao-Min

    2017-10-06

    To explore the possible underlying mechanism by investigating the effect of electroacupuncture (EA) treatment on the primary motor cortex and striatum in a unilateral 6-hydroxydopamine (6-OHDA) induced rat Parkinson's disease (PD) model. Male Sprague-Dawley rats were randomly divided into sham group (n=16), model group (n=14), and EA group (n=14). EA stimulation at Dazhui (GV 14) and Baihui (GV20) was applied to PD rats in the EA group for 4 weeks. Behavioral tests were conducted to evaluate the effectiveness of EA treatment. Metabolites were detected by 7.0 T proton nuclear magnetic resonance. Following 4 weeks of EA treatment in PD model rats, the abnormal behavioral impairment induced by 6-OHDA was alleviated. In monitoring changes in metabolic activity, ratios of myoinositol/creatine (Cr) and N-acetyl aspartate (NAA)/Cr in the primary motor cortex were significantly lower at the injected side than the non-injected side in PD rats (P=0.024 and 0.020). The ratios of glutamate + glutamine (Glx)/Cr and NAA/Cr in the striatum were higher and lower, respectively, at the injected side than the non-injected side (P=0.046 and 0.008). EA treatment restored the balance of metabolic activity in the primary motor cortex and striatum. In addition, the taurine/Cr ratio and Glx/Cr ratio were elevated in the striatum of PD model rats compared to sham-lesioned rats (P=0.026 and 0.000). EA treatment alleviated the excessive glutamatergic transmission by down-regulating the striatal Glx/Cr ratio (P=0.001). The Glx/Cr ratio was negatively correlated with floor plane spontaneous locomotion in PD rats (P=0.027 and P=0.0007). EA treatment is able to normalize the metabolic balance in the primary motor cortex and striatum of PD rats, which may contribute to its therapeutic effect on motor deficits. The striatal Glx/Cr ratio may serve as a potential indicator of PD and a therapeutic target of EA treatment.

  7. Neuroprotective Effect of Thymoquinone, the Nigella Sativa Bioactive Compound, in 6-Hydroxydopamine-Induced Hemi-Parkinsonian Rat Model

    PubMed Central

    Sedaghat, Reza; Roghani, Mehrdad; Khalili, Mohsen

    2014-01-01

    Parkinson disease (PD) is the most common movement disorder with progressive degeneration of midbrain dopaminergic neurons for which current treatments afford symptomatic relief with no-prevention of disease progression. Due to the neuroprotective property of the Nigella sativa bioactive compound thymoquinone (TQ), this study was undertaken to evaluate whether TQ could improve behavioral and cellular abnormalities and markers of oxidative stress in an experimental model of early PD in rat. Unilateral intrastriatal 6-hydroxydopamine (6-OHDA)-lesioned rats were daily pretreated p.o. with TQ at doses of 5 and/or 10 mg/Kg three times at an interval of 24 h. After 1 week, apomorphine caused contralateral rotations, a reduction in the number of neurons on the left side of the substantia nigra pars compacta (SNC) was observed, malondialdehyde (MDA) and nitrite level in midbrain homogenate increased and activity of superoxide dismutase (SOD) reduced in the 6-OHDA lesion group. TQ pretreatment significantly improved turning behavior, prevented loss of SNC neurons, and lowered level of MDA. These results suggest that TQ could afford neuroprotection against 6-OHDA neurotoxicity that is partly due to the attenuation of lipid peroxidation and this may provide benefits, along with other therapies, in neurodegenerative disorders including PD. PMID:24734075

  8. Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson's disease model.

    PubMed

    Park, Hi-Joon; Lim, Sabina; Joo, Wan-Seok; Yin, Chang-Shik; Lee, Hyang-Sook; Lee, Hye-Jung; Seo, Jung Chul; Leem, Kanghyun; Son, Yang-Sun; Kim, Youn-Jung; Kim, Chang-Ju; Kim, Yong-Sik; Chung, Joo-Ho

    2003-03-01

    Parkinson's disease (PD) is a chronic neurodegenerative disorder, and it has been suggested that treatments promoting survival and functional recovery of affected dopaminergic neurons could have a significant and long-term therapeutic value. In the present study, we investigated the neuroprotective effects of acupuncture on the nigrostriatal system in rat unilaterally lesioned with 6-hydroxydopamine (6-OHDA, 4 microg/microl, intrastriatal injection) using tyrosine hydroxylase (TH) and receptor for brain-derived neurotrophic factor, trkB, immunohistochemistries. Two weeks after the lesions were made, rats presented with asymmetry in rotational behavior (118.3 +/- 17.5 turns/h) following injection with apomorphine, a dopamine receptor agonist (0.5 mg/kg, sc). In contrast, acupunctural treatment at acupoints GB34 and LI3 was shown to significantly reduce this motor deficit (14.6 +/- 13.4 turns/h). Analysis via TH immunohistochemistry revealed a substantial loss of cell bodies in the substantia nigra (SN) (45.7% loss) and their terminals in the dorsolateral striatum ipsilateral to the 6-OHDA-induced lesion. However, acupunctural treatment resulted in the enhanced survival of dopaminergic neurons in the SN (21.4% loss) and their terminals in the dorsolateral striatum. Acupuncture also increased the expression of trkB significantly (35.6% increase) in the ipsilateral SN. In conclusion, we observed that only acupuncturing without the use of any drug has the neuroprotective effects against neuronal death in the rat PD model and these protective properties of acupuncture could be mediated by trkB.

  9. Moderate-Intensity Physical Exercise Protects Against Experimental 6-Hydroxydopamine-Induced Hemiparkinsonism Through Nrf2-Antioxidant Response Element Pathway.

    PubMed

    Aguiar, Aderbal Silva; Duzzioni, Marcelo; Remor, Aline Pertile; Tristão, Fabrine Sales Massafera; Matheus, Filipe C; Raisman-Vozari, Rita; Latini, Alexandra; Prediger, Rui Daniel

    2016-02-01

    Exercise improves the motor symptoms of patients with Parkinson disease in a palliative manner. Existing evidence demonstrates that exercise induces neuroprotection based on the neurotrophic properties. We investigated the effect of exercise on mitochondrial physiology and oxidative stress in an animal model of hemiparkinsonism. C57BL/6 mice completed a 6-week exercise program on a treadmill. We injected 6-hydroxydopamine (6-OHDA; 4 μg/2 μl) into the midstriatum. The animals progressively developed bradykinesia and R(-)-apomorphine-induced rotations that were attenuated by exercise. Transcriptional activation of protective genes is mediated by the antioxidant response element (ARE). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) binds to ARE. We investigated the Nrf2-ARE pathway in the striatum of animals. Exercise protected 6-OHDA-induced loss of tyrosine hydroxylase immunolabeling and activated the Nrf2-ARE pathway in the nigrostriatal pathway. Exercise stimulated mitochondrial biogenesis in the striatum of animals that was more resistant to oxidant 6-OHDA and nitric oxide donor (±)-S-nitroso-N-acetylpenicillamine. In mice, exercise activated Nrf2-ARE signaling in the nigrostriatal pathway that was protective against the development of hemiparkinsonism.

  10. Acetyl-l-carnitine protects dopaminergic nigrostriatal pathway in 6-hydroxydopamine-induced model of Parkinson's disease in the rat.

    PubMed

    Afshin-Majd, Siamak; Bashiri, Keyhan; Kiasalari, Zahra; Baluchnejadmojarad, Tourandokht; Sedaghat, Reza; Roghani, Mehrdad

    2017-02-12

    Parkinson's disease (PD) is a movement disorder and the second most common neurodegenerative disease worldwide in which nigrostriatal dopaminergic neurons within substantia nigra pars compacta (SNC) are lost, with clinical motor and non-motor symptoms including bradykinesia, resting tremor, rigidity, stooping posture and cognitive deficits. This study was undertaken to evaluate the neuroprotective potential of acetyl-l-carnitine (ALC) against unilateral striatal 6-hydroxydopamine (6-OHDA)-induced model of PD and to explore some involved mechanisms. In this experimental study, intrastriatal 6-OHDA-lesioned rats received ALC at doses of 100 or 200mg/kg/day for 1 week. ALC (200mg/kg) lowered apomorphine-induced rotational asymmetry and reduced the latency to initiate and the total time in the narrow beam test, reduced striatal malondialdehyde (MDA), increased catalase activity and glutathione (GSH) level, prevented reduction of nigral tyrosine hydroxylase (TH)-positive neurons and striatal TH-immunoreactivity, and lowered striatal glial fibrillary acidic protein (GFAP) and its immunoreactivity as an indicator of astrogliosis, and nuclear factor NF-kappa B and Toll-like receptor 4 (TLR4) as reliable markers of neuroinflammation. Meanwhile, ALC at both doses mitigated nigral DNA fragmentation as a valuable marker of apoptosis. The results of this study clearly suggest the neuroprotective effect of ALC in 6-OHDA-induced model of PD through abrogation of neuroinflammation, apoptosis, astrogliosis, and oxidative stress and it may be put forward as an ancillary therapeutic candidate for controlling PD.

  11. Transient transfection of human CDNF gene reduces the 6-hydroxydopamine-induced neuroinflammation in the rat substantia nigra.

    PubMed

    Nadella, Rasajna; Voutilainen, Merja H; Saarma, Mart; Gonzalez-Barrios, Juan A; Leon-Chavez, Bertha A; Jiménez, Judith M Dueñas; Jiménez, Sergio H Dueñas; Escobedo, Lourdes; Martinez-Fong, Daniel

    2014-12-16

    The anti-inflammatory effect of the cerebral dopamine neurotrophic factor (CDNF) was shown recently in primary glial cell cultures, yet such effect remains unknown both in vivo and in 6-hydroxydopamine (6-OHDA) models of Parkinson's disease (PD). We addressed this issue by performing an intranigral transfection of the human CDNF (hCDNF) gene in the critical period of inflammation after a single intrastriatal 6-OHDA injection in the rat. At day 15 after lesion, the plasmids p3xNBRE-hCDNF or p3xNBRE-EGFP, coding for enhanced green florescent protein (EGFP), were transfected into the rat substantia nigra (SN) using neurotensin (NTS)-polyplex. At day 15 post-transfection, we measured nitrite and lipoperoxide levels in the SN. We used ELISA to quantify the levels of TNF-α, IL-1β, IL-6, endogenous rat CDNF (rCDNF) and hCDNF. We also used qRT-PCR to measure rCDNF and hCDNF transcripts, and immunofluorescence assays to evaluate iNOS, CDNF and glial cells (microglia, astrocytes and Neuron/Glial type 2 (NG2) cells). Intact SNs were additional controls. In the SN, 6-OHDA triggered nitrosative stress, increased inflammatory cytokines levels, and activated the multipotent progenitor NG2 cells, which convert into astrocytes to produce rCDNF. In comparison with the hemiparkinsonian rats that were transfected with the EGFP gene or without transfection, 6-OHDA treatment and p3xNBRE-hCDNF transfection increased the conversion of NG2 cells into astrocytes resulting in 4-fold increase in the rCDNF protein levels. The overexpressed CDNF reduced nitrosative stress, glial markers and IL-6 levels in the SN, but not TNF-α and IL-1β levels. Our results show the anti-inflammatory effect of CDNF in a 6-OHDA rat of Parkinson's disease. Our results also suggest the possible participation of TNF-α, IL-1β and IL-6 in rCDNF production by astrocytes, supporting their anti-inflammatory role.

  12. Echinacoside Protects against 6-Hydroxydopamine-Induced Mitochondrial Dysfunction and Inflammatory Responses in PC12 Cells via Reducing ROS Production

    PubMed Central

    Wang, Yue-Hua; Xuan, Zhao-Hong; Tian, Shuo; Du, Guan-Hua

    2015-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons at the substantia nigra. Mitochondrial dysfunction and inflammatory responses are involved in the mechanism of cell damage in PD. 6-Hydroxydopamine (6-OHDA), a dopamine analog, specifically damages dopaminergic neurons. Echinacoside (ECH) is a phenylethanoid glycoside isolated from the stems of Cistanche salsa, showing a variety of neuroprotective effects in previous studies. The present study was to investigate its effect against 6-OHDA-induced neurotoxicity and possible mechanisms in PC12 cells. The results showed that 6-OHDA reduced cell viability, decreased oxidation-reduction activity, decreased mitochondrial membrane potential, and induced mitochondria-mediated apoptosis compared with untreated PC12 cells. However, echinacoside treatment significantly attenuated these changes induced by 6-OHDA. In addition, echinacoside also could significantly alleviate the inflammatory responses induced by 6-OHDA. Further research showed that echinacoside could reduce 6-OHDA-induced ROS production in PC12 cells. These results suggest that the underlying mechanism of echinacoside against 6-OHDA-induced neurotoxicity may be involve in attenuating mitochondrial dysfunction and inflammatory responses by reducing ROS production. PMID:25788961

  13. Effects of discontinuing a high-fat diet on mitochondrial proteins and 6-hydroxydopamine-induced dopamine depletion in rats

    PubMed Central

    Ma, Delin; Shuler, Jeffrey M.; Raider, Kayla D.; Rogers, Robert S.; Wheatley, Joshua L.; Geiger, Paige C.; Stanford, John A.

    2015-01-01

    Diet-induced obesity can increase the risk for developing age-related neurodegenerative diseases including Parkinson’s disease (PD). Increasing evidence suggests that mitochondrial and proteasomal mechanisms are involved in both insulin resistance and PD. The goal of this study was to determine whether diet intervention could influence mitochondrial or proteasomal protein expression and vulnerability to 6-Hydroxydopamine (6-OHDA)-induced nigrostriatal dopamine (DA) depletion in rats’ nigrostriatal system. After a 3 month high-fat diet regimen, we switched one group of rats to a low-fat diet for 3 months (HF-LF group), while the other half continued with the high-fat diet (HF group). A chow group was included as a control. Three weeks after unilateral 6-OHDA lesions, HF rats had higher fasting insulin levels and higher Homeostasis model assessment of insulin resistance (HOMA-IR), indicating insulin resistance. HOMA-IR was significantly lower in HF-LF rats than HF rats, indicating that insulin resistance was reversed by switching to a low-fat diet. Compared to the Chow group, the HF group exhibited significantly greater DA depletion in the substantia nigra but not in the striatum. DA depletion did not differ between the HF-LF and HF group. Proteins related to mitochondrial function (such as AMPK, PGC-1α), and to proteasomal function (such as TCF11/Nrf1) were influenced by diet intervention, or by 6-OHDA lesion. Our findings suggest that switching to a low-fat diet reverses the effects of a high-fat diet on systemic insulin resistance, and mitochondrial and proteasomal function in the striatum. Conversely, they suggest that the effects of the high-fat diet on nigrostriatal vulnerability to 6-OHDA-induced DA depletion persist. PMID:25862572

  14. Increasing CO[sub 2] concentration inhibits cytochrome c oxidase (cytox) in vitro, cytochrome pathway (cytpath) activity in plant mitochondria and dark respiration in plant tissue

    SciTech Connect

    Gonzalez-Meler, M.A.; Drake, B.G.; Jacob, J. ); Ribas-Carbo, M.; Siedow, J.N. ); Aranda, X.; Azcon-Bieto, J.; Palet, A. )

    1994-06-01

    Dark respiration is inhibited in many plant be exposure to elevated atmospheric CO[sub 2] concentration. The addition of 0.2mM free CO[sub 2] in the reaction medium decreased citpath activity in Pisum sativum and Glycine max mitochondria at pH 7.2, possibly by inhibiting cytox. Under similar conditions, activity of purified cytox from beef heart was also inhibited. Cytox activity extracted from plants grown in elevated CO[sub 2] for 7 years was lower than in those grown in normal ambient. The relationship among these effects and the rate of respiration as well as the role of the alternative pathway in each case will be discussed.

  15. Inhibition of Human Cytochrome P450 2c8-catalyzed Amodiaquine N-desethylation: Effect of Five Traditionally and Commonly Used Herbs.

    PubMed

    Muthiah, Yasotha Devi; Ong, Chin Eng; Sulaiman, Siti Amrah; Ismail, Rusli

    2016-01-01

    In Southeast Asia and many parts of the world, herbal products are increasingly used in parallel with modern medicine. This study aimed to investigate the effects of herbs commonly used in Southeast Asia on activity of cytochrome P450 2C8 (CYP2C8), an important human hepatic enzyme in drug metabolism. The selected herbs, such as Eurycoma longifolia Jack (ELJ), Labisia pumila (LP), Echinacea purpurea (EP), Andrographis paniculata (AP), and Ginkgo biloba (GB), were subjected to inhibition studies using an in vitro CYP2C8 activity marker, amodiaquine N-desethylase assay. Inhibition parameters, inhibitory concentration 50% (IC50), and Ki values were determined to study the potency and mode of inhibition. All herbs inhibited CYP2C8 with the following order of potency: LP > ELJ > GB > AP > EP. LP and ELJ inhibited potently at Ki's of 2 and 4 times the Ki of quercetin, the positive control. The inhibition by LP was uncompetitive in nature as compared to competitive or mixed type inhibition observed with other herbs. GB exhibited moderate inhibitory effect at a Ki6 times larger than quercetin Ki. AP and EP, on the other hand, showed only weak inhibition. The herbs we chose represented the more commonly used herbs in Southeast Asia where collision of tradition and modernization in healthcare, if not properly managed, may lead to therapeutic misadventures. We conclude that concurrent consumption of some herbs, in particular, LP and ELJ, may have relevance in drug-herb interactions via CYP2C8 inhibition in vivo. Herbs are increasingly used in parallel with modern medicines nowadays. In this study five commonly used herbs in Southeast Asia region, ELJ, LP, EP, AP and GB, were investigated for their in vitro inhibitory potency on CYP2C8, an important drug-metaboliz-ing human hepatic enzyme. All herbs inhibited CYP2C8 activity marker, amodiaquine N-desethylation, with potency order of LP > ELJ > GB >AP > EP. LP, ELJ and GB exhibited Ki values of 2, 4 and 6 times the Ki of

  16. Inhibition of Human Cytochrome P450 2c8-catalyzed Amodiaquine N-desethylation: Effect of Five Traditionally and Commonly Used Herbs

    PubMed Central

    Muthiah, Yasotha Devi; Ong, Chin Eng; Sulaiman, Siti Amrah; Ismail, Rusli

    2016-01-01

    Background: In Southeast Asia and many parts of the world, herbal products are increasingly used in parallel with modern medicine. Objective: This study aimed to investigate the effects of herbs commonly used in Southeast Asia on activity of cytochrome P450 2C8 (CYP2C8), an important human hepatic enzyme in drug metabolism. Materials and Methods: The selected herbs, such as Eurycoma longifolia Jack (ELJ), Labisia pumila (LP), Echinacea purpurea (EP), Andrographis paniculata (AP), and Ginkgo biloba (GB), were subjected to inhibition studies using an in vitro CYP2C8 activity marker, amodiaquine N-desethylase assay. Inhibition parameters, inhibitory concentration 50% (IC50), and Ki values were determined to study the potency and mode of inhibition. Results: All herbs inhibited CYP2C8 with the following order of potency: LP > ELJ > GB > AP > EP. LP and ELJ inhibited potently at Ki's of 2 and 4 times the Ki of quercetin, the positive control. The inhibition by LP was uncompetitive in nature as compared to competitive or mixed type inhibition observed with other herbs. GB exhibited moderate inhibitory effect at a Ki6 times larger than quercetin Ki. AP and EP, on the other hand, showed only weak inhibition. Conclusion: The herbs we chose represented the more commonly used herbs in Southeast Asia where collision of tradition and modernization in healthcare, if not properly managed, may lead to therapeutic misadventures. We conclude that concurrent consumption of some herbs, in particular, LP and ELJ, may have relevance in drug-herb interactions via CYP2C8 inhibition in vivo. SUMMARY Herbs are increasingly used in parallel with modern medicines nowadays. In this study five commonly used herbs in Southeast Asia region, ELJ, LP, EP, AP and GB, were investigated for their in vitro inhibitory potency on CYP2C8, an important drug-metaboliz-ing human hepatic enzyme. All herbs inhibited CYP2C8 activity marker, amodiaquine N-desethylation, with potency order of LP > ELJ > GB >AP

  17. Melatonin inhibits the caspase-1/cytochrome c/caspase-3 cell death pathway, inhibits MT1 receptor loss and delays disease progression in a mouse model of amyotrophic lateral sclerosis

    PubMed Central

    Zhang, Yi; Cook, Anna; Kim, Jinho; Baranov, Sergei V.; Jiang, Jiying; Smith, Karen; Cormier, Kerry; Bennett, Erik; Browser, Robert P.; Day, Arthur L.; Carlisle, Diane; Ferrante, Robert J.; Wang, Xin; Friedlander, Robert M.

    2013-01-01

    Caspase-mediated cell death contributes to the pathogenesis of motor neuron degeneration in the mutant SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS), along with other factors such as inflammation and oxidative damage. By screening a drug library, we found that melatonin, a pineal hormone, inhibited cytochrome c release in purified mitochondria and prevented cell death in cultured neurons. In this study, we evaluated whether melatonin would slow disease progression in SOD1G93A mice. We demonstrate that melatonin significantly delayed disease onset, neurological deterioration and mortality in ALS mice. ALS-associated ventral horn atrophy and motor neuron death were also inhibited by melatonin treatment. Melatonin inhibited Rip2/caspase-1 pathway activation, blocked the release of mitochondrial cytochrome c, and reduced the overexpression and activation of caspase-3. Moreover, for the first time, we determined that disease progression was associated with the loss of both melatonin and the melatonin receptor 1A (MT1) in the spinal cord of ALS mice. These results demonstrate that melatonin is neuroprotective in transgenic ALS mice, and this protective effect is mediated through its effects on the caspase-mediated cell death pathway. Furthermore, our data suggest that melatonin and MT1 receptor loss may play a role in the pathological phenotype observed in ALS. The above observations indicate that melatonin and modulation of Rip2/caspase-1/cytochrome c or MT1 pathways may be promising therapeutic approaches for ALS. PMID:23537713

  18. Melatonin inhibits the caspase-1/cytochrome c/caspase-3 cell death pathway, inhibits MT1 receptor loss and delays disease progression in a mouse model of amyotrophic lateral sclerosis.

    PubMed

    Zhang, Yi; Cook, Anna; Kim, Jinho; Baranov, Sergei V; Jiang, Jiying; Smith, Karen; Cormier, Kerry; Bennett, Erik; Browser, Robert P; Day, Arthur L; Carlisle, Diane L; Ferrante, Robert J; Wang, Xin; Friedlander, Robert M

    2013-07-01

    Caspase-mediated cell death contributes to the pathogenesis of motor neuron degeneration in the mutant SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS), along with other factors such as inflammation and oxidative damage. By screening a drug library, we found that melatonin, a pineal hormone, inhibited cytochrome c release in purified mitochondria and prevented cell death in cultured neurons. In this study, we evaluated whether melatonin would slow disease progression in SOD1(G93A) mice. We demonstrate that melatonin significantly delayed disease onset, neurological deterioration and mortality in ALS mice. ALS-associated ventral horn atrophy and motor neuron death were also inhibited by melatonin treatment. Melatonin inhibited Rip2/caspase-1 pathway activation, blocked the release of mitochondrial cytochrome c, and reduced the overexpression and activation of caspase-3. Moreover, for the first time, we determined that disease progression was associated with the loss of both melatonin and the melatonin receptor 1A (MT1) in the spinal cord of ALS mice. These results demonstrate that melatonin is neuroprotective in transgenic ALS mice, and this protective effect is mediated through its effects on the caspase-mediated cell death pathway. Furthermore, our data suggest that melatonin and MT1 receptor loss may play a role in the pathological phenotype observed in ALS. The above observations indicate that melatonin and modulation of Rip2/caspase-1/cytochrome c or MT1 pathways may be promising therapeutic approaches for ALS. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Inhibition against mosquito cytochrome P450 enzymes by rhinacanthin-A, -B, and -C elicits synergism on cypermethrin cytotoxicity in Spodoptera frugiperda cells.

    PubMed

    Pethuan, Sirikun; Duangkaew, Panida; Sarapusit, Songklod; Srisook, Ekaruth; Rongnoparut, Pornpimol

    2012-09-01

    Rhinacanthus nasutus (Acanthaceae) is a shrub reported to contain insecticidal activities. The current study was conducted to determine whether R. nasutus constituents could inhibit benzyloxyresorufin O-debenzylation (BROD) mediated by CYP6AA3 and CYP6P7. Both enzymes have shown pyrethroid degradation activity and been implicated to play role in pyrethroid resistance in Anopheles minumus (Theobald) mosquito, a malaria vector. Three compounds, rhinacanthin-A, -B, and -C that exhibited potent inhibitory activity were isolated and purified from aerial part of R. nasutus. Their kinetic parameters and modes of inhibition were determined. Rhinacanthin-B was the most potent inhibitor in in vitro inhibition assay and exhibited mechanism-based inhibition against both CYP6AA3 and CYP6P7. Rhinacanthin-C which is a major compound of R. nasutus reversibly inhibited both enzymes in vitro with 2-4 folds less inhibitory potency than rhinacanthin-B. In contrast, rhinacanthin-A reversibly inhibited CYP6AA3, but inhibition against CYP6P7 was a mechanism-based inhibition type. Where mechanism-based inhibition was found, the inhibition showed characteristic of time-, concentration-dependence, and requirement of NADPH. Using 3-(4, 5-dimethylthiazol-2-y-l)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity assay in intact Spodoptera frugiperda (Sf9) cells, the three compounds increased susceptibility of CYP6AA3- and CYP6P7-expressing cells to cypermethrin cytotoxicity because of inhibition effect on mosquito enzymes. The combined inhibition effect on mosquito cytochrome P450 enzyme and synergistic effect on cypermethrin cytotoxicity of the three R. nasutus compounds could be beneficial for resistance management strategies in mosquito vector control.

  20. Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release.

    PubMed

    Düssmann, Heiko; Perez-Alvarez, Sergio; Anilkumar, Ujval; Papkovsky, Dmitri B; Prehn, Jochen Hm

    2017-06-01

    The detection of intracellular molecular oxygen (O2) levels is important for understanding cell physiology, cell death, and drug effects, and has recently been improved with the development of oxygen-sensitive probes that are compatible with live cell time-lapse microscopy. We here provide a protocol for the use of the nanoparticle probe MitoImage-MM2 to monitor intracellular oxygen levels by confocal microscopy under baseline conditions, in response to mitochondrial toxins, and following mitochondrial cytochrome-c release. We demonstrate that the MitoImage-MM2 probe, which embeds Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin as oxygen sensor and poly(9,9-dioctylfluorene) as an O2-independent component, enables quantitative, ratiometric time-lapse imaging of intracellular O2. Multiplexing with tetra-methyl-rhodamine-methyl ester in HeLa cervical cancer cells showed significant increases in intracellular O2 accompanied by strong mitochondrial depolarization when respiratory chain complexes III or IV were inhibited by Antimycin A or sodium azide, respectively, and when cells were maintained at 'physiological' tissue O2 levels (5% O2). Multiplexing also allowed us to monitor intracellular O2 during the apoptotic signaling process of mitochondrial outer membrane permeabilization in HeLa expressing cytochrome-c-eGFP, and demonstrated that mitochondria post cytochrome-c release are able to retain their capacity to respire at physiological O2 despite a decrease in mitochondrial membrane potential.

  1. BENZYL ALCOHOL PROTECTS AGAINST ACETAMINOPHEN HEPATOTOXICITY BY INHIBITING CYTOCHROME P450 ENZYMES BUT CAUSES MITOCHONDRIAL DYSFUNCTION AND CELL DEATH AT HIGHER DOSES

    PubMed Central

    Du, Kuo; McGill, Mitchell R.; Xie, Yuchao; Jaeschke, Hartmut

    2015-01-01

    Acetaminophen (APAP) hepatotoxicity is a serious public health problem in western countries. Current treatment options for APAP poisoning are limited and novel therapeutic intervention strategies are needed. A recent publication suggested that benzyl alcohol (BA) protects against APAP hepatotoxicity and could serve as a promising antidote for APAP poisoning. To assess the protective mechanisms of BA, C56Bl/6J mice were treated with 400mg/kg APAP and/or 270mg/kg BA. APAP alone caused extensive liver injury at 6h and 24h post-APAP. This injury was attenuated by BA co-treatment. Assessment of protein adduct formation demonstrated that BA inhibits APAP metabolic activation. In support of this, in vitro experiments also showed that BA dose-dependently inhibits cytochrome P450 activities. Correlating with the hepatoprotection of BA, APAP-induced oxidant stress and mitochondrial dysfunction were reduced. Similar results were obtained in primary mouse hepatocytes. Interestingly, BA alone caused mitochondrial membrane potential loss and cell toxicity at high doses, and its protective effect could not be reproduced in primary human hepatocytes (PHH). We conclude that BA protects against APAP hepatotoxicity mainly by inhibiting cytochrome P450 enzymes in mice. Considering its toxic effect and the loss of protection in PHH, BA is not a clinically useful treatment option for APAP overdose patient. PMID:26522885

  2. Benzyl alcohol protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes but causes mitochondrial dysfunction and cell death at higher doses.

    PubMed

    Du, Kuo; McGill, Mitchell R; Xie, Yuchao; Jaeschke, Hartmut

    2015-12-01

    Acetaminophen (APAP) hepatotoxicity is a serious public health problem in western countries. Current treatment options for APAP poisoning are limited and novel therapeutic intervention strategies are needed. A recent publication suggested that benzyl alcohol (BA) protects against APAP hepatotoxicity and could serve as a promising antidote for APAP poisoning. To assess the protective mechanisms of BA, C56Bl/6J mice were treated with 400 mg/kg APAP and/or 270 mg/kg BA. APAP alone caused extensive liver injury at 6 h and 24 h post-APAP. This injury was attenuated by BA co-treatment. Assessment of protein adduct formation demonstrated that BA inhibits APAP metabolic activation. In support of this, in vitro experiments also showed that BA dose-dependently inhibits cytochrome P450 activities. Correlating with the hepatoprotection of BA, APAP-induced oxidant stress and mitochondrial dysfunction were reduced. Similar results were obtained in primary mouse hepatocytes. Interestingly, BA alone caused mitochondrial membrane potential loss and cell toxicity at high doses, and its protective effect could not be reproduced in primary human hepatocytes (PHH). We conclude that BA protects against APAP hepatotoxicity mainly by inhibiting cytochrome P450 enzymes in mice. Considering its toxic effect and the loss of protection in PHH, BA is not a clinically useful treatment option for APAP overdose patient. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    USDA-ARS?s Scientific Manuscript database

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethan...

  4. The cytochromes of Acanthamoeba castellanii.

    PubMed Central

    Edwards, S W; Chagla, A H; Griffiths, A J; Lloyd, D

    1977-01-01

    1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s). PMID:597258

  5. Characterization of the structural determinants required for potent mechanism-based inhibition of human cytochrome P450 1A1 by cannabidiol.

    PubMed

    Yamaori, Satoshi; Okushima, Yoshimi; Yamamoto, Ikuo; Watanabe, Kazuhito

    2014-05-25

    We previously demonstrated that cannabidiol (CBD) was a potent mechanism-based inhibitor of human cytochrome P450 1A1 (CYP1A1). However, the moiety of CBD that contributes to the potent mechanism-based inhibition of human CYP1A1 remains unknown. Thus, the effects of compounds structurally related to CBD on CYP1A1 activity were examined with recombinant human CYP1A1 in order to characterize the structural requirements for potent inactivation by CBD. When preincubated in the presence of NADPH for 20min, olivetol, which corresponds to the pentylresorcinol moiety of CBD, enhanced the inhibition of the 7-ethoxyresorufin O-deethylase activity of CYP1A1. In contrast, d-limonene, which corresponds to the terpene moiety of CBD, failed to inhibit CYP1A1 activity in a metabolism-dependent manner. Pentylbenzene, which lacks two free phenolic hydroxyl groups, also did not enhance CYP1A1 inhibition. On the other hand, preincubation of the CBD-2'-monomethyl ether (CBDM) and CBD-2',6'-dimethyl ether (CBDD) enhanced the inhibition of CYP1A1 activity. Inhibition by cannabidivarin (CBDV), which possessed a propyl side chain, was strongly potentiated by its preincubation. Orcinol, which has a methyl group, augmented CYP1A1 inhibition, whereas its derivative without an alkyl side chain, resorcinol, did not exhibit any metabolism-dependent inhibition. The preincubation of CBD-hydroxyquinone did not markedly enhance CYP1A1 inhibition. We further confirmed that olivetol, CBDM, CBDD, CBDV, and orcinol, as well as CBD (kinact=0.215min(-1)), inactivated CYP1A1 activity; their kinact values were 0.154, 0.0638, 0.0643, 0.226, and 0.0353min(-1), respectively. These results suggest that the methylresorcinol structure in CBD may have structurally important roles in the inactivation of CYP1A1.

  6. Investigation of drug-drug interaction via mechanism-based inhibition of cytochrome P450 3A by macrolides in dexamethasone-treated female rats.

    PubMed

    Kanazu, Takushi; Sato, Norihito; Kadono, Kyoko; Touchi, Akira; Takeda, Yuri; Yamaguchi, Yoshitaka; Baba, Takahiko

    2012-05-01

    The in vitro and in vivo inhibition of cytochrome P450 (CYP) 3A with mechanism-based inhibition (MBI) by macrolides was investigated using dexamethasone-treated female rats (DEX-female rats). In the in vitro CYP inhibition studies using erythromycin (ERM) and clarithromycin (CAM), similar inhibition responses were observed between human and DEX-female rat liver microsomes, however, there were fewer effects in intact male rats. The ex vivo study showed that midazolam (MDZ) metabolism in liver microsomes of DEX-female rats was reduced by ERM administration and the inhibitory effect was increased with increasing ERM doses, indicating that metabolite intermediate complex formation caused irreversible inhibition of CYP3A activity in DEX-female rats as well as in humans. In the in vivo studies, ERM and CAM significantly increased the area under the plasma concentration-time curve of MDZ and decreased the total clearance in DEX-female rats. It was concluded that the DDIs via MBI of CYP3A following macrolide administration in humans could be reproduced in female rats, suggesting that DEX-female rats can serve as an in vivo model for assessing this DDI in humans.

  7. Inhibition on human liver cytochrome P450 3A4 by constituents of fennel (Foeniculum vulgare): identification and characterization of a mechanism-based inactivator.

    PubMed

    Subehan; Zaidi, Syed F H; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2007-12-12

    Fennel, a seed of Foeniculum vulgare, is used as a culinary spice and traditional medicine. The methanolic extract of fennel showed a characteristic of mechanism-based inactivation on erythromycin N-demethylation mediated by human liver microsomal cytochrome P450 3A4 (CYP3A4). The present study was conducted to identify the fennel constituent having the inhibition. Thirteen compounds have been isolated from a methanol extract of fennel and tested for their inhibition on CYP3A4. Among them, 5-methoxypsoralen (5-MOP) showed the strongest inhibition with an IC50 value of 18.3 microM and a mixed type of inhibition. In addition, with the preincubation time of 20 min only 5-MOP showed preincubation time dependency; the IC50 value decreased from 18.3 microM with a preincubation time of 0 min to 4.6 microM with a preincubation time of 20 min. Further investigation on 5-MOP showed the characteristics of time-dependent inhibition, requirement of NADPH, lack of protecting effect of nucleophiles, and recovery of CYP3A4 activity by the competitive inhibitor. This result suggests that the inhibitory activity of CYP3A4 by 5-MOP was a mechanism-based inactivation. The kinetic parameter for mechanism-based inactivation was characterized by a KI value of 15.0 microM and a kinact value of 0.098 min(-1).

  8. In vitro assessment of cytochrome P450 inhibition and induction potential of tanespimycin and its major metabolite, 17-amino-17-demethoxygeldanamycin.

    PubMed

    Gan, Jinping; Liu-Kreyche, Peggy; Humphreys, W Griffith

    2012-01-01

    To assess the inhibition and induction potential of tanespimycin and its major metabolite, 17-amino-17-demethoxygeldanamycin (17-AG) on cytochrome P450 (CYP) enzymes. The inhibitory effect of tanespimycin and 17-AG on various CYP enzymes was determined in human liver microsomes. The inductive effects of tanespimycin and 17-AG on CYP1A2, CYP2B6, and CYP3A4/5 were determined in cultured primary human hepatocytes. Tanespimycin did not inhibit the activities of CYP1A2, 2A6, 2B6, and 2E1 up to a concentration of 60 μM, while it moderately inhibited CYP3A4/5 and 2C19, and weakly inhibited CYP2C8, 2C9, and 2D6. In addition, its inhibition on CYP3A4/5 was time-dependent. 17-AG moderately inhibited the activities of CYP3A4/5 and CYP2C19, but did not inhibit other CYPs up to a concentration of 30 μM. The inhibition of CYP3A4/5 by 17-AG was not time-dependent. Tanespimycin and 17-AG did not significantly induce the activities of CYP1A2, CYP2B6, or CYP3A4/5 in cultured human hepatocytes at concentrations up to 40 and 20 μM for tanespimycin and 17-AG, respectively. Tanespimycin together with its active metabolite, 17-AG are moderate inhibitors of CYP3A4/5 and CYP2C19, but not inducers of CYPs. Therefore, co-administration of tanespimycin has the potential to increase the exposure of substrates of CYP2C19 and CYP3A4/5.

  9. Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release

    PubMed Central

    Düssmann, Heiko; Perez-Alvarez, Sergio; Anilkumar, Ujval; Papkovsky, Dmitri B; Prehn, Jochen HM

    2017-01-01

    The detection of intracellular molecular oxygen (O2) levels is important for understanding cell physiology, cell death, and drug effects, and has recently been improved with the development of oxygen-sensitive probes that are compatible with live cell time-lapse microscopy. We here provide a protocol for the use of the nanoparticle probe MitoImage-MM2 to monitor intracellular oxygen levels by confocal microscopy under baseline conditions, in response to mitochondrial toxins, and following mitochondrial cytochrome-c release. We demonstrate that the MitoImage-MM2 probe, which embeds Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6–pentafluorophenyl)-porphyrin as oxygen sensor and poly(9,9-dioctylfluorene) as an O2-independent component, enables quantitative, ratiometric time-lapse imaging of intracellular O2. Multiplexing with tetra-methyl-rhodamine-methyl ester in HeLa cervical cancer cells showed significant increases in intracellular O2 accompanied by strong mitochondrial depolarization when respiratory chain complexes III or IV were inhibited by Antimycin A or sodium azide, respectively, and when cells were maintained at ‘physiological’ tissue O2 levels (5% O2). Multiplexing also allowed us to monitor intracellular O2 during the apoptotic signaling process of mitochondrial outer membrane permeabilization in HeLa expressing cytochrome-c-eGFP, and demonstrated that mitochondria post cytochrome-c release are able to retain their capacity to respire at physiological O2 despite a decrease in mitochondrial membrane potential. PMID:28569778

  10. Rapid screening of commercially available herbal products for the inhibition of major human hepatic cytochrome P450 enzymes using the N-in-one cocktail.

    PubMed

    Sevior, D K; Hokkanen, J; Tolonen, A; Abass, K; Tursas, L; Pelkonen, O; Ahokas, J T

    2010-04-01

    Self-administration of complementary products concurrently with conventional medication is increasingly common. The potential for cytochrome P450 (CYP) inhibition requires investigation. The N-in-one assay with ten probe substrates for nine CYPs was used with human liver microsomes to investigate ten products. CYP inhibition was measured in a single liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis. Estimated IC(50)-values were determined for the extracts that produced significant inhibition (less than 100 microg ml(-1)). Inhibition of CYP2C19 by dong quai (IC(50) = 13.7-14.3 microg ml(-1) for the methanolic extract) and CYP2D6 by goldenseal (IC(50) = 6.7 and 6.3 microg ml(-1) for the aqueous and methanolic extracts, respectively), are of particular concern as the potential for adverse interactions is high. The inhibition of CYP2C8 by horsetail (IC(50) = 93 microg ml(-1) for the aqueous extract) requires further investigation, as the potential for concurrent use with products that require CYP2C8 for metabolism is significant. CYP3A4 inhibition varied depending on the probe reaction being monitored. The earlier reported findings of inhibition by black cohosh, goldenseal and gotu kola were confirmed. The present work has shown that the N-in-one cocktail is a rapid and reliable method that can be used as an initial screen to help prioritize products that require more detailed investigations and it can also be applied to monitor product variability.

  11. Effect of antipsychotic drugs on human liver cytochrome P-450 (CYP) isoforms in vitro: preferential inhibition of CYP2D6.

    PubMed

    Shin, J G; Soukhova, N; Flockhart, D A

    1999-09-01

    The ability of antipsychotic drugs to inhibit the catalytic activity of five cytochrome P-450 (CYP) isoforms was compared using in vitro human liver microsomal preparations to evaluate the relative potential of these drugs to inhibit drug metabolism. The apparent kinetic parameters for enzyme inhibition were determined by nonlinear regression analysis of the data. All antipsychotic drugs tested competitively inhibited dextromethorphan O-demethylation, a selective marker for CYP2D6, in a concentration-dependent manner. Thioridazine and perphenazine were the most potent, with IC(50) values (2.7 and 1.5 microM) that were comparable to that of quinidine (0.52 microM). The estimated K(i) values for CYP2D6-catalyzing dextrorphan formation were ranked in the following order: perphenazine (0.8 microM), thioridazine (1.4 microM), chlorpromazine (6.4 microM), haloperidol (7.2 microM), fluphenazine (9.4 microM), risperidone (21.9 microM), clozapine (39.0 microM), and cis-thiothixene (65.0 microM). No remarkable inhibition of other CYP isoforms was observed except for moderate inhibition of CYP1A2-catalyzed phenacetin O-deethylation by fluphenazine (K(i) = 40.2 microM) and perphenazine (K(i) = 65.1). The estimated K(i) values for the inhibition of CYP2C9, 2C19, and 3A were >300 microM in almost all antipsychotics tested. These results suggest that antipsychotic drugs exhibit a striking selectivity for CYP2D6 compared with other CYP isoforms. This may reflect a remarkable commonality of structure between the therapeutic targets for these drugs, the transporters, and metabolic enzymes that distribute and eliminate them. Clinically, coadministration of these medicines with drugs that are primarily metabolized by CYP2D6 may result in significant drug interactions.

  12. Coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in cytochrome c oxidase. Studies with the carbon monoxide inhibited enzyme.

    PubMed

    Capitanio, N; Capitanio, G; Minuto, M; De Nitto, E; Palese, L L; Nicholls, P; Papa, S

    2000-05-30

    A study is presented on the coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in carbon monoxide inhibited cytochrome c oxidase isolated from bovine heart mitochondria. Detailed analysis of the coupling number for H(+) release per heme a, Cu(A) oxidized (H(+)/heme a, Cu(A) ratio) was based on direct measurement of the balance between the oxidizing equivalents added as ferricyanide to the CO-inhibited fully reduced COX, the equivalents of heme a, Cu(A), and added cytochrome c oxidized and the H(+) released upon oxidation and all taken up back by the oxidase upon rereduction of the metal centers. One of two reductants was used, either succinate plus a trace of mitochondrial membranes (providing a source of succinate-c reductase) or hexaammineruthenium(II) as the chloride salt. The experimental H(+)/heme a, Cu(A) ratios varied between 0.65 and 0.90 in the pH range 6.0-8.5. The pH dependence of the H(+)/heme a, Cu(A) ratios could be best-fitted by a function involving two redox-linked acid-base groups with pK(o)-pK(r) of 5.4-6.9 and 7.3-9.0, respectively. Redox titrations in the same samples of the CO-inhibited oxidase showed that Cu(A) and heme a exhibited superimposed E'(m) values, which decreased, for both metals, by around 20 mV/pH unit increase in the range 6.0-8.5. A model in which oxido-reduction of heme a and Cu(A) are both linked to the pK shifts of the two acid-base groups, characterized by the analysis of the pH dependence of the H(+)/heme a, Cu(A) ratios, provided a satisfactory fit for the pH dependence of the E'(m) of heme a and Cu(A). The results presented are consistent with a primary involvement of the redox Bohr effects shared by heme a and Cu(A) in the proton-pumping activity of cytochrome c oxidase.

  13. Inhibition of Cytochrome P450 (CYP3A4) Activity by Extracts from 57 Plants Used in Traditional Chinese Medicine (TCM).

    PubMed

    Ashour, Mohamed L; Youssef, Fadia S; Gad, Haidy A; Wink, Michael

    2017-01-01

    Herbal medicine is widely used all over the world for treating various health disorders. It is employed either alone or in combination with synthetic drugs or plants to be more effective. The assessment of the effect of both water and methanol extracts of 57 widely used plants from Traditional Chinese Medicine (TCM) against the main phase I metabolizing enzyme CYP3A4 in vitro for the first time. The inhibition of cytochrome P450 activity was evaluated using a luminescence assay. The principal component analysis (PCA) was used to correlate the inhibitory activity with the main secondary metabolites present in the plant extracts. Molecular modeling studies on CYP3A4 (PDB ID 4NY4) were carried out with 38 major compounds present in the most active plant extracts to validate the observed inhibitory effect. Aqueous extracts of Acacia catechu, Andrographis paniculata, Arctium lappa, Areca catechu, Bupleurum marginatum, Chrysanthemum indicum, Dysosma versipellis, and Spatholobus suberectus inhibited CYP3A4 is more than 85% (at a dose of 100 μg/mL). The corresponding methanol extracts of A. catechu, A. paniculata, A. catechu, Mahonia bealei, and Sanguisorba officinalis inhibited the enzyme by more than 50%. Molecular modeling studies revealed that two polyphenols, namely hesperidin and rutin, revealed the highest fitting scores in the active sites of the CYP3A4 with binding energies equal to -74.09 and -71.34 kcal/mol, respectively. These results provide evidence that many TCM plants can inhibit CYP3A4, which might cause a potential interference with the metabolism of other concomitantly administered herbs or drugs. In this study, the inhibitory activity of the aqueous and methanol extracts of 57 widely used plants from Traditional Chinese Medicine (TCM) against the main phase I metabolizing enzyme CYP3A4 was tested in vitro for the first time.Aqueous extracts of Acacia catechu, Andrographis paniculata, Arctium lappa, Areca catechu, Bupleurum marginatum, Dysosma

  14. Inhibition of Cytochrome P450 (CYP3A4) Activity by Extracts from 57 Plants Used in Traditional Chinese Medicine (TCM)

    PubMed Central

    Ashour, Mohamed L; Youssef, Fadia S; Gad, Haidy A; Wink, Michael

    2017-01-01

    Background: Herbal medicine is widely used all over the world for treating various health disorders. It is employed either alone or in combination with synthetic drugs or plants to be more effective. Objective: The assessment of the effect of both water and methanol extracts of 57 widely used plants from Traditional Chinese Medicine (TCM) against the main phase I metabolizing enzyme CYP3A4 in vitro for the first time. Materials and Methods: The inhibition of cytochrome P450 activity was evaluated using a luminescence assay. The principal component analysis (PCA) was used to correlate the inhibitory activity with the main secondary metabolites present in the plant extracts. Molecular modeling studies on CYP3A4 (PDB ID 4NY4) were carried out with 38 major compounds present in the most active plant extracts to validate the observed inhibitory effect. Results: Aqueous extracts of Acacia catechu, Andrographis paniculata, Arctium lappa, Areca catechu, Bupleurum marginatum, Chrysanthemum indicum, Dysosma versipellis, and Spatholobus suberectus inhibited CYP3A4 is more than 85% (at a dose of 100 μg/mL). The corresponding methanol extracts of A. catechu, A. paniculata, A. catechu, Mahonia bealei, and Sanguisorba officinalis inhibited the enzyme by more than 50%. Molecular modeling studies revealed that two polyphenols, namely hesperidin and rutin, revealed the highest fitting scores in the active sites of the CYP3A4 with binding energies equal to -74.09 and -71.34 kcal/mol, respectively. Conclusion: These results provide evidence that many TCM plants can inhibit CYP3A4, which might cause a potential interference with the metabolism of other concomitantly administered herbs or drugs. SUMMARY In this study, the inhibitory activity of the aqueous and methanol extracts of 57 widely used plants from Traditional Chinese Medicine (TCM) against the main phase I metabolizing enzyme CYP3A4 was tested in vitro for the first time.Aqueous extracts of Acacia catechu, Andrographis

  15. Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii.

    PubMed

    Alday, P Holland; Bruzual, Igor; Nilsen, Aaron; Pou, Sovitj; Winter, Rolf; Ben Mamoun, Choukri; Riscoe, Michael K; Doggett, J Stone

    2017-02-01

    Toxoplasma gondii is an apicomplexan parasite that causes fatal and debilitating brain and eye disease. Endochinlike quinolones (ELQs) are preclinical compounds that are efficacious against apicomplexan-caused diseases, including toxoplasmosis, malaria, and babesiosis. Of the ELQs, ELQ-316 has demonstrated the greatest efficacy against acute and chronic experimental toxoplasmosis. Although genetic analyses in other organisms have highlighted the importance of the cytochrome bc1 complex Qi site for ELQ sensitivity, the mechanism of action of ELQs against T. gondii and the specific mechanism of ELQ-316 remain unknown. Here, we describe the selection and genetic characterization of T. gondii clones resistant to ELQ-316. A T. gondii strain selected under ELQ-316 drug pressure was found to possess a Thr222-Pro amino acid substitution that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor. These findings provide further evidence for ELQ Qi site inhibition in T. gondii and greater insight into the interactions of Qi site inhibitors with the apicomplexan cytochrome bc1 complex. Copyright © 2017 American Society for Microbiology.

  16. SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

    SciTech Connect

    Klein, M.; Canoll, P.D.; Musacchio, J.M. )

    1991-01-01

    The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.

  17. Synthesis of cytochrome c oxidase 1 (SCO1) inhibits insulin sensitivity by decreasing copper levels in adipocytes.

    PubMed

    Wei, Xiang-Bo; Guo, Liang; Liu, Yang; Zhou, Shui-Rong; Liu, Yuan; Dou, Xin; Du, Shao-Yue; Ding, Meng; Peng, Wan-Qiu; Qian, Shu-Wen; Huang, Hai-Yan; Tang, Qi-Qun

    2017-09-23

    Dysregulation of insulin signaling leads to type 2 diabetes mellitus (T2DM) and other metabolic disorders. Obesity is an important contributor to insulin resistance, and although the understanding of this relationship has improved in recent years, the mechanism of obesity-induced insulin resistance is not completely understood. Disorders of copper metabolism tend to accompany the development of obesity, which increases the risk of insulin resistance. Synthesis of cytochrome c oxidase 1 (SCO1) functions in the assembly of cytochrome c oxidase (COX) and cellular copper homeostasis. However, the role of SCO1 in the regulation of metabolism remains unknown. Here, we found that obese mice had higher expression of SCO1 and lower levels of copper in white adipose tissue (WAT) than did the control mice. Overexpression of SCO1 in adipocytes was associated with copper deficiency. Copper increased insulin sensitivity by decreasing the level of phosphatase and tensin homolog (PTEN) protein. Ectopic expression of SCO1 led to insulin resistance and was accompanied by a decrease in intracellular copper level, and addition of copper abolished the inhibitory effect of SCO1 on insulin sensitivity. Our results demonstrated a novel role of SCO1 in modulating insulin sensitivity via the regulation of copper concentration in WAT and suggested a potential therapeutic target for T2DM. Copyright © 2017. Published by Elsevier Inc.

  18. Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo

    PubMed Central

    Ueng, Yune-Fang; Chen, Chien-Chih; Chung, Yu-Ting; Liu, Tsung-Yun; Chang, Yu-Ping; Lo, Wei-Sheng; Murayama, Norie; Yamazaki, Hiroshi; Souček, Pavel; Chau, Gar-Yang; Chi, Chin-Wen; Chen, Ruei-Ming; Li, Ding-Tzai

    2011-01-01

    BACKGROUND AND PURPOSE Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo. EXPERIMENTAL APPROACH Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS). KEY RESULTS CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC50 value decreased by 98%). This time-dependent inactivation (kinact 0.044 min−1; KI 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo. CONCLUSIONS AND IMPLICATIONS Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. ‘Poor metabolizers’ carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice. PMID:21418183

  19. Inhibition effects of Vernonia cinerea active compounds against cytochrome P450 2A6 and human monoamine oxidases, possible targets for reduction of tobacco dependence.

    PubMed

    Prasopthum, Aruna; Pouyfung, Phisit; Sarapusit, Songklod; Srisook, Ekaruth; Rongnoparut, Pornpimol

    2015-04-01

    The human cytochrome P450 2A6 (CYP2A6) and monoamine oxidases (MAO-A and MAO-B), catalyzing nicotine and dopamine metabolisms, respectively, are two therapeutic targets of nicotine dependence. Vernonia cinerea, a medicinal plant commonly used for treatment of diseases such as asthma and bronchitis, has been shown reducing tobacco dependence effect among tobacco users. In the present study, we found eight active compounds isolated from V. cinerea that comprise inhibitory activity toward CYP2A6 and MAO-A and MAO-B enzymes using activity-guided assays, with coumarin as substrate of CYP2A6 and kynuramine of MAOs. These compounds were three flavones (apigenin, chrysoeriol, luteolin), one flavonol (quercetin), and four hirsutinolide-type sesquiterpene lactones (8α-(2-methylacryloyloxy)-hirsutinolide-13-O-acetate, 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate, 8α-tigloyloxyhirsutinolide-13-O-acetate, and 8α-(4-hydroxytigloyloxy)-hirsutinolide-13-O-acetate). Modes and kinetics of inhibition against the three enzymes were determined. Flavonoids possessed strong inhibitory effect on CYP2A6 in reversible mode, while inhibition by hirsutinolides was mechanism-based (NADPH-, concentration-, and time-dependence) and irreversible. Inhibition by hirsutinolides could not be reversed by dialysis and by addition of trapping agents or potassium ferricyanide. Flavonoids inhibited MAOs with variable degrees and were more prominent in inhibition toward MAO-A than hirsutinolides, while two of hirsutinolides inhibited MAO-B approximately comparable to two flavonoids. These results could have implications in combination of drug therapy for smoking cessation.

  20. A Novel Rice Cytochrome P450 Gene, CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis1[C][W][OPEN

    PubMed Central

    Saika, Hiroaki; Horita, Junko; Taguchi-Shiobara, Fumio; Nonaka, Satoko; Nishizawa-Yokoi, Ayako; Iwakami, Satoshi; Hori, Kiyosumi; Matsumoto, Takashi; Tanaka, Tsuyoshi; Itoh, Takeshi; Yano, Masahiro; Kaku, Koichiro; Shimizu, Tsutomu; Toki, Seiichi

    2014-01-01

    Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species. PMID:24406793

  1. A novel rice cytochrome P450 gene, CYP72A31, confers tolerance to acetolactate synthase-inhibiting herbicides in rice and Arabidopsis.

    PubMed

    Saika, Hiroaki; Horita, Junko; Taguchi-Shiobara, Fumio; Nonaka, Satoko; Nishizawa-Yokoi, Ayako; Iwakami, Satoshi; Hori, Kiyosumi; Matsumoto, Takashi; Tanaka, Tsuyoshi; Itoh, Takeshi; Yano, Masahiro; Kaku, Koichiro; Shimizu, Tsutomu; Toki, Seiichi

    2014-11-01

    Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species. © 2014 American Society of Plant Biologists. All Rights Reserved.

  2. Cytochrome f

    SciTech Connect

    Soriano, G.M.; Smith, J.L.; Cramer, W.A.

    2001-07-17

    Cytochrome f (f, folium, leaf), a c-type cytochrome with a characteristic CysXXCysHis amino acid sequence for heme ligation, is the largest of the four major protein subunits of the membrane-embedded cytochrome b{sub 6}{sup f} complex of oxygenic photosynthesis. It contains 285-86 amino acids, consisting of a soluble 250-residue domain on the p-side (positive-side) or lumen-side of the membrane, a single trans-membrane 20-residue {alpha}-helix, and an n- or stromal-side segment consisting of 15 residues. These domains contain, respectively, the heme prosthetic group and intraprotein electron transfer pathway, the membrane anchor and a short segment that is important in the assembly of the b{sub 6}{sup f} complex. The function of the cytochrome f in oxygenic photosynthesis is to act as the terminal electron acceptor in the membrane-embedded cytochrome b{sub 6}{sup f} complex that provides the electron transport connection between the photosystem II and photosystem I reaction centers. Electron transfer through the complex is coupled to proton translocation and generation of a proton electrochemical potential that is utilized to drive the synthesis of ATP through the proton-motive ATP synthase. These functions of the cytochrome b{sub 6}{sup f} complex are analogous to those of the multisubunit cytochrome bc{sub 1} complex (ubiquinol:cytochrome c oxidoreductase) of the mitochondrial respiratory chain and photosynthetic bacteria. Both complexes contain four redox centers with very similar redox and structural properties: a covalently bound c-type heme in cytochrome f or c{sub 1}, the 2Fe-2S cluster of the Rieske ISP, and the two noncovalently bound hemes of cytochrome b. The structure properties have been defined in 3.0-3.1 {angstrom} structures of the b{sub 6}{sup f} complex from a thermophilic cyanobacterium and a green alga. These structures also defined a fifth redox prosthetic group, a novel covalently bound heme, tentatively called heme x. With the exception of

  3. In vitro metabolic interactions between black cohosh (Cimicifuga racemosa) and tamoxifen via inhibition of cytochromes P450 2D6 and 3A4

    PubMed Central

    Li, Jinghu; Gödecke, Tanja; Chen, Shao-Nong; Imai, Ayano; Lankin, David; Farnsworth, Norman R.; Pauli, Guido F.; van Breemen, Richard B.; Nikolić, Dejan

    2012-01-01

    Women who experience hot flashes as a side effect of tamoxifen therapy often try botanical remedies such as black cohosh to alleviate these symptoms. Since pharmacological activity of tamoxifen is dependent on the metabolic conversion into active metabolites by the action of cytochromes P450 2D6 and 3A4, the objective of this study was to evaluate whether black cohosh extracts can inhibit formation of active tamoxifen metabolites and possibly reduce its clinical efficacy.At 50 µg/ml, a 75% ethanolic extract of black cohosh inhibited formation of 4-hydroxy-tamoxifen by 66.3%, N-desmethyl tamoxifen by 74.6% and α-hydroxy tamoxifen by 80.3%. In addition, using midazolam and dextromethorphan as probe substrates, this extract inhibited CYP3A4 and CYP2D6 with IC50 values of 16.5 and 50.1 µg/ml, respectively.Eight triterpene glycosides were identified as competitive CYP3A4 inhibitors with IC50 values ranging from 2.3–5.1 µM, while the alkaloids protopine and allocryptopine were identified as competitive CYP2D6 inhibitors with Ki values of 78 and 122 nM, respectively.The results of this study suggests that co-administration of black cohosh with tamoxifen might interfere with the clinical efficacy of this drug. However, additional clinical studies are needed to determine the clinical significance of these in vitro results. PMID:21827327

  4. Anti-cytochrome P450 IIE1 (anti IIE1) and dimethyl sulfoxide inhibit acetaminophen and dimethylnitrosamine oxidation similarly

    SciTech Connect

    Jaw, S.; Jeffery, E.H. ); Roberts, D.W. )

    1991-03-11

    To evaluate specificity of dimethyl sulfoxide (DMSO), the authors compared anti IIE1 and DMSO inhibition of P450 oxidations. Hepatic microsomes from control and acetone-induced female Swiss-Webster mice were preincubated with polyclonal anti IIE1 or IgG for 20 min at 4C before addition of an NADPH-generating system, DMSO or buffer, and substrate (Ethylmorphine, EM; dimethylnitrosamine, DMN; or acetaminophen, AP; 1 mM final concentration). After 20 min at 37C, the incubations were terminated by adding 20% trichloroacetic acid or methanol. Formaldehyde was determined by the Nash method when using EM or DMN as substrate. AP-glutathione conjugate was determined by HPLC when using AP as substrate. Anti IIE1 and DMSO did not inhibit EM demethylation in control or acetone microsomes. However, DMSO inhibited DMN demethylation by 26% and 64% in control and 30% and 75% in acetone microsomes. Anti IIE1 inhibited DMN demethylation by 44% and 24% in control and acetone microsomes, respectively. DMSO inhibited AP metabolism by 31% and 56% and anti IIE1 inhibited AP metabolism by 33%, in control microsomes. The inhibitions of DMN and AP metabolism by anti IIE1 and DMSO were only additive at submaximal inhibitor concentrations and confirm that DMSO specifically inhibits IIE1 activity.

  5. Evaluation of In Vitro Cytochrome P450 Inhibition and In Vitro Fate of Structurally Diverse N-Oxide Metabolites: Case Studies with Clozapine, Levofloxacin, Roflumilast, Voriconazole and Zopiclone.

    PubMed

    Giri, Poonam; Naidu, Sneha; Patel, Nirmal; Patel, Harilal; Srinivas, Nuggehally R

    2017-08-01

    The role of metabolite(s) to elicit potential clinical drug-drug interaction (DDI) via cytochrome P450 enzymes (CYP) is gaining momentum. In this context, the role of N-oxides for in vitro CYP inhibition has not been evaluated. The objectives of this study were: (a) to examine in vitro CYP inhibition of N-oxides of clozapine, levofloxacin, roflumilast, voriconazole and zopiclone in a tiered approach and (b) evaluate in vitro fate of aforementioned N-oxides examined in recombinant CYPs, human microsomes and hepatocytes. CYP enzymes evaluated in the work included: CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 using standard procedures for incubation with appropriate probe substrates. The initial cutoff for CYP inhibition was ≥50% using 2 and 10 µM concentrations of various N-oxide metabolites (Tier 1). IC50 values were constructed for the CYP pathway(s) that showed ≥50% inhibition (Tier 2). In addition, co-incubation of N-oxides with parent was performed to evaluate potentiation of CYP inhibition (Tier 3). N-oxides of clozapine (CYP2B6/2C19) and voriconazole (CYP2C9/3A4) showed CYP inhibition ≥50%. Clozapine-N-oxide inhibited CYP2B6 and CYP2C19 pathways with IC50 of 8.3 and 8.7 µM, respectively. Voriconazole-N-oxide inhibited CYP2B6 and CYP2C19 pathways with IC50 of 10.5 and 11.2 µM, respectively. Co-incubation of clozapine-N-oxide with clozapine potentiated CYP2B6/2C19 pathways; however, incubation of voriconazole-N-oxide with voriconazole did not appear to potentiate the CYP pathways because parent caused an inhibition of almost 80%. None of the N-oxides appeared to further undergo biotransformation as judged by the in vitro metabolic fate experiments (stage 2). Clinical DDI potential of specific CYP enzymes needs to be considered arising due to circulatory concentrations of certain N-oxides depending on the dose size and/or frequency of dosing of the respective parent drugs.

  6. The inhibition of major human hepatic cytochrome P450 enzymes by 18 pesticides: comparison of the N-in-one and single substrate approaches.

    PubMed

    Abass, Khaled; Pelkonen, Olavi

    2013-08-01

    In the present study on human hepatic microsomes, the N-in-one assay with ten probe substrates for nine cytochrome-P450 enzymes (CYPs) was compared with the single substrate assays to investigate pesticides-CYP interactions. CYP inhibition was measured by liquid chromatography-tandem mass spectrometry (LC/MS-MS). As illustrated by the initial screening at 100 μM concentration of 18 pesticides, CYPs are more sensitive to organophosphates (OPs) than to other pesticide groups. Chlorpyrifos and fenitrothion were most effective in inhibiting CYP1A1/2, and CYP2B6. Profenofos was also inhibitory towards multiple CYPs. Pyrethroids, e.g. deltamethrin, fenvalerate and lambda-cyhalothrin, potently inhibited CYP2D6. CYP3A4 activity was moderately inhibited by fenvalerate and potently by alpha-cypermethrin. The correlations between IC50 values obtained from the N-in-one and single substrate approaches were highly significant for CYP2Cs (r(2)=0.94), CYP3A4, omeprazole-sulfoxidation, (r(2)=0.89), followed by CYP1A2 and CYP2B6 (r(2)=0.82), and CYP2D6 (r(2)=0.80). In contrast no correlation was observed with CYP2E1 and CYP3A4 (midazolam-1'-hydroxylation). The N-in-one screening assay seems useful and reliable for most CYP activities when a comprehensive and quick evaluation of potential interactions with CYPs is needed. However, at the present moment, it does not enable discrimination on the basis of mechanism of inhibition. A strict comparison between single and N-in-one assays is a prerequisite for more extensive routine use. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Enantioselective inhibition of Cytochrome P450-mediated drug metabolism by a novel antithrombotic agent, S002-333: Major effect on CYP2B6.

    PubMed

    Bhateria, Manisha; Ramakrishna, Rachumallu; Puttrevu, Santosh Kumar; Saxena, Anil K; Bhatta, Rabi Sankar

    2016-08-25

    A significant number of new chemical entities (NCEs) fail in drug discovery due to inhibition of Cytochrome P450 (CYP) enzymes. Therefore, to avert costly drug failure at the clinical phase it becomes indispensable to evaluate the CYP inhibition profile of NCEs early in drug discovery. In light of these concerns, we envisioned to investigate the inhibitory effects of S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide], a novel and potent antithrombotic agent, on nine major CYP enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4) of human liver microsomes (HLM). S002-333 exists as racemic mixture of S004-1032 (R-isomer) and S007-1558 (S-isomer), consequently, we further examined the enantioselective differences of S002-333 in the inhibition of human CYP enzymes. Of the CYP enzymes tested, CYP2B6-catalyzed bupropion 6-hydroxylation was inhibited by S002-333 (IC50 ∼ 9.25 ± 2.46 μM) in a stereoselective manner with (S)-isomer showing potent inhibition (IC50 ∼ 5.28 ± 1.25 μM) in contrast to (R)-isomer which showed negligible inhibition on CYP2B6 activity (IC50 > 50 μM). S002-333 and its (S)-isomer inhibited CYP2B6 activity in a non-competitive fashion with estimated Ki values of 10.1 ± 3.4 μM and 5.09 ± 1.05 μM, respectively. No shift in the IC50 value was observed for S002-333 and its isomers when preincubated for 30 min in the presence of NADPH suggesting that neither S002-333 nor its enantiomers are time-dependent inhibitors. Thus, the present findings signified that S002-333 is a potent stereoselective inhibitor of CYP2B6, whereas, inhibition for other CYPs was substantially negligible. These in vitro findings would be useful in deciding the development of S002-333 as a single-enantiomer or as a racemic mixture.

  8. In vitro drug-drug interactions of budesonide: inhibition and induction of transporters and cytochrome P450 enzymes.

    PubMed

    Chen, Nancy; Cui, Donghui; Wang, Qing; Wen, Zhiming; Finkelman, Richard D; Welty, Devin

    2017-07-21

    1. Budesonide is a glucocorticoid used in the treatment of several respiratory and gastrointestinal inflammatory diseases. Glucocorticoids have been demonstrated to induce cytochrome P450 (CYP) 3A and the efflux transporter P-glycoprotein (P-gp). This study aimed to evaluate the potential of budesonide to act as a perpetrator or a victim of transporter- or CYP-mediated drug-drug interactions (DDIs). 2. In vitro studies were conducted for P-gp, breast cancer resistance protein and organic anion and cation transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT2) in transporter-transfected cells. Changes in mRNA expression in human hepatocytes and enzyme activity in human liver microsomes by budesonide were determined for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A. 3. The data indicated that budesonide is a substrate of P-gp but is not a substrate or an inhibitor of the other transporters investigated. Budesonide is neither an inducer nor an inhibitor of major CYP enzymes. The effect of P-gp on budesonide disposition is anticipated to be low owing to CYP3A-mediated clearance. 4. Collectively, our data indicate there is a low risk of budesonide perpetrating clinical DDIs mediated by the transporters or CYPs studied.

  9. In vitro inhibition of cytochrome P-450 activities and quantification of constituents in a selection of commercial Rhodiola rosea products.

    PubMed

    Thu, Ole Kristian; Nilsen, Odd Georg; Hellum, Bent

    2016-12-01

    Rhodiola rosea L. (Crassulaceae) products are popular natural remedies with a worldwide distribution. Recent studies have revealed potent CYP inhibition by R. rosea extracts both in vitro and in vivo, but information on in vitro CYP inhibition by commercial products are lacking. Variations in commercial R. rosea product quality have also been published. This study evaluates the variation of in vitro CYP inhibition potential and product quality of six commercially available R. rosea products. Human CYPs isolated from baculovirus-infected cell system were incubated with testosterone (CYP3A4), dextromethorphan (CYP2D6) or phenacetin (CYP1A2). Positive CYP inhibitors ketoconazole (CYP3A4), quinidine (CYP2D6) and β-naphtoflavone (CYP1A2) were used as controls. Quantification of rosavin, rosarin, rosin, tyrosol and salidroside were used to evaluate R. rosea content. IC50 values ranged from 7.2-106.6 μg/mL for CYP3A4, 13.0-186.1 μg/mL for 2D6 and 10.7-116.0 μg/mL for 1A2. The tincture formulation of R. rosea was the strongest inhibitor giving the lowest IC50 values of 7.2 ± 0.7, 13 ± 1.7 and 10.7 ± 5.6 μg/mL, respectively. CYP3A4 was significantly more inhibited by the different products than CYP1A2 (p < .05). One of the six products did not contain any rosavin, rosarin or rosin and is not a R. rosea product. Constituent concentrations were not linked to enzyme inhibition. The present results show a large variation in inhibitory potential between the products. Several of the products demonstrate similar inhibition levels as the product Arctic Root already proven to inhibit CYP enzyme activity in man.

  10. Effect of genetic polymorphism on the inhibition of dopamine formation from p-tyramine catalyzed by brain cytochrome P450 2D6.

    PubMed

    Niwa, Toshiro; Shizuku, Marina; Yamano, Kaori

    2017-04-15

    The inhibitory effects of steroid hormones, including glucocorticoids such as cortisol, and related compounds on dopamine formation from p-tyramine, catalyzed by cytochrome P450 (CYP) 2D6.2 (Arg296Cys, Ser486Thr) and CYP2D6.10 (Pro34Ser, Ser486Thr) were compared with the effects of those catalyzed by CYP2D6.1 (wild type), to investigate the effect of a CYP2D6 polymorphism on neuroactive amine metabolism in the brain. Inhibition constants (Ki) or 50% inhibitory concentrations of six steroid hormones (cortisol, cortisone, corticosterone, dehydroepiandrosterone, progesterone, and pregnenolone) and quinidine and quinine-typical potent inhibitors of the human CYP2D6 and rat CYP2D subfamily, respectively-toward dopamine formation catalyzed by CYP2D6.1, CYP2D6.2, and CYP2D6.10 expressed in recombinant Escherichia coli were compared. Although most steroid hormones had no or minor inhibitory effects on the dopamine formation by all CYP2D6 variants, progesterone inhibited the metabolism and Ki value against CYP2D6.10 was approximately twice that for CYP2D6.1 and CYP2D6.2. Quinidine exhibited stronger inhibition than quinine; however, these two compounds inhibited the CYP2D6.10-mediated reaction more weakly than the CYP2D6.1 and CYP2D6.2 reactions. These results suggest that CYP2D6 polymorphism would affect drug interaction through dopamine formation in the brain. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Rough Set Theory as an Interpretable Method for Predicting the Inhibition of Cytochrome P450 1A2 and 2D6.

    PubMed

    Burton, Julien; Petit, Joachim; Danloy, Emeric; Maggiora, Gerald M; Vercauteren, Daniel P

    2013-07-01

    Early prediction of ADME properties such as the cytochrome P450 (CYP) mediated drug-drug interactions is an important challenge in the drug discovery area. In this study, we propose to couple an original data mining approach based on Rough Set Theory (RST) to a structural description of molecules. The latter was achieved by using two types of structural keys: (1) the MACCS keys and (2) a set of five in-house fingerprints based on properties of the electron density distributions of chemical groups. The compounds considered are involved in the inhibition of CYP1A2 and CYP2D6. RST allowed the extraction of rules further used as classifiers to predict the inhibitory profile of an independent set of molecules. The results reached prediction accuracies of 90.6 and 88.2 % for CYP1A2 and CYP2D6, respectively. In addition, these classifiers were analyzed to determine which structural fragments were most used for building the rules, revealing relationships between the occurrence of particular molecular fragments and CYP inhibition. The results assessed RST as a suitable tool to build strongly predictive models and infer structure-activity rules associated with potency.

  12. Peony-Glycyrrhiza Decoction, an Herbal Preparation, Inhibits Clozapine Metabolism via Cytochrome P450s, but Not Flavin-Containing Monooxygenase in In Vitro Models.

    PubMed

    Wang, Wei; Tian, Dan-Dan; Zheng, Bin; Wang, Di; Tan, Qing-Rong; Wang, Chuan-Yue; Zhang, Zhang-Jin

    2015-07-01

    Our previous studies have shown the therapeutic efficacy and underlying mechanisms of Peony-Glycyrrhiza Decoction (PGD), an herbal preparation, in treating antipsychotic-induced hyperprolactinemia in cultured cells, animal models, and human subjects. In the present study, we further evaluated pharmacokinetic interactions of PGD with clozapine (CLZ) in human liver microsomes (HLM), recombinantly expressed cytochrome P450s (P450s), and flavin-containing monooxygenases (FMOs). CLZ metabolites, N-demethyl-clozapine and clozapine-N-oxide, were measured. PGD, individual peony and glycyrrhiza preparations, and the two individual preparations in combination reduced production of CLZ metabolites to different extents in HLM. While the known bioactive constituents of PGD play a relatively minor role in the kinetic effects of PGD on P450 activity, PGD as a whole had a weak-to-moderate inhibitory potency toward P450s, in particular CYP1A2 and CYP3A4. FMOs are less actively involved in mediating CLZ metabolism and the PGD inhibition of CLZ. These results suggest that PGD has the capacity to suppress CLZ metabolism in the human liver microsomal system. This suppression is principally associated with the inhibition of related P450 activity but not FMOs. The present study provides in vitro evidence of herb-antipsychotic interactions.

  13. Arecoline inhibits the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1A1 activation in human hepatoma cells.

    PubMed

    Chang, Eddy Essen; Miao, Zhi-Feng; Lee, Wen-Jhy; Chao, How-Ran; Li, Lih-Ann; Wang, Ya-Fen; Ko, Ying-Chin; Tsai, Feng-Yuan; Yeh, Szu Ching; Tsou, Tsui-Chun

    2007-07-19

    In the present study, we investigated the effect of arecoline, a major areca nut alkaloid, on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced activation of cytochrome P4501A1 (CYP1A1) in a human hepatoma cell line Huh-7. We treated Huh-7 cells with 10nM TCDD in the presence of different concentrations of arecoline (50-300 microM). Our results indicated that arecoline attenuated the TCDD-induced CYP1A1 enzyme activation with an inhibitory effect on cell proliferation. By using real-time RT-PCR, we demonstrated that arecoline inhibited the TCDD-induced activations of CYP1A1 and AhR repressor (AhRR) mRNA expression in a similar pattern. Our results revealed that arecoline inhibited AhR mRNA expression with no direct effect on CYP1A1 enzyme activity. Therefore, in our present study, the observed inhibitory effect of arecoline on CYP1A1 activation was not due to the up-regulation of AhRR or direct inhibitory effect on CYP1A1. Taken together, here we have demonstrated that arecoline attenuates the TCDD-induced CYP1A1 activation mainly via down-regulation of AhR expression in human hepatoma cells, suggesting the possible involvement of arecoline in the AhR-mediated metabolism of environmental toxicants in liver.

  14. 12(R)-hydroxyicosatetraenoic acid: a cytochrome P450-dependent arachidonate metabolite that inhibits Na/sup +/, K/sup +/-ATPase in the cornea

    SciTech Connect

    Schwartzman, M.L.; Balazy, M.; Masferrer, J.; Abraham, N.G.; McGiff, J.C.; Murphy, R.C.

    1987-11-01

    When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, four polar metabolites (compounds A-D) were formed. Synthesis of these metabolites could be inhibited by carbon monoxide, SKF 525A, and anti-cytochrome c reductase antibodies. One of the metabolites, compound C, was found to inhibit partially purified Na/sup +/, K/sup +/-ATPase from the corneal epithelium in a dose-dependent manner. After compound C was purified by TLC and HPLC, it was found to have a UV absorption spectrum with a maximum absorbance at 236 nm suggesting the presence of a conjugated diene. Mass spectrometric analysis using positive- and negative-ionization modes was carried out on derivatized compound C. Abundant fragment ions were consistent with compound C being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon-12 of the icosanoid backbone; all deuterium atoms from (/sup 2/H/sub 8/)arachidonate were retained in the structure. Compound C was characterized as a 12-hydroxyicosatetraenoic acid. However, only 12(R) isomer was found to be an inhibitor of the Na/sup +/, K/sup +/-ATPase from the corneal epithelium, suggesting that the biologically active compound C was 12(R)-hydroxyy-5,8,10,14-icosatetraenoic acid. Such an inhibitor of Na/sup +/, K/sup +/-ATPase synthesized in the cornea may have an important role in regulating ocular transparency and aqueous human secretion.

  15. Acute toxicity of some synthetic cyanogens in rats: time-dependent cyanide generation and cytochrome oxidase inhibition in soft tissues after sub-lethal oral intoxication.

    PubMed

    Rao, Pooja; Singh, Poonam; Yadav, Shiv Kumar; Gujar, Niranjan L; Bhattacharya, Rahul

    2013-09-01

    Cyanogens include complex nitrile-containing compounds that can generate free cyanide of toxicological significance. Acute toxicity, time-dependent cyanide generation and cytochrome oxidase (CYTOX) inhibition in soft tissues, and urinary thiocyanate levels were measured after acute cyanogen intoxication in rats. Order of cyanogens in terms of LD₅₀ was: malononitrile (MCN)>propionitrile (PCN)≈sodium nitroprusside (SNP)>acrylonitrile (ACN)>succinonitrile (SCN)>acetonitrile (ATCN) for oral, and SNP>MCN>ACN>PCN>SCN>ATCN for intraperitoneal and subcutaneous routes. MCN was most toxic by oral (LD₅₀=66.4 mg/kg) and SNP by intraperitoneal (LD₅₀=16.7 mg/kg) and subcutaneous (LD₅₀=11.9 mg/kg) routes. Minimum survival time (25 min) was recorded after 4.0 LD₅₀ ATCN. Order of cyanogens (0.75 LD₅₀; oral) on the basis of maximum blood cyanide and time of peak cyanide generation were: ATCN>SNP>SCN>PCN>MCN>ACN, and MCN (30 min)inhibition and urinary thiocyanate levels. With the understanding of time-dependent toxicity of different cyanogens, suitable therapeutic windows can be designed for their management.

  16. Acute and Chronic Toxicity, Cytochrome P450 Enzyme Inhibition, and hERG Channel Blockade Studies with a Polyherbal, Ayurvedic Formulation for Inflammation

    PubMed Central

    Dey, Debendranath; Chaskar, Sunetra; Athavale, Nitin; Chitre, Deepa

    2015-01-01

    Ayurvedic plants are known for thousands of years to have anti-inflammatory and antiarthritic effect. We have recently shown that BV-9238, a proprietary formulation of Withania somnifera, Boswellia serrata, Zingiber officinale, and Curcuma longa, inhibits LPS-induced TNF-alpha and nitric oxide production from mouse macrophage and reduces inflammation in different animal models. To evaluate the safety parameters of BV-9238, we conducted a cytotoxicity study in RAW 264.7 cells (0.005–1 mg/mL) by MTT/formazan method, an acute single dose (2–10 g/kg bodyweight) toxicity study and a 180-day chronic study with 1 g and 2 g/kg bodyweight in Sprague Dawley rats. Some sedation, ptosis, and ataxia were observed for first 15–20 min in very high acute doses and hence not used for further chronic studies. At the end of 180 days, gross and histopathology, blood cell counts, liver and renal functions were all at normal levels. Further, a modest attempt was made to assess the effects of BV-9238 (0.5 µg/mL) on six major human cytochrome P450 enzymes and 3H radioligand binding assay with human hERG receptors. BV-9238 did not show any significant inhibition of these enzymes at the tested dose. All these suggest that BV-9238 has potential as a safe and well tolerated anti-inflammatory formulation for future use. PMID:25893199

  17. Curcuminoids inhibit multiple human cytochromes P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes, while piperine is a relatively selective CYP3A4 inhibitor

    PubMed Central

    Volak, Laurie P.; Ghirmai, Senait; Cashman, John R.; Court, Michael H.

    2008-01-01

    Curcuminoid extract and piperine are being evaluated for beneficial effects in Alzheimer’s disease, among other intractable disorders. Consequently, we studied the potential for herb-drug interactions involving cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes. The curcuminoid extract inhibited SULT > CYP2C19 > CYP2B6 > UGT > CYP2C9 > CYP3A activities with IC50 values ranging from 0.99 ± 0.04 to 25.3 ± 1.3 μM, while CYP2D6, CYP1A2, and CYP2E1 activities were less affected (IC50 values >60 μM). Inhibition of CYP3A activity by curcuminoid extract was consistent with competitive inhibition (Ki = 11.0 ± 1.3 μM), while inhibition of both CYP2C9 and CYP2C19 activities were consistent with mixed competitive-noncompetitive inhibition (10.6 ± 1.1 μM and 7.8 ± 0.9 μM, respectively). Piperine was a relatively selective noncompetitive inhibitor of CYP3A (IC50 5.5 ± 0.7 μM, Ki = 5.4 ± 0.3 μM) with less effect on other enzymes evaluated (IC50 >29 μM). Curcuminoid extract and piperine inhibited recombinant CYP3A4 much more potently (by >5-fold) than CYP3A5. Pure synthetic curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) were also evaluated for their effects on CYP3A, CYP2C9, UGT, and SULT activities. All three curcuminoids had similar effects on CYP3A, UGT, and SULT activity, but demethoxycurcumin (IC50 = 8.8 ± 1.2 μM) was more active against CYP2C9 than either curcumin or bisdemethoxycurcumin (IC50 >50 μM). Based on these data and expected tissue concentrations of inhibitors, we predict that an orally administered curcuminoid/piperine combination is most likely to inhibit CYP3A, CYP2C9, UGT, and SULT metabolism within the intestinal mucosa. PMID:18480186

  18. Total sleep deprivation inhibits the neuronal nitric oxide synthase and cytochrome oxidase reactivities in the nodose ganglion of adult rats.

    PubMed

    Chang, Hung-Ming; Wu, Un-In; Lin, Tzer-Bin; Lan, Chyn-Tair; Chien, Wei-Ching; Huang, Wei-Ling; Shieh, Jeng-Yung

    2006-08-01

    Sleep disorders are a form of stress associated with increased sympathetic activity, and they are a risk factor for the occurrence of cardiovascular disease. Given that nitric oxide (NO) may play an inhibitory role in the regulation of sympathetic tone, this study set out to determine the NO synthase (NOS) reactivity in the primary cardiovascular afferent neurons (i.e. nodose neurons) following total sleep deprivation (TSD). TSD was performed by the disc-on-water method. Following 5 days of TSD, all experimental animals were investigated for quantitative nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d, a co-factor of NOS) histochemistry, neuronal NOS immunohistochemistry and neuronal NOS activity assay. In order to evaluate the endogenous metabolic activity of nodose neurons, cytochrome oxidase (COX) reactivity was further tested. All the above-mentioned reactivities were objectively assessed by computerized image analysis. The clinical significance of the reported changes was demonstrated by alterations of mean arterial blood pressure (MAP). The results indicated that in normal untreated rats, numerous NADPH-d/NOS- and COX-reactive neurons were found in the nodose ganglion (NG). Following TSD, however, both the labelling and staining intensity of NADPH-d/NOS as well as COX reactivity were drastically reduced in the NG compared with normal untreated ganglions. MAP was significantly higher in TSD rats (136+/-4 mmHg) than in normal untreated rats (123+/-2 mmHg). NO may serve as an important sympathoinhibition messenger released by the NG neurons, and decrease of NOS immunoexpression following TSD may account for the decrease in NOS content. In association with the reduction of NOS activity, a defect in NOS expression in the primary cardiovascular afferent neurons would enhance clinical hypertension, which might serve as a potential risk factor in the development of TSD-relevant cardiovascular disturbances.

  19. Inhibition of Carcinogen-Activating Cytochrome P450 Enzymes by Xenobiotic Chemicals in Relation to Antimutagenicity and Anticarcinogenicity

    PubMed Central

    Shimada, Tsutomu

    2017-01-01

    A variety of xenobiotic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), aryl- and heterocyclic amines and tobacco related nitrosamines, are ubiquitous environmental carcinogens and are required to be activated to chemically reactive metabolites by xenobiotic-metabolizing enzymes, including cytochrome P450 (P450 or CYP), in order to initiate cell transformation. Of various human P450 enzymes determined to date, CYP1A1, 1A2, 1B1, 2A13, 2A6, 2E1, and 3A4 are reported to play critical roles in the bioactivation of these carcinogenic chemicals. In vivo studies have shown that disruption of Cyp1b1 and Cyp2a5 genes in mice resulted in suppression of tumor formation caused by 7,12-dimethylbenz[a]anthracene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, respectively. In addition, specific inhibitors for CYP1 and 2A enzymes are able to suppress tumor formation caused by several carcinogens in experimental animals in vivo, when these inhibitors are applied before or just after the administration of carcinogens. In this review, we describe recent progress, including our own studies done during past decade, on the nature of inhibitors of human CYP1 and CYP2A enzymes that have been shown to activate carcinogenic PAHs and tobacco-related nitrosamines, respectively, in humans. The inhibitors considered here include a variety of carcinogenic and/or non-carcinogenic PAHs and acethylenic PAHs, many flavonoid derivatives, derivatives of naphthalene, phenanthrene, biphenyl, and pyrene and chemopreventive organoselenium compounds, such as benzyl selenocyanate and benzyl selenocyanate; o-XSC, 1,2-, 1,3-, and 1,4-phenylenebis( methylene)selenocyanate. PMID:28443179

  20. Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition.

    PubMed

    Albertolle, Matthew E; Kim, Donghak; Nagy, Leslie D; Yun, Chul-Ho; Pozzi, Ambra; Savas, Üzen; Johnson, Eric F; Guengerich, F Peter

    2017-07-07

    Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    SciTech Connect

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Xie, Yuchao; Farhood, Anwar; Vinken, Mathieu; Jaeschke, Hartmut

    2013-12-15

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB

  2. In vitro metabolic interactions between black cohosh (Cimicifuga racemosa) and tamoxifen via inhibition of cytochromes P450 2D6 and 3A4.

    PubMed

    Li, Jinghu; Gödecke, Tanja; Chen, Shao-Nong; Imai, Ayano; Lankin, David C; Farnsworth, Norman R; Pauli, Guido F; van Breemen, Richard B; Nikolić, Dejan

    2011-08-09

    Women who experience hot flashes as a side effect of tamoxifen (TAM) therapy often try botanical remedies such as black cohosh to alleviate these symptoms. Since pharmacological activity of TAM is dependent on the metabolic conversion into active metabolites by the action of cytochromes P450 2D6 (CYP2D6) and 3A4, the objective of this study was to evaluate whether black cohosh extracts can inhibit formation of active TAM metabolites and possibly reduce its clinical efficacy. At 50 μg/mL, a 75% ethanolic extract of black cohosh inhibited formation of 4-hydroxy- TAM by 66.3%, N-desmethyl TAM by 74.6% and α-hydroxy TAM by 80.3%. In addition, using midazolam and dextromethorphan as probe substrates, this extract inhibited CYP3A4 and CYP2D6 with IC(50) values of 16.5 and 50.1 μg/mL, respectively. Eight triterpene glycosides were identified as competitive CYP3A4 inhibitors with IC(50) values ranging from 2.3-5.1 µM, while the alkaloids protopine and allocryptopine were identified as competitive CYP2D6 inhibitors with K(i) values of 78 and 122 nM, respectively. The results of this study suggests that co-administration of black cohosh with TAM might interfere with the clinical efficacy of this drug. However, additional clinical studies are needed to determine the clinical significance of these in vitro results.

  3. Development and validation of an in vitro, seven-in-one human cytochrome P450 assay for evaluation of both direct and time-dependent inhibition.

    PubMed

    Dahlinger, Dominik; Duechting, Sabrina; Nuecken, Daniela; Sydow, Konrad; Fuhr, Uwe; Frechen, Sebastian

    2016-01-01

    Direct and time-dependent inhibition (TDI) of cytochrome P450 enzymes (CYP) raises drug safety concerns and has major implications in drug development. This study describes the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based screening tool to simultaneously assess both the direct and the time-dependent inhibitory potential of xenobiotics on the seven major CYPs using a two-step approach. The in vitro cocktail of FDA recognized model substrates was incubated with human liver microsomes (HLM) and consisted of caffeine (CYP1A2), bupropion (CYP2B6), rosiglitazone (CYP2C8), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6) and midazolam (CYP3A4). Direct and time-dependent inhibitory profiles of direct and time-dependent reference inhibitors for each CYP were studied. For validation, the results were compared to those obtained with the traditional single substrate approach. Statistical uncertainty was quantified using the bootstrap method. The direct inhibition assay showed an acceptable fold bias of 1.35 (geometric mean fold absolute deviation, range 1.01-2.61) in the IC50 values for the cocktail assay compared to the single substrate results with no trend for under- or overestimation. Using a single point inactivation assay to assess TDI, we were able to identify all seven tested time-dependent reference inhibitors, without any false negatives. The presented design enhances throughput by assessing the seven major CYPs simultaneously and allows for detection of and discrimination between direct and time-dependent CYP inhibition via IC50 and single point inactivation experiments. For the latter, a threshold of 10% TDI is proposed for carrying out more detailed inactivation kinetic experiments. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

    PubMed

    Turrens, J F; Bickar, D; Lehninger, A L

    1986-06-01

    The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

  5. Use of In Vitro and Predictive In Silico Models to Study the Inhibition of Cytochrome P4503A by Stilbenes

    PubMed Central

    Basheer, Loai; Schultz, Keren; Fichman, Merav; Kerem, Zohar

    2015-01-01

    CYP3A4 is recognized as the main enzyme involved in the metabolism of drugs and xenobiotics in the human body and its inhibition may lead to undesirable consequences. Stilbenes, including resveratrol, belong to a group of dietary health-promoting compounds that also act as inhibitors of CYP3A4. The aim of this study was to examine the use of computer modeling of enzyme-ligand interactions to analyze and predict the inhibition of structurally related compounds. To this end, an aldehyde group was attached to resveratrol and the interactions of CYP3A4 with resveratrol, its aldehyde analogue (RA) and a known synthetic inhibitor were studied and compared in two biological models. Specifically, the metabolism of testosterone was examined in a human intestine cell line (Caco-2/TC7) and in rat liver microsomes (RLM). The results demonstrated a weak inhibitory effect of RA on CYP3A4, as compared to resveratrol itself, in both biological models. Human CYP3A4 was more susceptible to inhibition than the commonly used model isozyme from rat. Modeling of the binding site of CYP3A4 revealed a combination of three types of interactions: hydrophobic interactions, electrostatic interactions and hydrogen bonds. A docking simulation revealed that the RA lacked an important binding feature, as compared to resveratrol, and that that difference may be responsible for its lower level of affinity for CYP3A4. Software analysis of binding affinity may serve as a predictive tool for designing new therapeutic compounds in terms of inhibition of CYP3A4 and help to reveal the biochemical nature of the interactions of dietary compounds, herbal compounds and drugs whose metabolism is mediated by this enzyme. PMID:26485399

  6. In vitro evaluation of the inhibition and induction potential of olaparib, a potent poly(ADP-ribose) polymerase inhibitor, on cytochrome P450.

    PubMed

    McCormick, Alex; Swaisland, Helen; Reddy, Venkatesh Pilla; Learoyd, Maria; Scarfe, Graeme

    2017-07-25

    1. In vitro studies were conducted to evaluate potential inhibitory and inductive effects of the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, on cytochrome P450 (CYP) enzymes. Inhibitory effects were determined in human liver microsomes (HLM); inductive effects were evaluated in cultured human hepatocytes. 2. Olaparib did not inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6 or CYP2E1 and caused slight inhibition of CYP2C9, CYP2C19 and CYP3A4/5 in HLM up to a concentration of 100 μM. However, olaparib (17-500 μM) inhibited CYP3A4/5 with an IC50 of 119 μM. In time-dependent CYP inhibition assays, olaparib (10 μM) had no effect against CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 and a minor effect against CYP3A4/5. In a further study, olaparib (2-200 μM) functioned as a time-dependent inhibitor of CYP3A4/5 (KI, 72.2 μM and Kinact, 0.0675 min(-1)). Assessment of the CYP induction potential of olaparib (0.061-44 μM) showed minor concentration-related increases in CYP1A2 and more marked increases in CYP2B6 and CYP3A4 mRNA, compared with positive control activity; however, no significant change in CYP3A4/5 enzyme activity was observed. 3. Clinically significant drug-drug interactions due to olaparib inhibition or induction of hepatic or intestinal CYP3A4/5 cannot be excluded. It is recommended that olaparib is given with caution with narrow therapeutic range or sensitive CYP3A substrates, and that prescribers are aware that olaparib may reduce exposure to substrates of CYP2B6.

  7. Development of a highly reproducible system to evaluate inhibition of cytochrome P450 3A4 activity by natural medicines.

    PubMed

    Sato, Yu; Sasaki, Takamitsu; Takahashi, Shogo; Kumagai, Takeshi; Nagata, Kiyoshi

    2015-01-01

    In recent years, a number of natural medicines have been reported to have inductive or inhibitive effects on the activity of drug metabolizing enzymes, upon co-administration with prescribed medicines. However, information regarding natural medicine-drug interactions that influence drug metabolism is limited owing to the lack of efficient screening method for such interactions. Therefore, to understand whether P450 activity is affected by natural medicine in small intestines, we have established frozen recombinant P450-expressing cells infected with human CYP3A4 expressing adenovirus (Ad-CYP3A4) to evaluate the effect of natural medicines on CYP3A4 activity. Ad-CYP3A4 cells were created by infecting HepG2 cells with Ad-CYP3A4 at 10 multiplicity of infection (MOI) and these cells were stored using cryopreservation medium (fAd-CYP3A4 cells) to obtain long-term consistent data and stable supplies of cells expressing a constant level of CYP3A4 activity. The CYP3A4 activity in fAd-CYP3A4 cells remained unaffected at the end of each frozen period (0, 1, 2, and 6 months). Inhibitory effect on CYP3A4 activity by typical inhibitors (ketoconazole, hyperforin) and natural medicines (Cat's Claw, Devil's Claw, Feverfew, Peppermint Oil, Red Clover, and Siberian Eleuthero) were evaluated. The inhibitors had nearly equal IC50 values in fAd-CYP3A4 cells, Ad-CYP3A4 cells and recombinant CYP3A4 microsomes. Cat's Claw, Peppermint Oil and Siberian Eleuthero inhibited CYP3A4 activity more potently than 0.1 μM ketoconazole in fAd-CYP3A4 cells. In the present study, we have successfully developed a highly reproducible system to evaluate CYP3A4 inhibition in small intestines by natural medicines.

  8. Evaluation of the inhibition potential of plumbagin against cytochrome P450 using LC-MS/MS and cocktail approach

    PubMed Central

    Chen, Ang; Zhou, Xiaojing; Tang, Shuowen; Liu, Mingyao; Wang, Xin

    2016-01-01

    Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a natural naphthoquinone compound isolated from roots of Plumbago zeylanica L., has drawn a lot of attention for its plenty of pharmacological properties including antidiabetes and anti-cancer. The aim of this study was to investigate the effects of plumbagin on CYP1A2, CYP2B1/6, CYP2C9/11, CYP2D1/6, CYP2E1 and CYP3A2/4 activities in human and rat liver and evaluate the potential herb-drug interactions using the cocktail approach. All CYP substrates and their metabolites were analyzed using high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS). Plumbagin presented non-time-dependent inhibition of CYP activities in both human and rat liver. In humans, plumbagin was not only a mixed inhibitor of CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, but also a non-competitive inhibitor of CYP1A2, with Ki values no more than 2.16 μM. In rats, the mixed inhibition of CYP1A2 and CYP2D1, and competitive inhibition for CYP2B1, CYP2C11 and CYP2E1 with Ki values less than 9.93 μM were observed. In general, the relatively low Ki values of plumbagin in humans would have a high potential to cause the toxicity and drug interactions involving CYP enzymes. PMID:27329697

  9. Inhibition of cytochrome P450 3A by acetoxylated analogues of resveratrol in in vitro and in silico models

    NASA Astrophysics Data System (ADS)

    Basheer, Loai; Schultz, Keren; Kerem, Zohar

    2016-08-01

    Many dietary compounds, including resveratrol, are potent inhibitors of CYP3A4. Here we examined the potential to predict inhibition capacity of dietary polyphenolics using an in silico and in vitro approaches and synthetic model compounds. Mono, di, and tri-acetoxy resveratrol were synthesized, a cell line of human intestine origin and microsomes from rat liver served to determine their in vitro inhibition of CYP3A4, and compared to that of resveratrol. Docking simulation served to predict the affinity of the synthetic model compounds to the enzyme. Modelling of the enzyme’s binding site revealed three types of interaction: hydrophobic, electrostatic and H-bonding. The simulation revealed that each of the examined acetylations of resveratrol led to the loss of important interactions of all types. Tri-acetoxy resveratrol was the weakest inhibitor in vitro despite being the more lipophilic and having the highest affinity for the binding site. The simulation demonstrated exclusion of all interactions between tri-acetoxy resveratrol and the heme due to distal binding, highlighting the complexity of the CYP3A4 binding site, which may allow simultaneous accommodation of two molecules. Finally, the use of computational modelling may serve as a quick predictive tool to identify potential harmful interactions between dietary compounds and prescribed drugs.

  10. Inhibition of cytochrome P450 3A by acetoxylated analogues of resveratrol in in vitro and in silico models

    PubMed Central

    Basheer, Loai; Schultz, Keren; Kerem, Zohar

    2016-01-01

    Many dietary compounds, including resveratrol, are potent inhibitors of CYP3A4. Here we examined the potential to predict inhibition capacity of dietary polyphenolics using an in silico and in vitro approaches and synthetic model compounds. Mono, di, and tri-acetoxy resveratrol were synthesized, a cell line of human intestine origin and microsomes from rat liver served to determine their in vitro inhibition of CYP3A4, and compared to that of resveratrol. Docking simulation served to predict the affinity of the synthetic model compounds to the enzyme. Modelling of the enzyme’s binding site revealed three types of interaction: hydrophobic, electrostatic and H-bonding. The simulation revealed that each of the examined acetylations of resveratrol led to the loss of important interactions of all types. Tri-acetoxy resveratrol was the weakest inhibitor in vitro despite being the more lipophilic and having the highest affinity for the binding site. The simulation demonstrated exclusion of all interactions between tri-acetoxy resveratrol and the heme due to distal binding, highlighting the complexity of the CYP3A4 binding site, which may allow simultaneous accommodation of two molecules. Finally, the use of computational modelling may serve as a quick predictive tool to identify potential harmful interactions between dietary compounds and prescribed drugs. PMID:27530542

  11. Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus).

    PubMed

    Li, Hongxia; Boon, Jan P; Lewis, Wilma E; van den Berg, Martin; Nyman, Madeleine; Letcher, Robert J

    2003-03-01

    Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccumulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho-Cl substituents (CB-15 [4,4'-Cl2] and CB-77 [3,3',4,4'-Cl4]), vicinal meta and para-H atoms (CB-52 [2,2',5,5'-Cl4], and -101 [2,2',4,5,5'-Cl5]) or with both characteristics in combination with either only one ortho-Cl (CB-26 [2,3',5-Cl3], CB-31 [2,4',5-Cl3]) or two ortho-Cl substituents (CB-44 [2,2',3,5'-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations < or = 1.0 microM because these inhibitors became decreasingly selective at higher concentrations. At < 1.0 microM, ellipticine (CYPIAI/2 inhibitor) selectively inhibited CB-15, -26, -31, and -77 metabolism, with no significant inhibition of CB-44, -52, and -101 metabolism. Inhibition of CB-52 and -101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 microM and maximized at about 100% at 10 microM. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, -31, and -44 metabolism relative to CB-15, -77, and -52 at concentrations < or = 1.0 microM. Major testosterone metabolites formed in vitro were 2beta-(CYP3A), 6beta- (CYP3A, CYPIA), and 16beta- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory

  12. In vitro chemopreventive potential of fucophlorethols from the brown alga Fucus vesiculosus L. by anti-oxidant activity and inhibition of selected cytochrome P450 enzymes.

    PubMed

    Parys, Sabine; Kehraus, Stefan; Krick, Anja; Glombitza, Karl-Werner; Carmeli, Shmuel; Klimo, Karin; Gerhäuser, Clarissa; König, Gabriele M

    2010-02-01

    Within a project focusing on the chemopreventive potential of algal phenols, two phloroglucinol derivatives, belonging to the class of fucophlorethols, and the known fucotriphlorethol A were obtained from the ethanolic extract of the brown alga Fucus vesiculosus L. The compounds trifucodiphlorethol A and trifucotriphlorethol A are composed of six and seven units of phloroglucinol, respectively. The compounds were examined for their cancer chemopreventive potential, in comparison with the monomer phloroglucinol. Trifucodiphlorethol A, trifucotriphlorethol A as well as fucotriphlorethol A were identified as strong radical scavengers, with IC(50) values for scavenging of 1,1-diphenyl-2 picrylhydrazyl radicals (DPPH) in the range of 10.0-14.4 microg/ml. All three compounds potently scavenged peroxyl radicals in the oxygen radical absorbance capacity (ORAC) assay. In addition, the compounds were shown to inhibit cytochrome P450 1A activity with IC(50) values in the range of 17.9-33 microg/ml, and aromatase (Cyp19) activity with IC(50) values in the range of 1.2-5.6 microg/ml.

  13. Metabolic drug-drug interaction potential of macrolactin A and 7-O-succinyl macrolactin A assessed by evaluating cytochrome P450 inhibition and induction and UDP-glucuronosyltransferase inhibition in vitro.

    PubMed

    Bae, Soo Hyeon; Kwon, Min Jo; Park, Jung Bae; Kim, Doyun; Kim, Dong-Hee; Kang, Jae-Seon; Kim, Chun-Gyu; Oh, Euichaul; Bae, Soo Kyung

    2014-09-01

    Macrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), polyene macrolides containing a 24-membered lactone ring, show antibiotic effects superior to those of teicoplanin against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. MA and SMA are currently being evaluated as antitumor agents in preclinical studies in Korea. We evaluated the potential of MA and SMA for the inhibition or induction of human liver cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs) in vitro to assess their safety as new molecular entities. We demonstrated that MA and SMA are potent competitive inhibitors of CYP2C9, with Ki values of 4.06 μM and 10.6 μM, respectively. MA and SMA also weakly inhibited UGT1A1 activity, with Ki values of 40.1 μM and 65.3 μM, respectively. However, these macrolactins showed no time-dependent inactivation of the nine CYPs studied. In addition, MA and SMA did not induce CYP1A2, CYP2B6, or CYP3A4/5. On the basis of an in vitro-in vivo extrapolation, our data strongly suggested that MA and SMA are unlikely to cause clinically significant drug-drug interactions mediated via inhibition or induction of most of the CYPs involved in drug metabolism in vivo, except for the inhibition of CYP2C9 by MA. Similarly, MA and SMA are unlikely to inhibit the activity of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 enzymes in vivo. Although further investigations will be required to clarify the in vivo interactions of MA with CYP2C9-targeted drugs, our findings offer a clearer understanding and prediction of drug-drug interactions for the safe use of MA and SMA in clinical practice.

  14. Evaluation of the in vitro/in vivo drug interaction potential of BST204, a purified dry extract of ginseng, and its four bioactive ginsenosides through cytochrome P450 inhibition/induction and UDP-glucuronosyltransferase inhibition.

    PubMed

    Zheng, Yu Fen; Bae, Soo Hyeon; Choi, Eu Jin; Park, Jung Bae; Kim, Sun Ok; Jang, Min Jung; Park, Gyu Hwan; Shin, Wan Gyoon; Oh, Euichaul; Bae, Soo Kyung

    2014-06-01

    We evaluated the potential of BST204, a purified dry extract of ginseng, to inhibit or induce human liver cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) in vitro to assess its safety. In vitro drug interactions of four bioactive ginsenosides of BST204, S-Rg3, R-Rg3, S-Rh2, and R-Rh2, were also evaluated. We demonstrated that BST204 slightly inhibited CYP2C8, CYP2D6, CYP2C9, and CYP2B6 activities with IC50 values of 17.4, 26.8, 31.5, and 49.7μg/mL, respectively. BST204 also weakly inhibited UGT1A1, UGT1A9, and UGT2B7 activities with IC50 values of 14.5, 26.6, and 31.5μg/mL, respectively. The potential inhibition by BST204 of the three UGT activities might be attributable to S-Rg3, at least in part, as its inhibitory pattern was similar to that of BST204. However, BST204 showed no time-dependent inactivation of the nine CYPs studied. In addition, BST204 did not induce CYP1A2, 2B6, or 3A4/5. On the basis of an in vivo interaction studies, our data strongly suggest that BST204 is unlikely to cause clinically significant drug-drug interactions mediated via inhibition or induction of most CYPs or UGTs involved in drug metabolism in vivo. Our findings offer a clearer understanding and possibility to predict drug-drug interactions for the safe use of BST204 in clinical practice. Copyright © 2014. Published by Elsevier Ltd.

  15. Metabolic Drug-Drug Interaction Potential of Macrolactin A and 7-O-Succinyl Macrolactin A Assessed by Evaluating Cytochrome P450 Inhibition and Induction and UDP-Glucuronosyltransferase Inhibition In Vitro

    PubMed Central

    Bae, Soo Hyeon; Kwon, Min Jo; Park, Jung Bae; Kim, Doyun; Kim, Dong-Hee; Kang, Jae-Seon; Kim, Chun-Gyu; Oh, Euichaul

    2014-01-01

    Macrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), polyene macrolides containing a 24-membered lactone ring, show antibiotic effects superior to those of teicoplanin against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. MA and SMA are currently being evaluated as antitumor agents in preclinical studies in Korea. We evaluated the potential of MA and SMA for the inhibition or induction of human liver cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs) in vitro to assess their safety as new molecular entities. We demonstrated that MA and SMA are potent competitive inhibitors of CYP2C9, with Ki values of 4.06 μM and 10.6 μM, respectively. MA and SMA also weakly inhibited UGT1A1 activity, with Ki values of 40.1 μM and 65.3 μM, respectively. However, these macrolactins showed no time-dependent inactivation of the nine CYPs studied. In addition, MA and SMA did not induce CYP1A2, CYP2B6, or CYP3A4/5. On the basis of an in vitro-in vivo extrapolation, our data strongly suggested that MA and SMA are unlikely to cause clinically significant drug-drug interactions mediated via inhibition or induction of most of the CYPs involved in drug metabolism in vivo, except for the inhibition of CYP2C9 by MA. Similarly, MA and SMA are unlikely to inhibit the activity of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 enzymes in vivo. Although further investigations will be required to clarify the in vivo interactions of MA with CYP2C9-targeted drugs, our findings offer a clearer understanding and prediction of drug-drug interactions for the safe use of MA and SMA in clinical practice. PMID:24890600

  16. Benzo[a]pyrene-induced cytochrome P450 1A and DNA binding in cultured trout hepatocytes – inhibition by plant polyphenols

    PubMed Central

    Tsuji, Petra A.; Walle, Thomas

    2007-01-01

    Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP) mainly induce lung cancer in humans, but induce liver cancer in fishes. The chemoprevention of cancers through inhibition of molecular events via phytochemicals is a potentially beneficial area of research, and has been carried out in human cell cultures in the past. Carcinogenesis initiation events are thought to occur in similar ways in fish and humans. Our study investigated the feasibility of using cultured rainbow trout CRL-2301 liver cells as a model for BaP-induced carcinogenesis and its prevention by dietary phytochemicals. Treatment with 1 μM BaP resulted in extensive time-dependent covalent binding to cellular DNA and marked cytochrome P450 (CYP) 1A induction, for both about a 20-fold increase, which is similar to what has been observed in cultured human cells. A surprisingly high expression of epoxide hydrolase (EH) activity in these cells likely contributed substantially to the bioactivation of BaP. Two methoxylated flavones and the stilbene resveratrol were effective inhibitors of both the BaP-DNA binding and CYP 1A induction, in particular 5,7-dimethoxyflavone (5,7-DMF), supporting a role for these dietary compounds as cancer chemopreventive agents. Unlike in human liver or bronchial cells, the main mechanism of inhibition of BaP-induced CYP 1A activity in trout liver cells appears to be direct competition at the protein level. Different cellular responses in any particular model used can be expected and the effect of cell context on the biological responses to xenobiotics, including carcinogens as well as polyphenols, must be considered. The trout CRL-2301 cells' sensitivity to BaP treatment is a clear advantage when contemplating a model system for studies of PAH-induced carcinogenesis and cancer chemoprevention. However, extrapolation to human organs should be done cautiously. PMID:17583686

  17. Concurrent Cooperativity and Substrate Inhibition in the Epoxidation of Carbamazepine by Cytochrome P450 3A4 Active Site Mutants Inspired by Molecular Dynamics Simulations

    PubMed Central

    2015-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V. PMID:25545162

  18. Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations.

    PubMed

    Müller, Christian S; Knehans, Tim; Davydov, Dmitri R; Bounds, Patricia L; von Mandach, Ursula; Halpert, James R; Caflisch, Amedeo; Koppenol, Willem H

    2015-01-27

    Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V.

  19. Steroid-mediated inhibition of cAMP induced de novo synthesis of cytochrome P-450/sub 17 / in Leydig cell cultures

    SciTech Connect

    Hales, D.B.; Sha, L.; Payne, A.H.

    1987-05-01

    The present study was designed to investigate the mechanism by which testosterone (T), produced during cAMP induction of P-450/sub 17 /, modulates the rate of its de novo synthesis. Purified Leydig cells (LC) were maintained in culture for 7 days prior to the initiation of treatment. De novo synthesis was determined by TVS-methionine incorporation, immunoprecipitation with specific antibody, separation by SDS-gel electrophoresis and quantitation by laser densitometry. Treatment of LC with 0.05 mM 8-Br-cAMP (cA) results in a time-dependent increase in the rate of de novo synthesis of P-450/sub 17 / which is increased 2 fold when T production is inhibited by aminoglutethimide (AG). The addition of increasing concentrations of the androgen receptor antagonist, hydroxyflutamide (1-10 M), to cA treated LC enhances the rate of synthesis similar to that seen in cA-treated LC in which T production was inhibited by AG. The addition of increasing concentrations of T (0.05-5 M) or the androgen agonist, mibolerone (1-5 M), to cA + AG treated LC causes a dose-dependent reversal of the AG-enhanced increase in the rate of cA-induced de novo synthesis of P-450/sub 17 /. Addition of estradiol (1 M) or dexamethasone (1 M) was without effect. These data indicate that T produced during cA induction of P-450/sub 17 / negatively regulates the rate of synthesis of this cytochrome P-450 enzyme by an androgen receptor mediated mechanism.

  20. Inhibition of Cytochrome P450 1A2-Mediated Metabolism and Production of Reactive Oxygen Species by Heme Oxygenase-1 in Rat Liver Microsomes

    PubMed Central

    Reed, James R.; Cawley, George F.; Backes, Wayne L.

    2011-01-01

    Heme oxygenase-1 (HO-1) is induced in most cell types by many forms of environmental stress and is believed to play a protective role in cells exposed to oxidative stress. Metabolism by cytochromes P450 (P450) is highly inefficient as the oxidation of substrate is associated with the production of varying proportions of hydrogen peroxide and/or superoxide. This study tests the hypothesis that heme oxygenase-1 (HO-1) plays a protective role against oxidative stress by competing with P450 for binding to the common redox partner, the NADPH P450 reductase (CPR) and in the process, diminishing P450 metabolism and the associated production of reactive oxygen species (ROS). Liver microsomes were isolated from uninduced rats and rats that were treated with cadmium and/or β-napthoflavone (BNF) to induce HO-1 and/or CYP1A2. HO-1 induction was associated with slower rates of metabolism of the CYP1A2-specific substrate, 7-ethoxyresorufin. Furthermore, HO-1 induction also was associated with slower rates of hydrogen peroxide and hydroxyl radical production by microsomes from rats induced for CYP1A2. The inhibition associated with HO-1 induction was not dependent on the addition of heme to the microsomal incubations. The effects of HO-1 induction were less dramatic in the absence of substrate for CYP1A2, suggesting that the enzyme was more effective in inhibiting the CYP1A2-related activity than the CPR-related production of superoxide (that dismutates to form hydrogen peroxide). PMID:20942796

  1. Dynamic modeling of cytochrome P450 inhibition in vitro: impact of inhibitor depletion on IC₅₀ shift.

    PubMed

    Berry, Loren M; Zhao, Zhiyang; Lin, Min-Hwa Jasmine

    2013-07-01

    The impact of inhibitor depletion on the determination of shifted IC₅₀ (IC₅₀ determined after 30 minutes of preincubation with inhibitor) is examined. In addition, IC₅₀-shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as reversible and time-dependent inhibition. Anomalies such as a smaller-than-expected shift in IC₅₀ and even increases in IC₅₀ with preincubation were explained by the depletion of inhibitor during the preincubation. The IC₅₀-shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methods, it is recommended that IC₅₀-shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC₅₀-shift methods, shifted IC₅₀ should be calculated using observed inhibitor concentrations at the end of the incubation rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes, such as competitive inhibition, enzyme inactivation, and inhibitor depletion, can be used to describe accurately the observed IC₅₀ and shifted IC₅₀ curves. For compounds showing an IC₅₀ fold shift >1.5 based on the observed inhibitor concentrations, reanalyzing the IC₅₀-shift data using the mechanistic model appeared to allow for reasonable estimation of Ki, KI, and kinact directly from the IC₅₀ shift experiments.

  2. Metformin inhibits 7,12-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway

    SciTech Connect

    Maayah, Zaid H.; Ghebeh, Hazem; Alhaider, Abdulqader A.; El-Kadi, Ayman O.S.; Soshilov, Anatoly A.; Denison, Michael S.; Ansari, Mushtaq Ahmad; Korashy, Hesham M.

    2015-04-15

    Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted using 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in NAD(P)H:quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism

  3. Inhibition of cytochrome P450 by nefazodone in vitro: studies of dextromethorphan O- and N-demethylation

    PubMed Central

    SCHMIDER, JÜRGEN; GREENBLATT, DAVID J.; VON MOLTKE, LISA L.; HARMATZ, JEROLD S.; SHADER, RICHARD I.

    1996-01-01

    Nefazodone (NEF), a 5-HT2A/2C antagonist antidepressant, is extensively metabolized in the human body to hydroxy NEF (OH-NEF), p-hydroxy NEF (pOH-NEF), a dione metabolite, and via cleavage of the molecule to m-chlorophenyl-piperazine (mCPP) and BMY-33604. The latter is further metabolized to BMS-183695-01 (BMSa) and BMS-183562-01 (BMSb). To investigate the potential of NEF and its metabolites to interfere with the metabolism of other drugs, we tested these compounds for their ability to alter dextromethorphan (DMO) O-demethylation to dextrorphan (DOP; an index reaction for CYP2D6) and N-demethylation to 3-methoxy morphinan (MEM, a recently proposed index reaction of CYP3A3/4). The assay was performed in an in vitro system with human liver microsomes from three different donors. NEF, OH-NEF, pOH-NEF, mCPP and BMSb were weak inhibitors of DMO O and N-demethylation, with average Ki values ranging from 18 to 50 μm for DOP formation, and from 21 to >200 μm for MEM formation. The dione metabolite and BMSa did not produce detectable inhibition of either pathway. The findings for DMO O-demethylation, well-established as a CYP2D6-mediated reaction, indicate that NEF and metabolites are weak inhibitors of this reaction, with Ki values at least 100 times higher than fluoxetine (Ki=0.1 μm±0.09). The implications of results on DMO N-demethylation are not clear. In vivo data, as well as in vitro data based on ‘pure’ CYP3A3/4 substrates, provide evidence for clinically relevant CYP3A3/4 inhibition by NEF, OH-NEF, and pOH-NEF. Thus, formation of MEM by N-demethylation of DMO may not constitute a suitable index reaction to probe CYP3A3/4 activity. PMID:8730981

  4. IL-1β hampers glucose-stimulated insulin secretion in Cohen diabetic rat islets through mitochondrial cytochrome c oxidase inhibition by nitric oxide.

    PubMed

    Weksler-Zangen, Sarah; Aharon-Hananel, Genya; Mantzur, Carmit; Aouizerat, Tzemach; Gurgul-Convey, Ewa; Raz, Itamar; Saada, Ann

    2014-03-01

    A high-sucrose, low-copper-diet (HSD) induces inhibition of glucose-sensitive rats (CDs) but not Cohen diabetes-resistant rats (CDr). Copper-supplemented HSD increased activity of the copper-dependent mitochondrial respiratory chain enzyme cytochrome c oxidase (COX) and reversed hyperglycemia. This study examined the mechanism by which interleukin-1β modulates GSIS and the role of COX in this process. We measured COX activity, ATP content, GSIS, iNOS expression, and nitrite production with and without IL-1β, N(ω)-nitro-l-arginine, copper, or potassium cyanide in isolated islets of CDs and CDr fed different diets. We found reduced COX activity, ATP content, and GSIS in isolated islets of CDs rats fed a regular diet. These were severely reduced following HSD and were restored to regular diet levels on copper-supplemented HSD (P < 0.01 vs. CDr islets). Potassium cyanide chemically reduced COX activity, decreasing GSIS and thus reinforcing the link between islet COX activity and GSIS. Interleukin-1β (2.5 U/ml) reduced GSIS and COX activity in CDs islets. Exposure to 10 U/ml interleukin-1β decreased GSIS and COX activity in both CDs and CDr islets, inducing a similar nitrite production. Nevertheless, the effect on GSIS was more marked in CDs islets. A significant iNOS expression was detected in CDs on the HSD diet, which was reduced by copper supplementation. N(ω)-nitro-l-arginine and copper prevented the deleterious effect of interleukin-1β on COX activity and GSIS. We conclude that reduced islet COX activity renders vulnerability to GSIS inhibition on low-copper HSD through two interrelated pathways: 1) by further reducing the activity of COX that is essential for β-cell ATP-production and insulin secretion and 2) by inducing the expression of iNOS and nitric oxide-mediated COX inhibition. We suggest that islet COX activity must be maintained above a critical threshold to sustain adequate GSIS with exposure to low-copper HSD.

  5. A high-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry.

    PubMed

    Qin, Chong-Zhen; Ren, Xian; Tan, Zhi-Rong; Chen, Yao; Yin, Ji-Ye; Yu, Jing; Qu, Jian; Zhou, Hong-Hao; Liu, Zhao-Qian

    2014-02-01

    A sensitive and high-throughput inhibition screening liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7-hydroxycoumarin, CYP2A6; 4-hydroxytolbutamide, CYP2C9; 4'-hydroxymephenytoin, CYP2C19; α-hydroxymetoprolol, CYP2D6; and 1-hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C18 column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC50 values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13-103.37%), and inter-day (RSD < 6.20%) and intra-day (RSD < 6.13%) precision. Also, the incubation extracts of the sample were stable at room temperature (20 °C) for 48 h and for 96 h in the autosampler (4 °C). The presented method is the first HPLC-MS/MS method of this combination for simultaneous detection of the five metabolites 7-hydroxycoumarin, 4-hydroxytolbutamide, 4'-hydroxymephenytoin, α-hydroxymetoprolol and 1-hydroxymidazolam in a single-run process. It is possible that the high-quality and -throughput cocktail provides suitable information in drug discovery and screening for new drug entities.

  6. A single dose of methadone inhibits cytochrome P-4503A activity in healthy volunteers as assessed by the urinary cortisol ratio

    PubMed Central

    Boulton, David W; Arnaud, Philippe; DeVane, C Lindsay

    2001-01-01

    Aims To examine the effect of a single oral dose of methadone on cytochrome P450 (CYP) 3A activity using the urinary 6β-hydroxycortisol to cortisol ratio (UCR) as a marker of CYP3A activity. Methods A single oral dose (0.2 mg kg−1) of rac-methadone was administered to eight healthy female volunteers. Frequent blood samples and all urine over seven time periods was collected for 96 h following dosing. The UCR and the concentration of the major CYP3A metabolite of methadone, EDDP, were measured in urine. Methadone enantiomer concentrations were determined in plasma and urine. All quantifications were performed by validated high performance liquid chromatography assays. Results In all volunteers a significant decline of the UCR from immediately predose values was observed at the 4–8 and 8–12 h collection periods (P < 0.05, 95% CI for the differences: 0.4,16 and 0.6,16, respectively) with a return to immediately predose values after 2–3 days, suggesting methadone was an inhibitor of CYP3A. The UCR was found to be significantly correlated with the amount of EDDP excreted in the urine and with the area under the plasma concentration vs time profile for total (R + S) methadone, supporting in vitro data that CYP3A is primarily responsible for EDDP formation and has a significant influence on methadone disposition. Conclusions Methadone appears to be a CYP3A inhibitor in vivo following a single oral dose and measurements of the urinary cortisol ratio appear to be a useful index to follow this inhibition. PMID:11318772

  7. Interleukin-32γ attenuates ethanol-induced liver injury by the inhibition of cytochrome P450 2E1 expression and inflammatory responses.

    PubMed

    Lee, Dong Hun; Kim, Dae Hwan; Hwang, Chul Ju; Song, Sukgil; Han, Sang Bae; Kim, Youngsoo; Yoo, Hwan Soo; Jung, Young Suk; Kim, Soo Hyun; Yoon, Do Young; Hong, Jin Tae

    2015-05-01

    Alcohol abuse and alcoholism lead to alcoholic liver disease (ALD), which is a major type of chronic liver disease worldwide. Interleukin-32 (IL-32) is a novel cytokine involved in inflammation and cancer development. However, the role of IL-32 in chronic liver disease has not been reported. In the present paper, we tested the effect of IL-32γ on ethanol-induced liver injury in IL-32γ-overexpressing transgenic mice (IL-32γ mice) after chronic ethanol feeding. Male C57BL/6 and IL-32γ mice (10-12 weeks old) were fed on a Lieber-DeCarli diet containing 6.6% ethanol for 6 weeks. IL-32γ-transfected HepG2 and Huh7 cells, as well as primary hepatocytes from IL-32γ mice, were treated with or without ethanol. The hepatic steatosis and damage induced by ethanol administration were attenuated in IL-32γ mice. Ethanol-induced cytochrome P450 2E1 expression and hydrogen peroxide levels were decreased in the livers of IL-32γ mice, primary hepatocytes from IL-32γ mice and IL-32γ-overexpressing human hepatic cells. The ethanol-induced expression levels of cyclo-oxygenase-2 (COX-2) and IL-6 were reduced in the livers of IL-32γ mice. Because nuclear transcription factor κB (NF-κB) is a key redox transcription factor of inflammatory responses, we examined NF-κB activity. Ethanol-induced NF-κB activities were significantly lower in the livers of IL-32γ mice than in wild-type (WT) mice. Furthermore, reduced infiltration of natural killer cells, cytotoxic T-cells and macrophages in the liver after ethanol administration was observed in IL-32γ mice. These data suggest that IL-32γ prevents ethanol-induced hepatic injury via the inhibition of oxidative damage and inflammatory responses.

  8. Cytochrome P450 omega-hydroxylase inhibition reduces cardiomyocyte apoptosis via activation of ERK1/2 signaling in rat myocardial ischemia-reperfusion.

    PubMed

    Lv, Xiaojun; Wan, Jing; Yang, Jing; Cheng, Hao; Li, Ying; Ao, Ying; Peng, Rengxiu

    2008-10-31

    Cytochrome P450 (CYP) omega-hydroxylases and their arachidonic acid metabolites play important roles in myocardial ischemia-reperfusion injury. In this study we investigated the effects of several selective CYP omega-hydroxylase inhibitors on myocardial ischemia/reperfusion-induced myocardial apoptosis. Rats were subjected 30 min of ischemia and 2 h of reperfusion. Groups received either 17-octadecynoic acid (17-ODYA, 0.3 or 3 mg/kg), N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS, 0.4 or 0.8 mg/kg), N-hydroxy-N'-(4-butyl-2-methylphenyl) formamidine (HET0016, 0.1 or 1 mg/kg) or vehicle 10 min prior to ischemia. To further assess the role of mitogen-activated protein kinases (MAPKs) in the CYP omega-hydroxylase inhibitor-induced anti-apoptotic effect, rats also received PD98059 (1 mg/kg), SB203580 (1 mg/kg) or SP600125 (6 mg/kg) 15 min prior to ischemia, with subsets of rats also receiving HET0016 10 min prior to ischemia. Compared with vehicle group, 17-ODYA, DDMS and HET0016 significantly inhibited myocardial apoptosis as evidenced by decreased DNA ladder formation, terminal dUTP deoxynucleotidyltransferase nick end-labeling (TUNEL) positive nuclear staining. They also decreased caspase-3 activity and Bax protein expression but up-regulated the expression of Bcl-2. Conversely, exogenous 20-HETE administration exerted opposite effects. Moreover, HET0016 increased the activity of extracellular signal-related protein kinases 1 and 2 (ERK1/2) but had no significant effect on p38 MAPK or c-Jun N-terminal kinase (JNK) during ischemia/reperfusion. Pretreatment with PD98059, the inhibitor of ERK1/2, but not SB203580 or SP600125, almost completely blocked the effect exerted by HET0016. Taken together, these data suggest that CYP omega-hydroxylase inhibition exerts significant anti-apoptosis effects, at least in part, by activation of ERK1/2 in ischemia/reperfusion heart.

  9. Appetite suppressant drugs as inhibitors of human cytochromes P450: in vitro inhibition of P450-2D6 by D- and L-fenfluramine, but not phentermine.

    PubMed

    von Moltke, L L; Greenblatt, D J; Ciraulo, D A; Grassi, J M; Granda, B W; Duan, S X; Harmatz, J S; Shader, R I

    1998-08-01

    The activity of D-fenfluramine, L-fenfluramine, and phentermine as inhibitors of five human cytochromes P450 was evaluated using human liver microsomes in vitro. All three compounds produced negligible inhibition of P450-1A2, -2C9, -2E1, and -3A. Phentermine also did not inhibit P450-2D6. However, D- and L-fenfluramine significantly inhibited P450-2D6 activity as measured by dextromethorphan O-demethylation, with mean 50% inhibitory concentrations (15.1 microM) within one order of magnitude of that for fluoxetine (2.7 microM). Findings from the in vitro assay are consistent with clinical studies showing significant inhibition of desipramine clearance by coadministration of fenfluramine.

  10. Metformin inhibits 7,12-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway.

    PubMed

    Maayah, Zaid H; Ghebeh, Hazem; Alhaider, Abdulqader A; El-Kadi, Ayman O S; Soshilov, Anatoly A; Denison, Michael S; Ansari, Mushtaq Ahmad; Korashy, Hesham M

    2015-04-15

    Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted using 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism. Copyright

  11. The Interaction of Microsomal Cytochrome P450 2B4 with its Redox Partners, Cytochrome P450 Reductase and Cytochrome b5

    PubMed Central

    Im, Sang-Choul; Waskell, Lucy

    2010-01-01

    1 Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b5. In both instances, product formation occurs. When the second electron is donated by cytochrome b5, catalysis (product formation) is ∼ 10 to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼ 15% the coupling of NADPH consumption to product formation. Cytochrome b5 has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b5 on cytochrome P450 2B4 reactivity can explain how cytochrome b5 is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b5 to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochome b5 to cytochrome P450 reductase, cytochrome b5 inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b5 are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b5 stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase. PMID:21055385

  12. Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars

    USDA-ARS?s Scientific Manuscript database

    Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta and pre-implantation blastocyst. All catalyze estrogen synthesis, but the “gonadal” type enzyme is unique in also synthesizing a nonaromat...

  13. Effect of naphthalene on cytochrome oxidase activity

    SciTech Connect

    Harmon, H.J.

    1988-01-01

    Previous reports have demonstrated that naphthalene inhibits oxygen consumption in Daphnia magna tissue culture cells, and intact mitochondria and submitochondrial particles. These studies were extended to algal mitochondrial respiration as well as photosynthetic activity. The authors were able to demonstrate the specific site of apparent respiratory inhibition to be coenzyme Q (ubiquinone, UQ) and later to demonstrate the molecular basis of this inhibition at ubiquinone. The authors previously could not demonstrate an effect of naphthalene on cytochrome oxidase activity. However, the observation that naphthalene can stimulate respiration in algae prompted the reinvestigation of the effect of naphthalene on the kinetics of cytochrome oxidase. Cytochrome oxidase is a multi-subunit membranous protein responsible for the oxidation of cytochrome c and the reduction of molecular oxygen to water. Because of the complicated nature and mechanism of this enzyme, the potential exists for multiple and possibly opposite effects of naphthalene on its function.

  14. Orexin-A Protects Human Neuroblastoma SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Neurotoxicity: Involvement of PKC and PI3K Signaling Pathways.

    PubMed

    Pasban-Aliabadi, Hamzeh; Esmaeili-Mahani, Saeed; Abbasnejad, Mehdi

    2017-04-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by progressive and selective death of dopaminergic neurons. Multifunctional neuropeptide orexin-A is involved in many biological events of the body. It has been shown that orexin-A has protective effects in neurodegenerative disease such as PD. However, its cellular mechanisms have not yet been fully clarified. Here, we investigated the intracellular signaling pathway of orexin-A neuroprotection in 6-hydroxydopamine (6-OHDA)-induced SH-SY5H cells damage as an in vitro model of PD. The cells were incubated with 150 μM 6-OHDA, and the viability was examined by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay. Mitochondrial membrane potential and intracellular calcium were measured by fluorescent probes. Western blotting was also used to determine cyclooxygenase type 2 (COX-2), nuclear factor erythroid 2 related factor 2 (Nrf2), and HSP70 protein levels. The data showed that 6-OHDA has decreasing effects on cell viability, Nrf2, and HSP70 protein expression and increases the level of mitochondrial membrane potential, intracellular calcium, and COX-2 protein. Orexin-A (500 pM) significantly attenuated the 6-OHDA-induced cell damage. Furthermore, Orexin-A significantly prevented the mentioned effects of 6-OHDA on SH-SY5Y cells. Orexin 1 receptor antagonist (SB3344867), PKC, and PI3-kinase (PI3K) inhibitors (chelerythrin and LY294002, respectively) could suppress the orexin-A neuroprotective effect. In contrast, blockage of PKA by a selective inhibitor (KT5720) had no effects on the orexin protection. The results suggest that orexin-A protective effects against 6-OHDA-induced neurotoxicity are performed via its receptors, PKC and PI3K signaling pathways.

  15. Alcoholic extract of Bacopa monniera Linn. protects against 6-hydroxydopamine-induced changes in behavioral and biochemical aspects: a pilot study.

    PubMed

    Shobana, Chandrasekar; Kumar, Radhakrishnan Ramesh; Sumathi, Thangarajan

    2012-10-01

    Parkinson's disease is one of the commonest neurodegenerative diseases, and oxidative stress has been evidenced to play a vital role in its causation. In this study, we evaluated whether alcoholic extract of Bacopa monniera (AEBM), an antioxidant and memory enhancer can slow the neuronal injury in a 6-OHDA-rat model of Parkinson's. Rats were treated with 20 and 40 mg/kg bodyweight of AEBM for 3 weeks. On Day 21, 2 μl of 6-OHDA (12 μg in 0.01 % in ascorbic acid-saline) was infused into the right striatum, while the control group received 2 μl of vehicle. Three weeks after the 6-OHDA injection, the rats were tested for neurobehavioral activity (rotarod, locomotor activity, grip test, forced swim test, radial arm maze) and were killed after 6 weeks for the estimation of lipid peroxidation, reduced glutathione (GSH) content, activities of glutathione-S-transferase, glutathione reductase, glutathione peroxidase, superoxide dismutase (SOD), and catalase (CAT). The deficits in behavioral activity due to 6-OHDA lesioning were significantly and dose dependently restored by AEBM. Lesioning was followed by an increased lipid peroxidation and significant depletion of reduced GSH content in the substantia nigra, which was prevented with AEBM pretreatment. The activities of GSH-dependent enzymes, CAT and SOD in striatum were reduced significantly by lesioning, which were restored significantly and dose dependently by AEBM. This study indicates that the extract of B. monniera might be helpful in attenuating 6-OHDA-induced lesioning in rats.

  16. Protective effect of L-kynurenine and probenecid on 6-hydroxydopamine-induced striatal toxicity in rats: implications of modulating kynurenate as a protective strategy.

    PubMed

    Silva-Adaya, Daniela; Pérez-De La Cruz, Verónica; Villeda-Hernández, Juana; Carrillo-Mora, Paul; González-Herrera, Irma Gabriela; García, Esperanza; Colín-Barenque, Laura; Pedraza-Chaverrí, José; Santamaría, Abel

    2011-01-01

    The neuroactive metabolite at the kynunerine pathway, kynurenic acid (KYNA), is a well-known competitive antagonist at the co-agonist glycine site of the N-methyl-D-aspartate receptor (NMDAr), and also decreases the extracellular levels of glutamate by blocking α7-nicotinic acetylcholine receptor (α7-nAchr) located on glutamatergic terminals. KYNA has been often reported to be neuroprotective in different neurotoxic models. The systemic administration of L-kynurenine (L-KYN)--the precursor of KYNA--together with probenecid (PROB)--an inhibitor of organic acids transport--to rodents increases KYNA levels in the brain in a dose-dependent manner. The striatal infusion of the toxin 6-hydroxydopamine (6-OHDA) to rodents is one of the common models used to simulate Parkinson's disease (PD). Different studies have linked PD alterations with excessive glutamatergic transmission in the striatum since NMDAr antagonists exert beneficial effects in PD models. In this work we investigated the effect that a systemic administration of L-KYN+PROB exerted on the toxic model induced by 6-OHDA in rats. PROB (50 mg/kg, i.p.) + L-KYN (75 mg/kg, i.p.) were given to rats for seven consecutive days. On day two of treatment, the animals were infused with a single injection of 6-OHDA (20 μg/2 μl) into the right striatum. Fourteen days post-lesion, rotation behavior was assessed as a marker of motor impairment. The total levels of dopamine (DA) were also estimated in striatal tissue samples of 6-OHDA-treated animals as a neurochemical marker of damage. In addition, twenty eight days post-lesion, the striatal damage was assessed by hematoxylin/eosin staining and immunohistochemistry against glial fibrillary acidic protein (GFAP) in the same animals. Neurodegeneration was also assessed by Fluoro Jade staining. 6-OHDA infusion increased rotation behavior, striatal reactive gliosis and neurodegeneration, while DA levels were decreased. For all markers evaluated, we observed protective effects of L-KYN+PROB on the dopaminergic damage induced by 6-OHDA. Our results suggest that this strategy was useful to mitigate dopaminergic toxicity in the hemiparkinsonian model. The combined use of L-KYN and PROB is a valuable tool to modulate glutamatergic and cholinergic activities, presumably by means of increased levels of endogenous KYNA.

  17. In vivo visualization and monitoring of viable neural stem cells using noninvasive bioluminescence imaging in the 6-hydroxydopamine-induced mouse model of Parkinson disease.

    PubMed

    Im, Hyung-Jun; Hwang, Do Won; Lee, Han Kyu; Jang, Jaeho; Lee, Song; Youn, Hyewon; Jin, Yeona; Kim, Seung U; Kim, E Edmund; Kim, Yong Sik; Lee, Dong Soo

    2013-06-01

    Transplantation of neural stem cells (NSCs) has been proposed as a treatment for Parkinson disease (PD). The aim of this study was to monitor the viability of transplanted NSCs expressing the enhanced luciferase gene in a mouse model of PD in vivo. The PD animal model was induced by unilateral injection of 6-hydroxydopamine (6-OHDA). The behavioral test using apomorphine-induced rotation and positron emission tomography with [18F]N-(3-fluoropropyl)-2'-carbomethoxy-3'-(4-iodophenyl)nortropane ([18F]FP-CIT) were conducted. HB1.F3 cells transduced with an enhanced firefly luciferase retroviral vector (F3-effLuc cells) were transplanted into the right striatum. In vivo bioluminescence imaging was repeated for 2 weeks. Four weeks after transplantation, [18F]FP-CIT PET and the rotation test were repeated. All 6-OHDA-injected mice showed markedly decreased [18F]FP-CIT uptake in the right striatum. Transplanted F3-effLuc cells were visualized on the right side of the brain in all mice by bioluminescence imaging. The bioluminescence intensity of the transplanted F3-effLuc cells gradually decreased until it was undetectable by 10 days. The behavioral test showed that stem cell transplantation attenuated the motor symptoms of PD. No significant change was found in [18F]FP-CIT imaging after cell transplantation. We successfully established an in vivo bioluminescence imaging system for the detection of transplanted NSCs in a mouse model of PD. NSC transplantation induced behavioral improvement in PD model mice.

  18. Effect of different doses of estrogen on the nigrostriatal dopaminergic system in two 6-hydroxydopamine-induced lesion models of Parkinson's disease.

    PubMed

    Cordellini, Marcela Ferreira; Piazzetta, Giovana; Pinto, Karin Cristine; Delattre, Ana Márcia; Matheussi, Francesca; Carolino, Ruither O G; Szawka, Raphael Escorsim; Anselmo-Franci, Janete A; Ferraz, Anete Curte

    2011-06-01

    Parkinson's disease results from a degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNpc) and it is more prevalent in men than in women. Estrogen has neuroprotective action of the nigrostriatal dopaminergic (NSDA) neurons. It was investigated whether differences in plasma 17β-estradiol (E2) levels alter the degree of neuroprotection in NSDA neurons. Ovariectomized rats, implanted with subcutaneous capsules containing 400, 800 or 1,600 μg of E2 or corn oil, were injected with 1 μg of 6-OHDA in the SNpc or the medial forebrain bundle (MFB). Striatal dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and plasma E2 levels were measured. Only at 400 μg, E2 protected striatal DA against lesion of the MFB. In the SNpc, E2 failed to prevent DA depletion, but increased DOPAC/DA ratio in the striatum. In an NSDA moderate lesion, E2 has a neuroprotective action. In a severe lesion, E2 could stimulate DA activity in remaining neurons.

  19. Allogeneic/xenogeneic transplantation of peptide-labeled mitochondria in Parkinson's disease: restoration of mitochondria functions and attenuation of 6-hydroxydopamine-induced neurotoxicity.

    PubMed

    Chang, Jui-Chih; Wu, Shey-Lin; Liu, Ko-Hung; Chen, Ya-Hui; Chuang, Chieh-Sen; Cheng, Fu-Chou; Su, Hong-Lin; Wei, Yau-Huei; Kuo, Shou-Jen; Liu, Chin-San

    2016-04-01

    Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated allogeneic mitochondrial delivery (PMD) has been demonstrated in vitro, the in vivo therapeutic efficacy of PMD in Parkinson's disease (PD) has yet to be determined. In this study, we compared the functionality of mitochondrial transfer with or without Pep-1 conjugation in neurotoxin (6-hydroxydopamine, 6-OHDA)-induced PC12 cells and PD rat models. We injected mitochondria into the medial forebrain bundle (MFB) of the PD rats after subjecting the nigrostriatal pathway to a unilateral 6-OHDA lesion for 21 days, and we verified the effectiveness of the mitochondrial graft in enhancing mitochondrial function in the soma of the substantia nigra (SN) neuron through mitochondrial transport dynamics in the nigrostriatal circuit. The result demonstrated that only PMD with allogeneic and xenogeneic sources significantly sustained mitochondrial function to resist the neurotoxin-induced oxidative stress and apoptotic death in the rat PC12 cells. The remaining cells exhibited a greater capability of neurite outgrowth. Furthermore, allogeneic and xenogeneic transplantation of peptide-labeled mitochondria after 3 months improved the locomotive activity in the PD rats. This increase was accompanied by a marked decrease in dopaminergic neuron loss in the substantia nigra pars compacta (SNc) and consistent enhancement of tyrosine hydroxylase-positive immunoreaction of dopaminergic neurons in the SNc and striatum. We also observed that in the SN dopaminergic neuron in the treated PD rats, mitochondrial complex I protein and mitochondrial dynamics were restored, thus ameliorating the oxidative DNA damage. Moreover, we determined signal translocation of graft allogeneic mitochondria from the MFB to the calbindin-positive SN neuron, which demonstrated the regulatory role of mitochondrial transport in alleviating 6-OHDA-induced degeneration of dopaminergic neurons.

  20. Phytic Acid Protects against 6-Hydroxydopamine-Induced Dopaminergic Neuron Apoptosis in Normal and Iron Excess Conditions in a Cell Culture Model.

    PubMed

    Xu, Qi; Kanthasamy, Anumantha G; Reddy, Manju B

    2011-02-07

    Iron may play an important role in Parkinson's disease (PD) since it can induce oxidative stress-dependent neurodegeneration. The objective of this study was to determine whether the iron chelator, phytic acid (IP6) can protect against 6-hydroxydopamine- (6-OHDA-) induced apoptosis in immortalized rat mesencephalic dopaminergic cells under normal and iron-excess conditions. Caspase-3 activity was increased about 6-fold after 6-OHDA treatment (compared to control; P < .001) and 30 μmol/L IP6 pretreatment decreased it by 38% (P < .05). Similarly, a 63% protection (P < .001) against 6-OHDA induced DNA fragmentation was observed with IP6 pretreatment. Under iron-excess condition, a 6-fold increase in caspase-3 activity (P < .001) and a 42% increase in DNA fragmentation (P < .05) with 6-OHDA treatment were decreased by 41% (P < .01) and 27% (P < .05), respectively, with 30 μmol/L IP6. Together, our data suggest that IP6 protects against 6-OHDA-induced cell apoptosis in both normal and iron-excess conditions, and IP6 may offer neuroprotection in PD.

  1. Phytic Acid Protects against 6-Hydroxydopamine-Induced Dopaminergic Neuron Apoptosis in Normal and Iron Excess Conditions in a Cell Culture Model

    PubMed Central

    Xu, Qi; Kanthasamy, Anumantha G.; Reddy, Manju B.

    2011-01-01

    Iron may play an important role in Parkinson's disease (PD) since it can induce oxidative stress-dependent neurodegeneration. The objective of this study was to determine whether the iron chelator, phytic acid (IP6) can protect against 6-hydroxydopamine- (6-OHDA-) induced apoptosis in immortalized rat mesencephalic dopaminergic cells under normal and iron-excess conditions. Caspase-3 activity was increased about 6-fold after 6-OHDA treatment (compared to control; P < .001) and 30 μmol/L IP6 pretreatment decreased it by 38% (P < .05). Similarly, a 63% protection (P < .001) against 6-OHDA induced DNA fragmentation was observed with IP6 pretreatment. Under iron-excess condition, a 6-fold increase in caspase-3 activity (P < .001) and a 42% increase in DNA fragmentation (P < .05) with 6-OHDA treatment were decreased by 41% (P < .01) and 27% (P < .05), respectively, with 30 μmol/L IP6. Together, our data suggest that IP6 protects against 6-OHDA-induced cell apoptosis in both normal and iron-excess conditions, and IP6 may offer neuroprotection in PD. PMID:21331377

  2. Protective effects of Althaea officinalis L. extract in 6-hydroxydopamine-induced hemi-Parkinsonism model: behavioral, biochemical and histochemical evidence.

    PubMed

    Rezaei, Maryam; Alirezaei, Masoud

    2014-05-01

    It is well known that Parkinson's disease (PD) is the second most common neurodegenerative disorder in humans. In this regard, the neuroprotective effect of Althaea officinalis (AO) has already been reported. Therefore, this study examined whether administration of AO extract would improve behavioral, biochemical and structural abnormalities in an experimental animal model of PD in rats. For this purpose, we induced hemi-Parkinsonism by unilateral intranigral injection of 6-hydroxydopamine (6-OHDA, 8 μg/5 μl saline-ascorbate). The rats were pretreated i.p. with AO extract (10 mg/kg) started 6 days before surgery and continued until the 3rd day post-surgery. Regarding oxidative stress, brain MDA concentration (as a lipid peroxidation marker) increased significantly in the 6-OHDA-administered group in comparison with rats pretreated with AO extract. It was found that AO treatment attenuated rotational behavior in the 6-OHDA-administered group and protected the neurons of substantia nigra pars compacta against 6-OHDA toxicity. Overall, AO extract administration indicated neuroprotective effects against 6-OHDA-induced hemi-Parkinsonism in rats.

  3. Neuroprotective Role of MicroRNA-22 in a 6-Hydroxydopamine-Induced Cell Model of Parkinson's Disease via Regulation of Its Target Gene TRPM7.

    PubMed

    Yang, Chao Ping; Zhang, Zhen Hua; Zhang, Li Hua; Rui, Han Chen

    2016-12-01

    Parkinson's disease (PD), the second most prevalent neurodegenerative disorder with only symptomatic treatment available, is characterized by a progressive loss of dopaminergic neurons in the midbrain. Ample evidence indicated that microRNAs (miRs) could regulate post-transcriptional gene expression and neuronal disease. In the present study, we have evaluated the effects and mechanism of miR-22 in PC12 pheochromocytoma cells treated with 6-hydroxydopamine (6-OHDA) to mimic PD. RT-PCR results showed that the expression of miR-22 is downregulated in 6-OHDA-treated PC12 cells, and the overexpression of miR-22 significantly promoted the survival and proliferation of 6-OHDA-induced PC12 cells, whereas miR-22 inhibitor reversed these effects. In addition, PC12 cells were treated with miR-22 mimics or inhibitor following 6-OHDA administration, which medicated ROS production and upregulation or downregulation of caspase-3 activity, respectively. A luciferase reporter assay revealed that transient receptor potential melastatin 7 (TRPM7) is a direct target gene of miR-22, and miR-22 overexpression markedly downregulated the level of TRPM7. Strikingly, further analysis showed that miR-22 mediated 6-OHDA-induced PC12 cell survival and proliferation by targeting TRPM7. Taken together, the present study showed that miR-22 overexpression exhibited neuroprotective and reversal effects on the 6-OHDA-induced PC12 cell growth and apoptosis by targeting TRPM7.

  4. Carnosic acid protects against 6-hydroxydopamine-induced neurotoxicity in in vivo and in vitro model of Parkinson's disease: involvement of antioxidative enzymes induction.

    PubMed

    Wu, Chi-Rei; Tsai, Chia-Wen; Chang, Shu-Wei; Lin, Chia-Yuan; Huang, Li-Chun; Tsai, Chia-Wen

    2015-01-05

    The neuroprotective effects of carnosic acid (CA), a phenolic diterpene isolated from rosemary (Rosmarinus officinalis), have been widely investigated in recent years, however, its protection in in vivo still unclear. In this study, we investigated the behavioral activity and neuroprotective effects of CA in a rat model of Parkinson's disease (PD) induced by 6-hydroxydopamine (6-OHDA). Rats were treated with 20mg/kg body weight of CA for 3 weeks before 6-OHDA exposure. Results indicated that CA improved the locomotor activity and reduced the apomorphine-caused rotation in 6-OHDA-stimulated rats. Significant protection against lipid peroxidation and GSH reduction was observed in the 6-OHDA rats pretreated with CA. Pretreatment with CA increased the protein expression of γ-glutamate-cysteine ligase catalytic subunit, γ-glutamate-cysteine ligase modifier subunit, superoxide dismutase, and glutathione reductase compared with 6-OHDA-stimulated rats and SH-SY5Y cells. Immunoblots showed that the reduction of the Bcl-2/Bax ratio, the induction of caspase 3 cleavage, and the induction of poly(ADP-ribose) polymerase (PARP) cleavage by 6-OHDA was reversed in the presence of SB203580 (a p38 inhibitor) or SP600125 (a JNK inhibitor) in SH-SY5Y cells. Rats treated with CA reversed the 6-OHDA-mediated the activation of c-Jun NH2-terminal kinase and p38, the down-regulation of the Bcl-2/Bax ratio, the up-regulation of cleaved caspase 3/caspase 3 and cleaved PARP/PARP ratio, and the down-regulation of tyrosine hydroxylase protein. However, BAM7, an activator of Bax, attenuated the effect of CA on apoptosis in SH-SY5Y cells. These results suggest that CA protected against 6-OHDA-induced neurotoxicity is attributable to its anti-apoptotic and anti-oxidative action. The present findings may help to clarify the possible mechanisms of rosemary in the neuroprotection of PD.

  5. Pharmacognostical Analysis and Protective Effect of Standardized Extract and Rizonic Acid from Erythrina velutina against 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

    PubMed

    Silva, Aline H; Fonseca, Francisco Noé; Pimenta, Antônia T A; Lima, MaryAnne S; Silveira, Edilberto Rocha; Viana, Glauce S B; Vasconcelos, Silvânia M M; Leal, Luzia Kalyne A M

    2016-01-01

    Erythrina velutina is a tree common in the northeast of Brazil extensively used by traditional medicine for the treatment of central nervous system disorders. To develop a standardized ethanol extract of E. velutina (EEEV) and to investigate the neuroprotective potential of the extract and rizonic acid (RA) from E. velutina on neuronal cells. The plant drug of E. velutina previously characterized was used for the production of EEEV. Three methods were evaluated in order to obtain an extract with higher content of phenols. The neuroprotective effect of standardized EEEV (HPLC-PDA) and RA was investigated on SH-SY5Y cell exposure to the neurotoxin 6-hydroxydopamine (6-OHDA). The powder of the plant drug was classified as moderately coarse and several quality control parameters were determined. EEEV produced by percolation gave the highest phenol content when related to others extractive methods, and its HPLC-PDA analysis allowed to identify four flavonoids and RA, some reported for the first time for the species. EEEV and RA reduced significantly the neurotoxicity induced by 6-OHDA in SH-SY5Y cells determined by the MTT assay and the nitrite concentration. EEEV also showed a free radical scavenging activity. This is the first pharmacological study about E. velutina which used a controlled standardized extract since the preparation of the herbal drug. This extract and RA, acting as an antioxidant, presents a neuroprotective effect suggesting that they have potential for future development as a therapeutic agent in neurodegenerative disease as Parkinson. The powder of Erythrina velutina was classified as moderately coarse and several quality-control parameters were determined.Ethanolic extract from E. velutina (EEEV) produced by percolation gave the highest phenol content when related to others extractive methods and its HPLC-PDA analysis of EEEV allowed to identify four flavonoids and rizonic acid (RA), some reported for the first time for the species.The EEEV and RA reduced significantly the neurotoxicity induced by 6-OHDA in SH-SY5Y cells determined by the MTT assay and the nitrite concentration.The EEEV also showed a free radical scavenging activity. Abbreviations used: ±: More or less, %: Percentage, °C: Degree Celsius, <: Less than, μg: Microgram, μL: Microliter, μM: Micromol, [1D] MNR: One-dimensional nuclear magnetic resonance spectroscopy, [2D] MNR:Two-dimensional nuclear magnetic resonance spectroscopy, 6-OHDA: [6-] Hydroxydopamine. Abs: Absorbance, CFU: Colony forming units, CH2Cl2: Dichloromethane, CHCl3: Chloroform cmCentimeter, DMEM/F12: Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12. DMSO: Dimethyl sulfoxide, DPPH: 1,1-Diphenyl-2-picrylhydrazyl, EAG: Gallic acid equivalents, EEEV: Ethanolic extract of Erythrina velutina, EtOAc: Ethyl acetate, g: Gram, h: Hour, H2O: Water, HPLC: High-performance liquid chromatography, H REIMS: Hydrogen rapid evaporative ionization mass spectrometry, Kg: Kilogram M: Molar, m: Metro, MeOH: Methanol, mg: Milligram, min: Minute, mL: Milliliter, mm: Millimeter, MTT: Bromide 3 [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium, N: Normal, NBT: Nitroblue tetrazolium, nm: Nanometer, PDA: Photodiode array detector, TPC: Total polyphenol content, RA: Rizonic acid, RP: Reverse phase, SOD: Superoxide dismutase, v/v: Volume per volume, Vs: Versus W: Watts.

  6. Pharmacognostical Analysis and Protective Effect of Standardized Extract and Rizonic Acid from Erythrina velutina against 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells

    PubMed Central

    Silva, Aline H.; Fonseca, Francisco Noé; Pimenta, Antônia T. A.; Lima, MaryAnne S.; Silveira, Edilberto Rocha; Viana, Glauce S. B.; Vasconcelos, Silvânia M. M.; Leal, Luzia Kalyne A. M.

    2016-01-01

    Background: Erythrina velutina is a tree common in the northeast of Brazil extensively used by traditional medicine for the treatment of central nervous system disorders. Objective: To develop a standardized ethanol extract of E. velutina (EEEV) and to investigate the neuroprotective potential of the extract and rizonic acid (RA) from E. velutina on neuronal cells. Materials and methods: The plant drug of E. velutina previously characterized was used for the production of EEEV. Three methods were evaluated in order to obtain an extract with higher content of phenols. The neuroprotective effect of standardized EEEV (HPLC-PDA) and RA was investigated on SH-SY5Y cell exposure to the neurotoxin 6-hydroxydopamine (6-OHDA). Results: The powder of the plant drug was classified as moderately coarse and several quality control parameters were determined. EEEV produced by percolation gave the highest phenol content when related to others extractive methods, and its HPLC-PDA analysis allowed to identify four flavonoids and RA, some reported for the first time for the species. EEEV and RA reduced significantly the neurotoxicity induced by 6-OHDA in SH-SY5Y cells determined by the MTT assay and the nitrite concentration. EEEV also showed a free radical scavenging activity. Conclusion: This is the first pharmacological study about E. velutina which used a controlled standardized extract since the preparation of the herbal drug. This extract and RA, acting as an antioxidant, presents a neuroprotective effect suggesting that they have potential for future development as a therapeutic agent in neurodegenerative disease as Parkinson. SUMMARY The powder of Erythrina velutina was classified as moderately coarse and several quality-control parameters were determined.Ethanolic extract from E. velutina (EEEV) produced by percolation gave the highest phenol content when related to others extractive methods and its HPLC–PDA analysis of EEEV allowed to identify four flavonoids and rizonic acid (RA), some reported for the first time for the species.The EEEV and RA reduced significantly the neurotoxicity induced by 6-OHDA in SH-SY5Y cells determined by the MTT assay and the nitrite concentration.The EEEV also showed a free radical scavenging activity. Abbreviations used: ±: More or less, %: Percentage, °C: Degree Celsius, <: Less than, μg: Microgram, μL: Microliter, μM: Micromol, [1D] MNR: One-dimensional nuclear magnetic resonance spectroscopy, [2D] MNR:Two-dimensional nuclear magnetic resonance spectroscopy, 6-OHDA: [6-] Hydroxydopamine. Abs: Absorbance, CFU: Colony forming units, CH2Cl2: Dichloromethane, CHCl3: Chloroform cmCentimeter, DMEM/F12: Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12. DMSO: Dimethyl sulfoxide, DPPH: 1,1-Diphenyl-2-picrylhydrazyl, EAG: Gallic acid equivalents, EEEV: Ethanolic extract of Erythrina velutina, EtOAc: Ethyl acetate, g: Gram, h: Hour, H2O: Water, HPLC: High-performance liquid chromatography, H REIMS: Hydrogen rapid evaporative ionization mass spectrometry, Kg: Kilogram M: Molar, m: Metro, MeOH: Methanol, mg: Milligram, min: Minute, mL: Milliliter, mm: Millimeter, MTT: Bromide 3 [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium, N: Normal, NBT: Nitroblue tetrazolium, nm: Nanometer, PDA: Photodiode array detector, TPC: Total polyphenol content, RA: Rizonic acid, RP: Reverse phase, SOD: Superoxide dismutase, v/v: Volume per volume, Vs: Versus W: Watts PMID:27867274

  7. Human induced pluripotent stem cell-derived neurons improve motor asymmetry in a 6-hydroxydopamine-induced rat model of Parkinson's disease.

    PubMed

    Han, Fabin; Wang, Wei; Chen, Baoxing; Chen, Chao; Li, Sen; Lu, Xianjie; Duan, Jing; Zhang, Yang; Zhang, Yu Alex; Guo, Wennian; Li, Guangyao

    2015-05-01

    Since human embryonic stem cells and human fetal neural stem cells have immune rejection and ethical issues, recent advancements in induced pluripotent stem cells (iPS cells) provide new possibilities to study autologous cell therapy for Parkinson's disease (PD). We isolated human skin fibroblasts from normal individuals and patients with PD; we generated iPS cells by transfecting these human skin fibroblasts with retroviral reprogramming factors of OCT4, SOX2, KLF4 and c-MYC and induced iPS cells to differentiate neural stem cells (NSCs) and then into neurons and dopamine neurons in vitro. We found that iPS cell-derived NSC transplant into the striatum of the 6-hydroxydopamine (OHDA)-induced PD rats improved their functional defects of rotational asymmetry at 4, 8, 12 and 16 weeks after transplantation. iPS cell-derived NSCs were found to survive and integrate into the brain of transplanted PD rats and differentiated into neurons, including dopamine neurons in vivo. Transplantation of iPS cell-derived NSCs has therapeutic potential for PD. Our study provided experimental proof for future clinical application of iPS cells in cell-based treatment of PD. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. Fibroblast growth factor 1attenuates 6-hydroxydopamine-induced neurotoxicity: an in vitro and in vivo investigation in experimental models of parkinson’s disease

    PubMed Central

    Wei, Xiaojie; He, Songbin; Wang, Zhouguang; Wu, Jiamin; Zhang, Jinjing; Cheng, Yi; Yang, Jie; Xu, Xinlong; Chen, Zaifeng; Ye, Junmin; Chen, Li; Lin, Li; Xiao, Jian

    2014-01-01

    Parkinson’s disease (PD) is a degenerative disorder of the central nervous system and is characterized by motor system disorders resulting in loss of dopamine producing brain cells. Acidic fibroblast growth factor, also called FGF1, promotes the survival of neurons. The aims of the present study were to confirm FGF1 could protect neurons cultures from 6-hydroxydopamine (6-OHDA) toxicity in vitro and in vivo. Our results demonstrated FGF1 administration improved the motor function recovery, increased the TH-positive neurons survival and up-regulated the levels of neurotransmitters in PD rats. Meanwhile, FGF1 prevents the death of DA neuron at least in part by reducing the levels of α-synuclein and ER stress. The administration of FGF1 activated downstream signals PI3K/Akt and ERK1/2. In conclusion, FGF1 diminished α-synuclein neurotoxicity by down regulating ER stress mediators and the level of apoptosis, and these effects may underlying the activation of the PI3K/Akt and ERK1/2 signal pathway. PMID:25628778

  9. Lever pressing responses under a fixed-ratio schedule of mice with 6-hydroxydopamine-induced dopamine depletion in the nucleus accumbens.

    PubMed

    Tsutsui, Yuji; Nishizawa, Kayo; Kai, Nobuyuki; Kobayashi, Kazuto

    2011-02-02

    In order to investigate the relationship between dopamine transmission in the nucleus accumbens and operant behavior in mice, mice with 6-hydroxydopamine (6-OHDA)-induced dopamine depletion in the nucleus accumbens were tested for their performance in lever pressing tasks under FR schedules with 8 ratios from FR5 to FR120. The mice were given one 20-mg food pellet per completed FR schedule in FR5, FR10, and FR20; they were given 2 pellets in FR40, and one more cumulatively in the rest of the schedules. Before the 6-OHDA injection surgery, all mice were trained to press a lever under all FR schedules. Then, 6-OHDA or ascorbate was injected into the nucleus accumbens. Postoperatively, the mice were tested under each FR schedule, with 3 sessions per schedule. 6-OHDA-treated mice exhibited an increase in lever pressing latency, i.e., the time interval between the last presentation of the reward and the next lever press, and a decrease in inter-response intervals, i.e., the time interval between 2 lever presses excluding lever pressing latency, irrespective of the FR ratios. Furthermore, in these 6-OHDA-treated mice, the number of lever presses during the first 300s of the session decreased under FR schedules with low ratios (5, 10, and 20). Open field activity, food motivation, and the amount of food consumed were not affected by dopamine depletion in the nucleus accumbens. These results suggest that the dopamine system in the nucleus accumbens had an important role in the control of lever pressing latency and inter-response intervals under FR reinforcement schedules. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Stimulation of cellular XTT reduction by cytochrome oxidase inhibitors.

    PubMed

    Kunimoto, S; Nosaka, C; Takeuchi, T

    1999-06-01

    XTT reducing activity by CHO and L1210 cells was found to be stimulated by the presence of cytochrome oxidase inhibitors such as NaN3 or KCN. Among the other respiratory chain inhibitors, antimycin A (a complex III inhibitor) and chlorpromazine inhibited cellular XTT reduction, and rotenone and malonate showed slight inhibition and no effect, respectively. It is suggested that XTT reduction is coupled with the respiratory chain via cytochrome c, which is located between complexes III and IV (cytochrome oxidase).

  11. Inhibition of cytochrome P-450 with 2-diethylamino-ethyl-2,2-diphenylpropylacetate (SKF-525A) reduces immunotoxicity of chlorinated carbohydrates.

    PubMed

    Zabrodskii, P F; Mandych, V G; Germanchuk, V G

    2006-09-01

    Experiments on outbred albino rats showed that single intraperitoneal injection of cytochrome P-450 inhibitor 2-diethylaminoethyl-2,2-diphenylpropylacetate (SKF-525A) in a dose of 50 mg/kg before acute poisoning with 1,2-dichloroethane and trichloroethane in a dose of 1.0 LD(50), metabolized in the body to compounds with higher toxicity (the phenomenon of "lethal synthesis") reduced their immunotoxicity by decreasing the formation of their biotransformation products.

  12. Application of Osmotic Pumps for Sustained Release of 1-Aminobenzotriazole and Inhibition of Cytochrome P450 Enzymes in Mice: Model Comparison with the Hepatic P450 Reductase Null Mouse.

    PubMed

    Stringer, Rowan A; Ferreira, Suzie; Rose, Jonathan; Ronseaux, Sebastien

    2016-08-01

    The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longer-term model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  13. In silico prediction of cytochrome P450 2D6 and 3A4 inhibition using Gaussian kernel weighted k-nearest neighbor and extended connectivity fingerprints, including structural fragment analysis of inhibitors versus noninhibitors.

    PubMed

    Jensen, Berith F; Vind, Christian; Padkjaer, Søren B; Brockhoff, Per B; Refsgaard, Hanne H F

    2007-02-08

    Inhibition of cytochrome P450 (CYP) enzymes is unwanted because of the risk of severe side effects due to drug-drug interactions. We present two in silico Gaussian kernel weighted k-nearest neighbor models based on extended connectivity fingerprints that classify CYP2D6 and CYP3A4 inhibition. Data used for modeling consisted of diverse sets of 1153 and 1382 drug candidates tested for CYP2D6 and CYP3A4 inhibition in human liver microsomes. For CYP2D6, 82% of the classified test set compounds were predicted to the correct class. For CYP3A4, 88% of the classified compounds were correctly classified. CYP2D6 and CYP3A4 inhibition were additionally classified for an external test set on 14 drugs, and multidimensional scaling plots showed that the drugs in the external test set were in the periphery of the training sets. Furthermore, fragment analyses were performed and structural fragments frequent in CYP2D6 and CYP3A4 inhibitors and noninhibitors are presented.

  14. Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes.

    PubMed

    Berry, E A; Trumpower, B L

    1985-02-25

    An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.

  15. Further Characterization of the Metabolism of Desloratadine and Its Cytochrome P450 and UDP-glucuronosyltransferase Inhibition Potential: Identification of Desloratadine as a Relatively Selective UGT2B10 Inhibitor.

    PubMed

    Kazmi, Faraz; Yerino, Phyllis; Barbara, Joanna E; Parkinson, Andrew

    2015-09-01

    Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a nonsedating antihistamine used for the treatment of seasonal allergies and hives. Previously we reported that the formation of 3-hydroxydesloratadine, the major human metabolite of desloratadine, involves three sequential reactions, namely N-glucuronidation by UGT2B10 followed by 3-hydroxylation by CYP2C8 followed by deconjugation (rapid, nonenzymatic hydrolysis of the N-glucuronide). In this study we assessed the perpetrator potential of desloratadine based on in vitro studies of its inhibitory effects on cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes (HLM). Desloratadine (10 µM) caused no inhibition (<15%) of CYP1A2, CYP2C8, CYP2C9, or CYP2C19 and weak inhibition (32-48%) of CYP2B6, CYP2D6, and CYP3A4/5. In cryopreserved human hepatocytes (CHH), which can form the CYP2C8 substrate desloratadine N-glucuronide, desloratadine did not inhibit the CYP2C8-dependent metabolism of paclitaxel or amodiaquine. Assessment of UGT inhibition identified desloratadine as a potent and relatively selective competitive inhibitor of UGT2B10 (Ki value of 1.3 μM). Chemical inhibition of UGT enzymes in HLM demonstrated that nicotine (UGT2B10 inhibitor) but not hecogenin (UGT1A4 inhibitor) completely inhibited the conversion of desloratadine (1 µM) to 3-hydroxydesloratadine in HLM fortified with both NADPH and UDP-glucuronic acid. 3-Hydroxydesloratadine formation correlated well with levomedetomidine glucuronidation (UGT2B10 marker activity) with a panel of individual CHH (r(2) = 0.72). Overall, the results of this study confirm the role of UGT2B10 in 3-hydroxydesloratadine formation and identify desloratadine as a relatively selective in vitro inhibitor of UGT2B10.

  16. Cytochromes of Aquatic Fungi

    PubMed Central

    Gleason, Frank H.; Unestam, Torgny

    1968-01-01

    The cytochrome systems of two classes of aquatic fungi, the Oomycetes and Chytridiomycetes, were studied by means of reduced-minus-oxidized difference spectra at room and at low temperature. At room temperature, all of these fungi have a c-type cytochrome with an absorption maximum at 551 mμ and a b-type cytochrome at 564 mμ. The Oomycetes have a-type cytochromes at 605 mμ, and the Chytridiomycetes have a-type cytochromes at 606 mμ (Blastocladiales) or at 609 mμ (Monoblepharidales). Additional b-type cytochromes are found at 557 mμ in the Oomycetes and at approximately 560 mμ in the Chytridiomycetes. The data obtained from spectra at low temperature are consistent with these conclusions. Thus, the difference spectra reveal variation between the cytochrome systems of these two classes of aquatic fungi. PMID:5650068

  17. The respiratory system of the marine bacterium Beneckea natriegens. I. Cytochrome composition.

    PubMed

    Weston, J A; Knowles, C J

    1974-02-22

    (1) The cytochrome composition of Beneckea natriegens grown under aerobic conditions has been examined. (2) Cell-free extracts obtained by sonication were separated into particulate and supernatant fractions by centrifugation at 150,000 x g. (3) The particulate fraction contained cytochromes b562, b557, b or c554, c549.5, c547, and low concentrations of cytochromes a1 and a2. (Subscripts refer to the wavelength optima of the b and c type cytochrome alpha-peaks in low temperature (77 degrees K) difference spectra.) Also present was a second cytochrome c549.5 which is capable of binding carbon monoxide (cytochrome c549.5(CO)) and which is also found in the supernatant fraction. (4) Reduced plus CO minus reduced difference spectra had spectral peaks corresponding to cytochrome o and two c type cytochromes, and low concentrations of cytochromes a1 and a2. (5) Action spectra for the relief of CO inhibition showed that cytochrome a2, the CO binding c type cytochrome(s) and possibly cytochrome o, but not cytochrome a1, had oxidase activity in intact cells. In cells grown to the late stationary phase, when cytochrome a2 and particularly cytochrome a1 were induced, the primary functual oxidase was cytochrome a1.

  18. 2-Diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) revisited: comparative cytochrome P450 inhibition in human liver microsomes by SKF525A, its metabolites, and SKF-acid and SKF-alcohol.

    PubMed

    Franklin, Michael R; Hathaway, Laura B

    2008-12-01

    When incubated with human liver microsomes, 2-diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) undergoes cytochrome P450 (P450)-dependent oxidative N-deethylation to the secondary amine metabolite 2-ethylaminoethyl-2,2-diphenylvalerate (SKF8742). P450-selective inhibitors indicated CYP3As catalyzed this reaction, and the deethylation rate correlated best with the CYP3A activity across a range of human liver microsomes. SKF525A and its metabolite and primary amine analog all inhibited CYP2B6-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A-selective reactions to varying degrees but had little effect on CYP1A2, CYP2A6, and CYP2E1 reactions. Only the inhibition of CYP3A showed major enhancement when the inhibitors were preincubated with NADPH-fortified microsomes, and the extent of metabolic intermediate (MI) complex formation approximated typical CYP3A content. Two "lost with time" SKF525A derivatives devoid of the ethylamine moiety, 2,2-diphenylpropylethanol (SKF-Alcohol) and 2,2-diphenylpropylacetic acid (SKF-Acid) did not form an MI complex and were identified as selective inhibitors of CYP2C9. Although without detectable metabolism, their CYP2C9 inhibition fitted best with a competitive mechanism. Thus, not all the human P450s are inhibited by SKF525A and related compounds, and the mechanisms contributing to those that are inhibited vary with the isoform. P450 MI-complex formation only seems to play a role with CYP3As.

  19. Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii.

    PubMed

    Jurtshuk, P; Mueller, T J; Wong, T Y

    1981-09-14

    A membrane-bound cytochrome oxidase for Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using as ascorbate-TMPD oxidation assay. The oxidase was 'solubilized' from a sonic-type electron-transport particle (R3 fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27-70% (NH4)2SO4. The highly purified cytochrome oxidase has a V of 60-78 microgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN3 and NH2OH; NaNO2 (but not NaNO3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c4 and cytochrome o; cytochromes a1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c4-o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a3 oxidase of mammalian mitochondria.

  20. Structure-Function Relationships of Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 Flavonoid Derivatives

    PubMed Central

    Shimada, Tsutomu; Tanaka, Katsuhiro; Takenaka, Shigeo; Murayama, Norie; Martin, Martha V.; Foroozesh, Maryam K.; Yamazaki, Hiroshi; Guengerich, F. Peter; Komori, Masayuki

    2010-01-01

    Structure-function relationships for inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced Reverse Type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, e.g. 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 µM to 0.09, 0.21, 0.25, and 0.27 µM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 µM. The introduction of a 4’-methoxy- or 3’,4’-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 µM, respectively. The above hydroxyl- and/or methoxy-groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always towards P450 1A2, where 3-, 5-, or 7-hydroxyflavone, and 4’-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-,5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3-and 5-hydroxyflavone. Flavone and several other flavonoids produced Type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities towards P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids towards these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids. PMID

  1. Structure-function relationships of inhibition of human cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives.

    PubMed

    Shimada, Tsutomu; Tanaka, Katsuhiro; Takenaka, Shigeo; Murayama, Norie; Martin, Martha V; Foroozesh, Maryam K; Yamazaki, Hiroshi; Guengerich, F Peter; Komori, Masayuki

    2010-12-20

    Structure-function relationships for the inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced reverse type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, for example, 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 μM to 0.09, 0.21, 0.25, and 0.27 μM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 μM. The introduction of a 4'-methoxy- or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 μM, respectively. The above hydroxyl and/or methoxy groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always toward P450 1A2, where 3-, 5-, or 7-hydroxyflavone and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-, 5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3- and 5-hydroxyflavone. Flavone and several other flavonoids produced type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities toward P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids toward these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.

  2. Suicide Inhibition of Cytochrome P450 Enzymes by Cyclopropylamines via a Ring-Opening Mechanism: Proton-Coupled Electron Transfer Makes a Difference

    PubMed Central

    Zhang, Xiaoqian; Li, Xiao-Xi; Liu, Yufang; Wang, Yong

    2017-01-01

    N-benzyl-N-cyclopropylamine (BCA) has been attracting great interests for decades for its partial suicide inactivation role to cytochrome P450 (P450) via a ring-opening mechanism besides acting as a role of normal substrates. Understanding the mechanism of such partial inactivation is vital to the clinical drug design. Thus, density functional theoretical (DFT) calculations were carried out on such P450-catalyzed reactions, not only on the metabolic pathway, but on the ring-opening inactivation one. Our theoretical results demonstrated that, in the metabolic pathway, besides the normal carbinolamine, an unexpected enamine was formed via the dual hydrogen abstraction (DHA) process, in which the competition between rotation of the H-abstracted substrate radical and the rotation of hydroxyl group of the protonated Cpd II moiety plays a significant role in product branch; In the inactivation pathway, the well-noted single electron transfer (SET) mechanism-involved process was invalidated for its high energy barrier, a proton-coupled electron transfer [PCET(ET)] mechanism plays a role. Our results are consistent with other related theoretical works on heteroatom-hydrogen (X-H, X = O, N) activation and revealed new features. The revealed mechanisms will play a positive role in relative drug design. PMID:28197402

  3. Suicide Inhibition of Cytochrome P450 Enzymes by Cyclopropylamines via a Ring-opening Mechanism: Proton-Coupled Electron Transfer Makes a Difference

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaoqian; Li, Xiao-Xi; Liu, Yufang; Wang, Yong

    2017-01-01

    N-benzyl-N-cyclopropylamine (BCA) has been attracting great interests for decades for its partial suicide inactivation role to cytochrome P450 (P450) via a ring-opening mechanism besides acting as a role of normal substrates. Understanding the mechanism of such partial inactivation is vital to the clinical drug design. Thus, density functional theoretical (DFT) calculations were carried out on such P450-catalyzed reactions, not only on the metabolic pathway, but on the ring-opening inactivation one. Our theoretical results demonstrated that, in the metabolic pathway, besides the normal carbinolamine, an unexpected enamine was formed via the dual hydrogen abstraction (DHA) process, in which the competition between rotation of the H-abstracted substrate radical and the rotation of hydroxyl group of the protonated Cpd II moiety plays a significant role in product branch; In the inactivation pathway, the well-noted single electron transfer (SET) mechanism-involved process was invalidated for its high energy barrier, a proton-coupled electron transfer (PCET(ET)) mechanism plays a role. Our results are consistent with other related theoretical works on heteroatom-hydrogen (X-H, X = O, N) activation and revealed new features. The revealed mechanisms will play a positive role in relative drug design.

  4. Suicide Inhibition of Cytochrome P450 Enzymes by Cyclopropylamines via a Ring-Opening Mechanism: Proton-Coupled Electron Transfer Makes a Difference.

    PubMed

    Zhang, Xiaoqian; Li, Xiao-Xi; Liu, Yufang; Wang, Yong

    2017-01-01

    N-benzyl-N-cyclopropylamine (BCA) has been attracting great interests for decades for its partial suicide inactivation role to cytochrome P450 (P450) via a ring-opening mechanism besides acting as a role of normal substrates. Understanding the mechanism of such partial inactivation is vital to the clinical drug design. Thus, density functional theoretical (DFT) calculations were carried out on such P450-catalyzed reactions, not only on the metabolic pathway, but on the ring-opening inactivation one. Our theoretical results demonstrated that, in the metabolic pathway, besides the normal carbinolamine, an unexpected enamine was formed via the dual hydrogen abstraction (DHA) process, in which the competition between rotation of the H-abstracted substrate radical and the rotation of hydroxyl group of the protonated Cpd II moiety plays a significant role in product branch; In the inactivation pathway, the well-noted single electron transfer (SET) mechanism-involved process was invalidated for its high energy barrier, a proton-coupled electron transfer [PCET(ET)] mechanism plays a role. Our results are consistent with other related theoretical works on heteroatom-hydrogen (X-H, X = O, N) activation and revealed new features. The revealed mechanisms will play a positive role in relative drug design.

  5. Modulation of cytochromes P450 with xanthone-based molecules: from aromatase to aldosterone synthase and steroid 11β-hydroxylase inhibition.

    PubMed

    Gobbi, Silvia; Hu, Qingzhong; Negri, Matthias; Zimmer, Christina; Belluti, Federica; Rampa, Angela; Hartmann, Rolf W; Bisi, Alessandra

    2013-02-28

    Imidazolylmethylflavones previously reported by us as aromatase inhibitors proved to be able to interact with aldosterone synthase (CYP11B2), a cytochrome P450 enzyme involved in the biosynthesis of the mineralcorticoid hormone aldosterone, and were used to obtain a pharmacophore model for this enzyme. Here, in the search for potential ligands for CYP11B2 and the related CYP11B1, a virtual screening of a small compounds library of our earlier synthesized aromatase inhibitors was performed and, according to the results and the corresponding biological data, led to the design and synthesis of a series of xanthones derivatives carrying an imidazolylmethyl substituent in position 1 and different substituents in position 4. Some very potent inhibitors were obtained; in particular, the 4-chlorine derivative was active in the low nanomolar or subnanomolar range on CYP11B2 and CYP11B1, respectively, proving that xanthone can be considered as an excellent scaffold, whose activity can be directed to different targets when appropriately functionalized.

  6. Kinetics and selectivity of mechanism-based inhibition of guinea pig hepatic and pulmonary cytochrome P450 by N-benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole.

    PubMed

    Sinal, C J; Bend, J R

    1996-09-01

    The time dependence for mechanism-based inactivation of cytochrome P450 (P450)-dependent 7-pentoxyresorufin O-depentylation (PROD), 7-ethoxyresorufin O-deethylation (EROD), and 7-methoxyresorufin O-demethylation (MROD) activities by N-benzyl-1-aminobenzotriazole (BBT) and N-alpha-methylbenzyl-1-aminobenzotriazole (alpha MB) was investigated in hepatic and pulmonary microsomes from phenobarbital-treated guinea pigs. In the presence of NADPH, both compounds inhibited P450-dependent catalytic activity in a time- and concentration-dependent manner. Inactivation of hepatic PROD activity was more rapid (t1/2 = 13.2 vs. 155 min) for 0.1 microM alpha MB when compared with equimolar BBT. On the other hand, hepatic EROD inactivation was more rapid (t1/2 = 8.1 vs. 11 min) with 0.1 microM BBT, compared with equimolar alpha MB. Inactivation of pulmonary PROD activity was the most rapid and potent, with an apparent half-life for inactivation of t1/2 = 0.94 and 32.2 min for 0.025 microM alpha MB and BBT, respectively. Incubation of hepatic microsomes for 45 min in the presence of NADPH and 10 microM BBT or alpha MB resulted in > 90% inhibition of PROD, EROD, and MROD activities. After washing by repeated sedimentation and resuspension, inhibition of PROD (78%; 93% for BBT and alpha MB, respectively), EROD (80% and 50%), and MROD (15% and 3%) activities was reversed to varying degrees. We conclude that BBT and alpha MB are rapidly metabolized to products that inhibit individual P450 isozymes by both mechanism-based (P4502B and P4501A1) and reversible (P4501A2) mechanisms. Of the two inhibitors, alpha MB is relatively more potent and selective for guinea pig lung P4502B isozyme(s).

  7. Ferulic acid attenuated acetaminophen-induced hepatotoxicity though down-regulating the cytochrome P 2E1 and inhibiting toll-like receptor 4 signaling-mediated inflammation in mice

    PubMed Central

    Yuan, Junhui; Ge, Kuang; Mu, Junhuan; Rong, Jiang; Zhang, Li; Wang, Bin; Wan, Jingyuan; Xia, Gong

    2016-01-01

    Ferulic acid (FA), a phenolic acid which is abundant in vegetables and fruits, has been reported to exert anti-oxidative and anti-inflammatory activities. In the present study, the pharmacological effects and the underlying mechanisms of FA in mice with acetaminophen-induced hepatotoxicity were investigated. Our results revealed that FA pretreatment inhibited the augments of serum aminotransferases in a dose-dependent manner and attenuated the hepatic histopathological abnormalities and hepatocellular apoptosis in acetaminophen (APAP) exposed mice. Moreover, FA inhibited the expression of cytochrome P450 2E1 (CYP2E1), enhanced the activities of superoxide dismutase (SOD) and catalase (CAT) as well as the contents of glutathione (GSH). Furthermore, FA markedly attenuated acetaminophen-induced serum tumor necrosis factor (TNF)-α and interleukin (IL)-1β production, suppressed Toll-like receptor (TLR) 4 expression and dampened p38 mitogen-activated (MAPK) and nuclear factor kappa (NF-κB) activation. These data suggested that FA could effectively protect against APAP-induced liver injury by down-regulated expression of CYP 2E1 and the suppression of TLR4-mediated inflammatory responses. PMID:27830004

  8. Influence of polyhalogenated aromatic hydrocarbons on the induction, activity, and stabilization of cytochrome P450

    SciTech Connect

    Voorman, R.

    1987-01-01

    In the course of experiments evaluating the metabolism of polybrominated biphenyls by cytochrome P450 isozymes induced by 3,4,5,3',4',5'-hexabromobiphenyl (HBB), it was discovered that the inducer remained closely associated with cytochrome P450d. Subsequent purification of cytochromes from HBB treated rates revealed a 0.5:1 association of HBB to cytochrome P450d but virtually none with cytochrome P450c or cytochrome b5. Immunochemical quantitation of cytochrome P450d in the same microsomes yielded a ratio of P450d:HBB that approached unity. Measurement of cytochrome P450d estradiol 2-hydroxylase indicated non-competitive or mixed type inhibition caused by HBB at a concentration of 10-1000 nM. Inhibition was specific to cytochrome P450d since estradiol 2-hydroxylase catalyzed by cytochrome P450h was unaffected by HBB. The ability of HCB and isosafrole to stabilize cytochrome P450d, and thus indirectly influence regulation of the enzyme, was evaluated by treating rats with a dose of TCDD sufficient to produce maximum induction of cytochromes P450c and P450d via the Ah receptor, yet insufficient to bind to the enzyme. Subsequent treatment of these animals with HCB or isosafrole and a radiolabeled amino acid, revealed a significant increase in cytochrome P450d specific content relative to cytochrome P450c and significant retention of the radiolabel in P450d relative to rats treated only with TCDD.

  9. Inhibition of cytochrome P450 3A by clarithromycin uniformly affects the pharmacokinetics and pharmacodynamics of oxycodone in young and elderly volunteers.

    PubMed

    Liukas, Antti; Hagelberg, Nora M; Kuusniemi, Kristiina; Neuvonen, Pertti J; Olkkola, Klaus T

    2011-06-01

    The aim of this study was to investigate the effect of the cytochrome P450 3A4 inhibitor clarithromycin on the pharmacokinetics and pharmacodynamics of oral oxycodone in young and elderly subjects. Ten young and 10 elderly healthy subjects participated in this placebo-controlled, randomized, 2-phase crossover study. Subjects took clarithromycin 500 mg or placebo twice daily for 5 days. On day 4, subjects ingested an oral dose of 10 mg oxycodone. Plasma concentrations of oxycodone and its oxidative metabolites were measured for 48 hours, and pharmacological response for 12 hours. Clarithromycin decreased the apparent clearance of oxycodone by 53% in young and 48% in elderly subjects (P < 0.001) and prolonged its elimination half-life. The mean area under the plasma concentration-time curve (AUC0-∞) of oxycodone was increased by 2.0-fold (range, 1.3-fold to 2.7-fold) (P < 0.001) in young and 2.3-fold (range, 1.1-fold to 3.8-fold) (P < 0.001) in elderly subjects. The formation of noroxycodone was decreased by 74% in young and 71% in elderly subjects (P < 0.001). The ratio of AUC0-∞ of oxycodone during the clarithromycin phase compared with the one with placebo did not differ between the age groups. Clarithromycin did not alter the pharmacological response to oxycodone. Clarithromycin increased the exposure to oral oxycodone, but the magnitude of this effect was not age related. Although the pharmacological response to oxycodone was not significantly influenced by clarithromycin, dose reductions may be necessary in the most sensitive patients to avoid adverse effects when oxycodone is used concomitantly with clarithromycin.

  10. Inhibitive effect of cremophor RH40 or tween 80-based self-microemulsiflying drug delivery system on cytochrome P450 3A enzymes in murine hepatocytes.

    PubMed

    Rao, Zichao; Si, Luqin; Guan, Yanbin; Pan, Hongping; Qiu, Jun; Li, Gao

    2010-10-01

    This study examined the effect of self-microemulsiflying drug delivery system (SMEDDS) containing Cremophor RH40 or Tween 80 at various dilutions on cytochrome P450 3A (CYP3A) enzymes in rat hepatocytes, with midazolam serving as a CYP3A substrate. The particle size and zeta potential of microemulsions were evaluated upon dilution with aqueous medium. In vitro release was detected by a dialysis method in reverse. The effects of SMEDDS at different dilutions and surfactants at different concentrations on the metabolism of MDZ were investigated in murine hepatocytes. The cytotoxicity of SMEDDS at different dilutions was measured by LDH release and MTT technique. The effects of SMEDDS on the CYP3A enzymes activity were determined by Western blotting. Our results showed that dilution had less effect on the particle size and zeta potential in the range from 1:25 to 1:500. The MDZ was completely released in 10 h. A significant decrease in the formation of 1'-OH-MDZ in rat hepatocytes was observed after treatment with both SMEDDS at dilutions ranging from 1:50 to 1:250 and Cremophor RH 40 or Tween 80 at concentrations ranging from 0.1% to 1% (w/v), with no cytotoxicity observed. A significant decrease in CYP3A protein expression was observed in cells by Western blotting in the presence of either Cremophor RH40 or Tween 80-based SMEDDS at the dilutions ranging from 1:50 to 1:250. This study suggested that the excipient inhibitor-based formulation is a potential protective platform for decreasing metabolism of sensitive drugs that are CYP3A substrates.

  11. Canine cytochrome P-450 pharmacogenetics.

    PubMed

    Court, Michael H

    2013-09-01

    The cytochrome P-450 (CYP) drug metabolizing enzymes are essential for the efficient elimination of many clinically used drugs. These enzymes typically display high interindividual variability in expression and function resulting from enzyme induction, inhibition, and genetic polymorphism thereby predisposing patients to adverse drug reactions or therapeutic failure. There are also substantial species differences in CYP substrate specificity and expression that complicate direct extrapolation of information from humans to veterinary species. This article reviews the available published data regarding the presence and impact of genetic polymorphisms on CYP-dependent drug metabolism in dogs in the context of known human-dog CYP differences. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Ketoconazole inhibition of the bifunctional cytochrome P450c17 does not affect androgen formation from the endogenous lyase substrate. The catalytic site remains refractory in the course of intermediary hydroxyprogesterone processing.

    PubMed

    Kühn-Velten, W N; Lessmann, M

    1992-12-15

    The inhibition of the bifunctional steroidogenic cytochrome P450c17 (CYP17: steroid-17 alpha-hydroxylase/steroid-17,20-lyase) by the imidazole-type fungicide, [(+/-)-cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl- methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) (ketoconazole), was investigated with the aim of differentiating between effects on androgen formation from exogenously added and endogenously produced 17 alpha-hydroxyprogesterone. Using microsomal membranes from rat testis, turnover of progesterone by P450c17 was competitively inhibited by ketoconazole with KI = 0.40 microM. Ketoconazole did not affect the linear relationship between the ratio of productive events (corresponding to androgen formation rates) versus abortive events (corresponding to 17 alpha-hydroxyprogesterone formation rates) and the sum of catalytic events. This was an indication that this inhibitor did not interfere with intermediate processing by P450c17. Androgen formation from exogenous but not from endogenous 17 alpha-hydroxyprogesterone was competitively inhibited by ketoconazole. The simultaneous conversion of 1 microM each of [3H]progesterone and 17 alpha-hydroxy[14C]progesterone was also reduced by ketoconazole. Calculation of 3H/14C ratios in the 17 alpha-hydroxyprogesterone and androgen fractions revealed that the endogenous 17 alpha-hydroxyprogesterone pool was metabolized to androgens at rates 6.4, 11.6, 17.6 and 21.2-fold faster than the exogenous pool in the presence of 0.5, 1, 2 and 4 microM ketoconazole, respectively; this value was only 4.0 in controls. It is concluded that ketoconazole inhibits turnover of steroid ligands only when they approach the P450c17 active site in a substrate-state and that inhibition of androgen formation from progesterone is due to inhibition of the first catalytic step only. A model is described in which the P450c17 active site is refractory towards ketoconazole when the intermediary steroid is retained and being

  13. Inhibition of P-glycoprotein, multidrug resistance-associated protein 2 and cytochrome P450 3A4 improves the oral absorption of octreotide in rats with portal hypertension.

    PubMed

    Sun, Xiao-Yu; Duan, Zhi-Jun; Liu, Zhen; Tang, Shun-Xiong; Li, Yang; He, Shou-Cheng; Wang, Qiu-Ming; Chang, Qing-Yong

    2016-12-01

    The aim of the present study was to increase the intestinal transport of octreotide (OCT) by targeting the first-pass impact to identify a potential method for decreasing portal vein pressure (PVP) using oral OCT. Thus, the bioavailability of intestinally absorbed OCT was evaluated in normal rats and rats with portal hypertension (PH) that had been administered P-glycoprotein/multidrug resistance-associated protein 2/cytochrome P450 3A4 (P-gp/MRP2/CYP3A4) inhibitors. The mRNA and protein expression levels of P-gp, MRP2 and CYP3A4 were evaluated in normal and PH rats with or without OCT and the inhibitors using RT-PCR, western blot and immunohistochemical analyses. The potential effects of the inhibitor administration on PVP were also examined. The results suggest that P-gp, MRP2 and CYP3A4 play important roles in prohibiting the enteral absorption of OCT, particularly under a PH environment. Moreover, inhibitors of P-gp, MRP2 and CYP3A4 decrease the first-pass effects of OCT and effectively reduce PVP under PH conditions. Therefore, the present results suggest P-gp, MRP2 and CYP3A4 are key factors in the intestinal absorption of OCT. The inhibition of P-gp, MRP2 and CYP3A4 can markedly decrease the first-pass effects of OCT, and their use may facilitate the use of orally administered OCT.

  14. Inhibition of P-glycoprotein, multidrug resistance-associated protein 2 and cytochrome P450 3A4 improves the oral absorption of octreotide in rats with portal hypertension

    PubMed Central

    Sun, Xiao-Yu; Duan, Zhi-Jun; Liu, Zhen; Tang, Shun-Xiong; Li, Yang; He, Shou-Cheng; Wang, Qiu-Ming; Chang, Qing-Yong

    2016-01-01

    The aim of the present study was to increase the intestinal transport of octreotide (OCT) by targeting the first-pass impact to identify a potential method for decreasing portal vein pressure (PVP) using oral OCT. Thus, the bioavailability of intestinally absorbed OCT was evaluated in normal rats and rats with portal hypertension (PH) that had been administered P-glycoprotein/multidrug resistance-associated protein 2/cytochrome P450 3A4 (P-gp/MRP2/CYP3A4) inhibitors. The mRNA and protein expression levels of P-gp, MRP2 and CYP3A4 were evaluated in normal and PH rats with or without OCT and the inhibitors using RT-PCR, western blot and immunohistochemical analyses. The potential effects of the inhibitor administration on PVP were also examined. The results suggest that P-gp, MRP2 and CYP3A4 play important roles in prohibiting the enteral absorption of OCT, particularly under a PH environment. Moreover, inhibitors of P-gp, MRP2 and CYP3A4 decrease the first-pass effects of OCT and effectively reduce PVP under PH conditions. Therefore, the present results suggest P-gp, MRP2 and CYP3A4 are key factors in the intestinal absorption of OCT. The inhibition of P-gp, MRP2 and CYP3A4 can markedly decrease the first-pass effects of OCT, and their use may facilitate the use of orally administered OCT. PMID:28105103

  15. Binding of Diverse Environmental Chemicals with Human Cytochromes P450 2A13, 2A6, and 1B1 and Enzyme Inhibition

    PubMed Central

    Shimada, Tsutomu; Kim, Donghak; Murayama, Norie; Tanaka, Katsuhiro; Takenaka, Shigeo; Nagy, Leslie D.; Folkman, Lindsay M.; Foroozesh, Maryam K.; Komori, Masayuki; Yamazaki, Hiroshi; Guengerich, F. Peter

    2014-01-01

    A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e. the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2’,5’-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2’-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2’-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family

  16. Binding of diverse environmental chemicals with human cytochromes P450 2A13, 2A6, and 1B1 and enzyme inhibition.

    PubMed

    Shimada, Tsutomu; Kim, Donghak; Murayama, Norie; Tanaka, Katsuhiro; Takenaka, Shigeo; Nagy, Leslie D; Folkman, Lindsay M; Foroozesh, Maryam K; Komori, Masayuki; Yamazaki, Hiroshi; Guengerich, F Peter

    2013-04-15

    A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e., the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2',5'-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2'-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2'-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family 1 P450

  17. In vitro metabolism of N'-Nitrosonornicotine catalyzed by cytochrome P450 2A13 and its inhibition by nicotine, N'-Nitrosoanatabine and N'-Nitrosoanabasine.

    PubMed

    Liu, Xingyu; Zhang, Jie; Wang, Limeng; Yang, Bicheng; Zhang, Chen; Liu, Wei; Zhou, Jun

    2016-12-25

    N'-Nitrosonornicotine (NNN) is considered to be one of the most carcinogenic compounds of the four conventionally measured tobacco-specific N-nitrosamines (TSNAs). In order to evaluate the significance of metabolic activation for the carcinogenic potential of NNN, its catalysis by different phase I enzymes and its interaction with nicotine and nicotine-derived TSNAs need to be investigated. Using an in vitro model system, NNN was found to interact with various cytochrome P450 enzymes, predominantly CYP2A13. Mass-spectrometric analysis confirmed the presence of various predicted NNN metabolites, including 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and 4-hydroxy-4-(3-pyridyl)-butyric acid (hydroxy acid) but little amount of 4-oxo-4-(3-pyridyl) butanal (OPB), which was somewhat different from in vitro NNK metabolism. Addition of nicotine, N'-Nitrosoanatabine (NAT), N'-Nitrosoanabasine (NAB) resulted in a competitive inhibition for NNN metabolism. The inhibition constant Ki value was calculated as 0.98 μM (nicotine), 1.37 μM (NAT), 0.71 μM (NAB) for HPB formation, 1.35 μM (nicotine), 1.35 μM (NAT), 1.01 μM (NAB) for hydroxy acid formation and 8.40 μM (nicotine), 3.40 μM (NAT), 3.04 μM (NAB) for OPB formation, respectively. These results implied that CYP2A13 is the most efficient enzyme to metabolize NNN in vitro and structurally similar tobacco constitutes including nicotine, NAT and NAB influence the metabolic activation of NNN, which may further interfere in its carcinogenicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. The cytochrome bd respiratory oxygen reductases.

    PubMed

    Borisov, Vitaliy B; Gennis, Robert B; Hemp, James; Verkhovsky, Michael I

    2011-11-01

    Cytochrome bd is a respiratory quinol: O₂ oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O₂ and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O₂-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O₂, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. 2011 Elsevier B.V. All rights reserved.

  19. The cytochrome bd respiratory oxygen reductases

    PubMed Central

    Borisov, Vitaliy B.; Gennis, Robert B.; Hemp, James; Verkhovsky, Michael I.

    2011-01-01

    Summary Cytochrome bd is a respiratory quinol:O2 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O2 and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O2-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O2, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. PMID:21756872

  20. Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

    PubMed

    Ponnamperuma, K; Croteau, R

    1996-05-01

    Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

  1. The role of cytochrome P4501A activity inhibition in three- to five-ringed polycyclic aromatic hydrocarbons embryotoxicity of marine medaka (Oryzias melastigma).

    PubMed

    Mu, Jing-li; Wang, Xin-hong; Jin, Fei; Wang, Ju-ying; Hong, Hua-sheng

    2012-07-01

    The mode of action of PAHs that causes fish developmental malformations is unclear. The embryotoxicity of marine medaka (Oryzias melastigma) was investigated after individual exposure to three- to five-ring PAHs Phe, Py, and BaP or co-exposure with α-ANF for 18 days. We found that the relationships between EROD induction and developmental deformities of embryos showed a various pattern under different exposure scenarios of Phe, Py, and BaP, which suggested possibly different modes of action in determining the developmental toxicities. As for co-exposure scenarios of each PAH combined with ANF, it showed potentially synergistic effects. The inhibited CYP1A mediated enzyme activity by ANF after co-exposure did not effectively alleviate developmental toxicity of embryo. It showed potentially synergistic effects after co-exposure of marine fish embryos to CYP1A inhibitors and PAH-type CYP1A inducers. Heart deformities in the early life stages of marine medaka were recommended as a biomarker for indicating the extent of PAH pollution.

  2. Reverse type I binding spectra of human cytochrome P450 1B1 induced by flavonoid, stilbene, pyrene, naphthalene, phenanthrene, and biphenyl derivatives that inhibit catalytic activity: a structure-function relationship study.

    PubMed

    Shimada, Tsutomu; Tanaka, Katsuhiro; Takenaka, Shigeo; Foroozesh, Maryam K; Murayama, Norie; Yamazaki, Hiroshi; Guengerich, F Peter; Komori, Masayuki

    2009-07-01

    Fifty-one chemicals including derivatives of 16 flavonoids, three stilbenes, six pyrenes, seven naphthalenes, seven phenanthrenes, 10 biphenyls, 17beta-estradiol, and estrone were examined for their abilities to induce reverse type I binding spectra with human cytochrome P450 (P450) 1B1 and to inhibit 7-ethoxyresorufin O-deethylation (EROD) activities catalyzed by P450 1B1. Forty-nine chemicals showed reverse type I spectra with P450 1B1, and we found that 3,5,7-trihydroxyflavone, 3',4'-dimethoxy-5,7-dihydroxyflavone, 4'-methoxy-5,7-dihydroxyflavone, alpha- and beta-naphthoflavones, 2,4,3',5'-tetramethoxystilbene, pyrene, and several acetylenic pyrenes and phenanthrenes were strong inducers of the spectra and also potent inhibitors of EROD activities catalyzed by P450 1B1. The spectral dissociation constant (K(s)) and the magnitude of the binding (DeltaA(max)/K(s)) of 49 chemicals were correlated with the inhibition potencies of EROD activities by these chemicals [correlation coefficients (r) of 0.72 and 0.74, respectively]. The K(s) and DeltaA(max)/K(s) values were more correlated with IC(50) values when compared in a group of derivatives of flavonoids, stilbenes, and estrogens (r = 0.81 and 0.88, respectively) or a group of derivatives of pyrenes, naphthalenes, phenanthrenes, and biphenyls (r = 0.88 and 0.91, respectively). Among 14 flavonoids examined, 3,5,7-trihydroxyflavone and 4'-methoxy- and 3',4'-dimethoxy-5,7-dihydroxyflavone were more active than flavone in interacting with P450 1B1, but the respective 7,8-dihydroxyflavones were less active. Pyrene itself was highly active in interacting with P450 1B1, but its binding was slightly decreased when substituted with acetylenic groups. In contrast, substitution of naphthalene with methyl and ethyl propargyl ethers led to more interaction with P450 1B1 than with naphthalene itself. Similarly, substitution on phenanthrene or biphenyl with acetylenic groups and propargyl ethers increased affinities to P450 1B1

  3. Reverse Type I Binding Spectra of Human Cytochrome P450 1B1 Induced by Flavonoid, Stilbene, Pyrene, Naphthalene, Phenanthrene, and Biphenyl Derivatives That Inhibit Catalytic Activity: A Structure-Function Relationship Study

    PubMed Central

    Shimada, Tsutomu; Tanaka, Katsuhiro; Takenaka, Shigeo; Foroozesh, Maryam K.; Murayama, Norie; Yamazaki, Hiroshi; Guengerich, F. Peter; Komori, Masayuki

    2013-01-01

    Fifty-one chemicals including derivatives of sixteen flavonoids, three stilbenes, six pyrenes, seven naphthalenes, seven phenanthrenes, ten biphenyls, 17β-estradiol, and estrone were examined for their abilities to induce Reverse Type I binding spectra with human cytochrome P450 (P450) 1B1 and to inhibit 7-ethoxyresorufin O-deethylation (EROD) activities catalyzed by P450 1B1. Forty nine chemicals showed Reverse Type I spectra with P450 1B1 and we found that 3,5,7-trihydroxyflavone, 3′,4′-dimethoxy-5,7-dihydroxyflavone, 4′-methoxy-5,7-dihydroxyflavone, α- and β-naphthoflavones, 2,4,3′,5′-tetramethoxystilbene, pyrene, and several acetylenic pyrenes and phenanthrenes were strong inducers of the spectra and also potent inhibitors of EROD activities catalyzed by P450 1B1. Spectral dissociation constant (Ks) and the magnitude of the binding (ΔAmax/Ks) of 49 chemicals were correlated with the inhibition potencies of EROD activities by these chemicals (correlation coefficients (r) of 0.72 and 0.74, respectively). The Ks and ΔAmax/Ks values were more correlated with IC50 values when compared in a group of derivatives of flavonoids, stilbenes, and estrogens (r=0.81 and 0.88, respectively) or a group of derivatives of pyrenes, naphthalenes, phenanthrenes, and biphenyls (r=0.88 and 0.91, respectively). Among 14 flavonoids examined, 3,5,7-trihydroxyflavone and 4′-methoxy- and 3′,4′-dimethoxy-5,7-dihydroxyflavone were more active than flavone in interacting with P450 1B1, but the respective 7,8-dihydroxyflavones were less active. Pyrene itself was highly active in interacting with P450 1B1, but its binding was slightly decreased when substituted with acetylenic groups. In contrast, substitution of naphthalene with methyl- and ethyl propargyl ethers led to more interaction with P450 1B1 than with naphthalene itself. Similarly, substitution on phenanthrene or biphenyl with acetylenic groups and propargyl ethers increased affinities to P450 1B1. These results

  4. 1-[2-(4-Benzyloxyphenoxy)Ethyl]Imidazole inhibits monoamine oxidase B and protects against neuronal loss and behavioral impairment in rodent models of Parkinson's disease.

    PubMed

    Chung, Jin Yong; Lee, Ji Won; Ryu, Choon Ho; Min, Hye Kyung; Yoon, Yeo Jin; Lim, Mi Jung; Park, Cheol Hyoung

    2015-08-01

    Monoamine oxidase B (MAO-B) is well known as a therapeutic target for Parkinson's disease (PD). MAO-B inhibitors retain antiparkinsonism abilities to improve motor function and prevent neuronal loss by decreasing dopamine metabolism and oxidative stress in the brain. From the study to find novel antiparkinsonism drugs that can inhibit MAO-B activity, neuronal loss, and behavioral deficits in the mouse model of PD, we identified that 1-[2-(4-benzyloxyphenoxy)ethyl]imidazole (BPEI) or safinamide strongly and selectively inhibited MAO-B activities in a dose-dependent manner (IC50 of BPEI and safinamide for MAO-B were 0.016 and 0.0021 µM and for MAO-A were 70.0 and 370 µM, respectively). In ex vivo studies after an administration (30 mg/kg, i.p.) of BPEI or safinamide to normal mice, the MAO-B activity in the brain was reduced by up to 90.6% or 82.4% at 1.0 hr. BPEI (20 mg/kg, i.p.) or safinamide (20 mg/kg, i.p.) significantly reversed the behavioral impairments, dopamine levels in the striatum, and neuronal loss in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice compared with the MPTP-alone-treated group. In the 6-hydroxydopamine-induced PD rat model, behavioral improvement by levodopa sparing activity was observed in the BPEI- or safinamide-treated (20 mg/kg, i.p.) rats. Moreover, BPEI revealed additional curative activities for nonmotor symptoms of PD such as pain, anxiety, epilepsy, and depression in rodent disease models. Therefore, BPEI has broad therapeutic potential for treating motor symptoms via strong and selective inhibitory effects on MAO-B, with additional benefits for comorbid symptoms in PD. © 2015 Wiley Periodicals, Inc.

  5. Cytochrome c: functions beyond respiration.

    PubMed

    Ow, Yong-Ling P; Green, Douglas R; Hao, Zhenyue; Mak, Tak W

    2008-07-01

    Cytochrome c is primarily known for its function in the mitochondria as a key participant in the life-supporting function of ATP synthesis. However, when a cell receives an apoptotic stimulus, cytochrome c is released into the cytosol and triggers programmed cell death through apoptosis. The release of cytochrome c and cytochrome-c-mediated apoptosis are controlled by multiple layers of regulation, the most prominent players being members of the B-cell lymphoma protein-2 (BCL2) family. As well as its role in canonical intrinsic apoptosis, cytochrome c amplifies signals that are generated by other apoptotic pathways and participates in certain non-apoptotic functions.

  6. In vitro antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone induced apoptosis against COLO320 cells through cytochrome c release caspase mediated pathway with PI3K/AKT and COX-2 inhibition.

    PubMed

    Balachandran, C; Emi, N; Arun, Y; Yamamoto, N; Duraipandiyan, V; Inaguma, Yoko; Okamoto, Akinao; Ignacimuthu, S; Al-Dhabi, N A; Perumal, P T

    2016-04-05

    The present study investigated the anticancer activity of 2,3-dihydroxy-9,10-anthraquinone against different cancer cells such as MCF-7, COLO320, HepG-2, Skov-3, MOLM-14, NB-4, CEM, K562, Jurkat, HL-60, U937, IM-9 and Vero. 2,3-dihydroxy-9,10-anthraquinone showed good antiproliferative activity against COLO320 cells when compared to other tested cells. The cytotoxicity results showed 79.8% activity at the dose of 2.07 μM with IC50 value of 0.13 μM at 24 h in COLO320 cells. So we chose COLO320 cells for further anticancer studies. mRNA expression was confirmed by qPCR analysis using SYBR green method. Treatment with 2,3-dihydroxy-9,10-anthraquinone was found to trigger intrinsic apoptotic pathway as indicated by down regulation of Bcl-2, Bcl-xl; up regulation of Bim, Bax, Bad; release of cytochrome c and pro-caspases cleaving to caspases. Furthermore, 2,3-dihydroxy-9,10-anthraquinone stopped at G0/G1 phase with modulation in protein levels of cyclins. On the other hand PI3K/AKT signaling plays an important role in cell metabolism. We found that 2,3-dihydroxy-9,10-anthraquinone inhibits PI3K/AKT activity after treatment. Also, COX-2 enzyme plays a major role in colorectal cancer. Our results showed that the treatment significantly reduced COX-2 enzyme in COLO320 cells. These results indicated antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone involving apoptotic pathways, mitochondrial functions, cell cycle checkpoint and controlling the over expression genes during the colorectal cancer. Molecular docking studies showed that the compound bound stably to the active sites of Bcl-2, COX-2, PI3K and AKT. This is the first report of anticancer mechanism involving 2,3-dihydroxy-9,10-anthraquinone in COLO320 cells. The present results might provide helpful suggestions for the design of antitumor drugs toward colorectal cancer treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Preventive effect of rikkunshito on gastric motor function inhibited by L-dopa in rats.

    PubMed

    Wang, Lixin; Mogami, Sachiko; Karasawa, Hiroshi; Yamada, Chihiro; Yakabi, Seiichi; Yakabi, Koji; Hattori, Tomohisa; Taché, Yvette

    2014-05-01

    We previously reported that ghrelin prevented l-dopa (LD)-induced inhibition of gastric emptying (GE) of a non-nutrient solution in rats. Parkinson's disease treatment involves the combined administration of l-dopa with the enzyme l-amino acid decarboxylase inhibitor, carbidopa (CD) to reduce peripheral formation of dopamine. We investigated the effect LD/CD given orogastrically (og) on GE of a non-nutrient or nutrient meal and whether og pretreatment with rikkunshito, a kampo medicine clinically used to treat gastroparesis, influenced LD/CD effect on GE and postprandial antral and duodenal motility in conscious rats. LD/CD (20/2 mgkg(-1)) decreased significantly GE to 26.3 ± 6.0% compared to 61.2 ± 3.2% in og vehicle monitored 20-min after a non-nutrient meal and to 41.9 ± 5.8% compared to 72.9 ± 5.2% in og vehicle monitored 60 min after a nutrient meal. Rikkunshito (0.5 or 1.0 g kg(-1)) reduced the LD/CD (20/2 mg kg(-1)) inhibition of GE of non-nutrient meal (36.9 ± 7.4% and 46.6 ± 4.8% respectively vs. 12.1 ± 7.4% in og vehicle plus LD/CD) while having no effect alone (56.6 ± 8.5%). The ghrelin antagonist, [d-Lys(3)]-GHRP-6 (1 mg kg(-1)) injected intraperitoneally partially reversed rikkunshito preventive effect on LD/CD-inhibited GE. Rikkunshito (1.0 g kg(-1)) blocked LD/CD (20/2 mg kg(-1))-induced delayed GE of a nutrient meal and the reduction of postprandial antral motility. In 6-hydroxydopamine-induced Parkinson's disease rat model, rikkunshito (1.0 g kg(-1), og) also prevented LD/CD-inhibited gastric emptying of a nutrient meal and enhanced fasting plasma levels of acylated ghrelin. These data indicate that oral rikkunshito alleviates the delayed GE induced by LD/CD in naïve and PD rat model in part through ghrelin-related mechanisms.

  8. Simulation of multihaem cytochromes.

    PubMed

    Soares, Cláudio M; Baptista, António M

    2012-03-09

    This article presents an overview of the simulation studies of the behaviour of multihaem cytochromes using theoretical/computational methodologies, with an emphasis on cytochrome c(3). It starts with the first studies using rigid molecules and continuum electrostatic models, where protonation and redox events were treated as independent. The gradual addition of physical details is then described, from the inclusion of proton isomerism, to the proper treatment of the thermodynamics of electron-proton coupling, to the explicit inclusion of the solvent and protein structural reorganization into the models, culminating with the method for molecular dynamics simulations at constant pH and reduction potential, where the solvation, conformational, protonation and redox features are all simulated in a fully integrated and coupled way. We end with a discussion of the strategies used to study the interaction between multihaem cytochromes, taking into account the further coupling effect introduced by the molecular association. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Interactions of Avocado (Persea americana) Cytochrome P-450 with Monoterpenoids

    PubMed Central

    Hallahan, David L.; Nugent, Jonathan H. A.; Hallahan, Beverly J.; Dawson, Glenn W.; Smiley, Diane W.; West, Jevon M.; Wallsgrove, Roger M.

    1992-01-01

    The microsomal fraction of avocado (Persea americana) mesocarp is a rich source of cytochrome P-450 active in the demethylation of xenobiotics. Cytochrome P-450 from this tissue has been purified and well characterized at the molecular level (DP O'Keefe, KJ Leto [1989] Plant Physiol 89: 1141-1149; KR Bozak, H Yu, R Sirevag, RE Christoffersen [1990] Proc Natl Acad Sci USA 87: 3904-3908). Despite this extensive characterization, the role of the enzyme in vivo was not established. Optical and electron paramagnetic resonance binding studies described here suggest that the monoterpenoids, nerol and geraniol, are substrates of avocado cytochrome P-450 (spectral dissociation constant of 7.2 and 35 micromolar, respectively). Avocado microsomes have been shown to catalyze the hydroxylation of these monoterpenoids, and both nerol and geraniol have been shown to inhibit the activity of avocado cytochrome P-450 toward the artificial substrate 7-ethoxycoumarin, with nerol a competitive inhibitor of this activity. PMID:16668790

  10. Building Structure Feature-based Models for Predicting Isoform-specific Human Cytochrome P-450 (hCYP 3A4, 2D6 and 2C9) Inhibition Assay Results in ToxCast

    EPA Science Inventory

    EPA’s ToxCast project is using high-throughput screening (HTS) to profile and prioritize chemicals for further testing. ToxCast Phase I evaluated 309 unique chemicals, the majority pesticide actives, in over 500 HTS assays. These included 3 human cytochrome P450 (hCYP3A4, hCYP2...

  11. Building Structure Feature-based Models for Predicting Isoform-specific Human Cytochrome P-450 (hCYP 3A4, 2D6 and 2C9) Inhibition Assay Results in ToxCast

    EPA Science Inventory

    EPA’s ToxCast project is using high-throughput screening (HTS) to profile and prioritize chemicals for further testing. ToxCast Phase I evaluated 309 unique chemicals, the majority pesticide actives, in over 500 HTS assays. These included 3 human cytochrome P450 (hCYP3A4, hCYP2...

  12. Reduction of Hg2+ with reduced mammalian cytochrome c by cytochrome c oxidase purified from a mercury-resistant acidithiobacillus ferrooxidans strain, MON-1.

    PubMed

    Sugio, Tsuyoshi; Fujii, Mitsuko; Ninomiya, Yumika; Kanao, Tadayoshi; Negishi, Atsunori; Takeuchi, Fumiaki

    2008-07-01

    Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 muM and 5 muM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 muM, and 70% by 10 muM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.

  13. The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase.

    PubMed

    Bickar, D; Turrens, J F; Lehninger, A L

    1986-11-05

    When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two-electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c.

  14. Cytochrome C — EDRN Public Portal

    Cancer.gov

    CYCS, or cytochrome C, is an electron carrier protein that is an important part of the electron transport chain in mitochondria. The cytochrome C protein is a small heme protein that associates with the inner membrane of the mitochondrion where it accepts electrons from cytochrome b and transfers them to the cytochrome oxidase complex. Cytochrome C also plays a role in apoptosis.

  15. Alterations of BDNF and trkB mRNA Expression in the 6-Hydroxydopamine-Induced Model of Preclinical Stages of Parkinson’s Disease: An Influence of Chronic Pramipexole in Rats

    PubMed Central

    Berghauzen-Maciejewska, Klemencja; Wardas, Jadwiga; Kosmowska, Barbara; Głowacka, Urszula; Kuter, Katarzyna; Ossowska, Krystyna

    2015-01-01

    Our recent study has indicated that a moderate lesion of the mesostriatal and mesolimbic pathways in rats, modelling preclinical stages of Parkinson’s disease, induces a depressive-like behaviour which is reversed by chronic treatment with pramipexole. The purpose of the present study was to examine the role of brain derived neurotrophic factor (BDNF) signalling in the aforementioned model of depression. Therefore, we investigated the influence of 6-hydoxydopamine (6-OHDA) administration into the ventral region of the caudate-putamen on mRNA levels of BDNF and tropomyosin-related kinase B (trkB) receptor. The BDNF and trkB mRNA levels were determined in the nigrostriatal and limbic structures by in situ hybridization 2 weeks after the operation. Pramipexole (1 mg/kg sc twice a day) and imipramine (10 mg/kg ip once a day) were injected for 2 weeks. The lesion lowered the BDNF and trkB mRNA levels in the hippocampus [CA1, CA3 and dentate gyrus (DG)] and amygdala (basolateral/lateral) as well as the BDNF mRNA content in the habenula (medial/lateral). The lesion did not influence BDNF and trkB expression in the caudate-putamen, substantia nigra, nucleus accumbens (shell and core) and ventral tegmental area (VTA). Chronic imipramine reversed the lesion-induced decreases in BDNF mRNA in the DG. Chronic pramipexole increased BDNF mRNA, but decreased trkB mRNA in the VTA in lesioned rats. Furthermore, it reduced BDNF and trkB mRNA expression in the shell and core of the nucleus accumbens, BDNF mRNA in the amygdala and trkB mRNA in the caudate-putamen in these animals. The present study indicates that both the 6-OHDA-induced dopaminergic lesion and chronic pramipexole influence BDNF signalling in limbic structures, which may be related to their pro-depressive and antidepressant activity in rats, respectively. PMID:25739024

  16. The ameliorative effect of Monascus purpureus NTU 568-fermented rice extracts on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells and the rat model of Parkinson's disease.

    PubMed

    Tseng, Wei-Ting; Hsu, Ya-Wen; Pan, Tzu-Ming

    2016-02-01

    Oxidative stress and neuroinflammation underlie the major pathogenesis in Parkinson's disease (PD). Antioxidants are known to protect against the degeneration of dopaminergic neurons. Monascus purpureus-fermented rice, a traditional Chinese medicine as well as a health food, includes multifunctional metabolites. The present study was designed to investigate the effects of the antioxidant-containing M. purpureus NTU 568-fermented rice extract (extracted with 50% ethanol, so called R50E) in 6-hydrodopamine (6-OHDA)-induced neurotoxicity in vitro and in vivo. In vitro, treatment with R50E reduced 6-OHDA-induced SH-SY5Y cell death. In vivo, two doses of R50E (5.5 and 11.0 mg kg(-1)) were administered for a period of 28 days following 6-OHDA-induced lesioning. The administration of R50E reduced parkinsonian motor dysfunction and the number of tyrosine hydroxylase (TH)-immunoreactive neurons present in 6-OHDA-induced lesioned rats. Moreover, the administration of R50E reversed the elevation of reactive oxygen species (ROS) and malondialdehyde (MDA) levels and promoted the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione reductase, and glutathione peroxidase via down-regulation of p47 phox, NOX1, and NOX2 expression in the 6-OHDA-lesion rats. Furthermore, treatment with R50E attenuated nitric oxide (NO) and tumor necrosis factor (TNF-α) levels in the 6-OHDA-lesion rats. In conclusion, R50E may prevent neurodegeneration via anti-oxidative and anti-inflammatory mechanisms, suggesting its potential therapeutic value for PD treatment. This is the first study for evaluating the neuroprotective effects of red mold fermented products in PD models.

  17. Similar L-dopa-stimulated motor activity in mice with adult-onset 6-hydroxydopamine-induced symmetric dopamine denervation and in transcription factor Pitx3 null mice with perinatal-onset symmetric dopamine denervation.

    PubMed

    Li, Li; Sagot, Ben; Zhou, Fu-Ming

    2015-07-30

    The transcription factor Pitx3 null mutant (Pitx3Null) mice have a constitutive perinatal-onset and symmetric bilateral dopamine (DA) loss in the striatum. In these mice l-3,4-dihydroxyphenylalanine (l-dopa) induces apparently normal horizontal movements (walking) but also upward movements consisting of the vertical body trunk and waving paws that are absent in normal animals and in animals with the classic unilateral 6-hydroxydopamine (6-OHDA) lesion-induced DA denervation. Thus, a concern is that the perinatal timing of the DA loss and potential developmental abnormalities in Pitx3Null mice may underlie these upward movements, thus reducing the usefulness as a DA denervation model. Here we show that in normal wild-type (Pitx3WT) mice with adult-onset symmetric, bilateral 6-OHDA-induced DA lesion in the dorsal striatum, l-dopa induces normal horizontal movements and upward movements that are qualitatively identical to those in Pitx3Null mice. Furthermore, after unilateral 6-OHDA lesion of the residual DA innervation in the striatum in Pitx3Null mice, l-dopa induces contraversive rotation that is similar to that in Pitx3WT mice with the classic unilateral 6-OHDA lesion. These results indicate that in Pitx3Null mice, the bilateral symmetric DA denervation in the dorsal striatum is sufficient for expressing the l-dopa-induced motor phenotype and the perinatal timing of their DA loss is not a determining factor, providing further evidence that Pitx3Null mice are a convenient and suitable mouse model to study the consequences of DA loss and dopaminergic replacement therapy in Parkinson's disease.

  18. β-asarone and levodopa co-administration increase striatal dopamine level in 6-hydroxydopamine induced rats by modulating P-glycoprotein and tight junction proteins at the blood-brain barrier and promoting levodopa into the brain.

    PubMed

    Huang, Liping; Deng, Minzhen; He, Yuping; Lu, Shiyao; Ma, Ruanxin; Fang, Yongqi

    2016-06-01

    Levodopa (L-dopa) is widely considered as one of the most effective drug constituents in the treatment of Parkinson's disease (PD), but the blood-brain barrier (BBB) permeability of L-dopa is <5%, which causes low efficacy. Neuroprotective effects of β-asarone on 6-hydroxydopamine (6-OHDA)-induced PD rats were demonstrated by our previous studies. Co-administration of β-asarone and L-dopa has not been explored until being investigated on PD rats in this study. PD rats were divided into four groups: untreated, L-dopa-treated, β-asarone-treated and co-administered-treated groups. All of the treatments were administered to the rats twice per day for 30 days. The L-dopa, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), S100β and neuron-specific enolase (NSE) levels were subsequently determined. The P-glycoprotein (P-gp), zonula occludens-1 (ZO-1), claudin-5, occludin and actin expression was also assessed in cortex. Changes in BBB ultrastructure were observed using transmission electron microscopy. Our results showed that the co-administered treatment increased levels of L-dopa, DA, DOPAC and HVA in striatum, and S100β in plasma, but down-regulated NSE, P-gp, ZO-1, occludin, actin and claudin-5 in cortex. Crevices were observed between capillary endothelial cells at intercellular tight junction of the striatum in co-administered-treated group, while the endothelial cells in untreated group were tightly jointing each other. In addition, the correlations of L-dopa or DA and P-gp or tight junction proteins respectively were significantly negative in co-administered- and β-asarone-treated groups. These findings suggest that co-administered treatment may enhance the L-dopa BBB permeability and attenuate brain injury, which may be beneficial to PD treatment.

  19. Relationships among rat ultrasonic vocalizations, behavioral measures of striatal dopamine loss, and striatal tyrosine hydroxylase immunoreactivity at acute and chronic time points following unilateral 6-hydroxydopamine-induced dopamine depletion.

    PubMed

    Grant, Laura M; Barnett, David G; Doll, Emerald J; Leverson, Glen; Ciucci, Michelle

    2015-09-15

    Voice deficits in Parkinson disease (PD) emerge early in the disease process, but do not improve with standard treatments targeting dopamine. Experimental work in the rat shows that severe and chronic unilateral nigrostriatal dopamine depletion with 6-OHDA results in decreased intensity, bandwidth, and complexity of ultrasonic vocalizations. However, it is unclear if mild/acute dopamine depletion, paralleling earlier stages of PD, results in vocalization deficits, or to what degree vocalization parameters are correlated with other dopamine-dependent indicators of lesion severity or percent of tyrosine hydroxylase (%TH) loss. Here, we assayed ultrasonic vocalizations, forelimb asymmetry, and apomorphine rotations in rats with a range of unilateral dopamine loss resulting from 6-OHDA or vehicle control infusions to the medial forebrain bundle at acute (72 h) and chronic (4 weeks) time points post-infusion. The %TH loss was evaluated at 4 weeks. At 72 h, forelimb asymmetry and %TH loss were significantly correlated, while at 4 weeks, all measures of lesion severity were significantly correlated with each other. Call complexity was significantly correlated with all measures of lesion severity at 72 h but only with %TH loss at 4 weeks. Bandwidth was correlated with forelimb asymmetry at both time points. Duration was significantly correlated with all dopamine depletion measures at 4 weeks. Notably, not all parameters were affected universally or equally across time. These results suggest that vocalization deficits may be a sensitive index of acute and mild catecholamine loss and further underscores the need to characterize the neural mechanisms underlying vocal deficits in PD.

  20. Neuronal activity in the medial associative-limbic and lateral motor part of the rat subthalamic nucleus and the effect of 6-hydroxydopamine-induced lesions of the dorsolateral striatum.

    PubMed

    Lindemann, Christoph; Alam, Mesbah; Krauss, Joachim K; Schwabe, Kerstin

    2013-10-01

    Lesions of the rat nigrostriatal dopamine system by injection of 6-hydroxydopamine (6-OHDA) lead to abnormal neuronal activity in the basal ganglia (BG) motor loop similar to that found in Parkinson's disease (PD). In the BG motor loop the subthalamic nucleus (STN) represents an important structure, which, however, also comprises areas of the BG associative and limbic loops. We were interested whether neuronal activity would differ between the STN medial associative-limbic and lateral motor part, and whether selective 6-OHDA-induced lesions of the dorsolateral striatum, the entrance region of the BG motor loop, would differently affect these subregions. In male Sprague-Dawley rats 6-OHDA (n = 12) or vehicle (n = 10) was bilaterally injected in the dorsolateral striatum. Four weeks later extracellular single-unit activity and local field potentials were recorded in medial and lateral STN neurons of urethane-anesthetized rats. In sham-lesioned rats the discharge rate and burst activity were higher in the lateral compared to the medial STN. Similar differences were found for other neuronal activity measures (coefficient of variation of interspike interval, skewness, kurtosis, approximate entropy). After 6-OHDA injection neuronal burst activity was enhanced, while the discharge rate was not affected. In addition, in 6-OHDA-lesioned rats β-band oscillatory activity was enhanced, with no difference between STN subregions. We found important differences of neuronal activity between STN subregions, indicating functional segregation. However, selective 6-OHDA lesions of the dorsolateral striatum also had a pronounced effect on the medial STN subregion, indicating interaction between BG loops. © 2013 Wiley Periodicals, Inc.

  1. Effects of zingerone [4-(4-hydroxy-3-methoxyphenyl)-2-butanone] and eugenol [2-methoxy-4-(2-propenyl)phenol] on the pathological progress in the 6-hydroxydopamine-induced Parkinson's disease mouse model.

    PubMed

    Kabuto, Hideaki; Yamanushi, Tomoko T

    2011-12-01

    Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the nigrostriatal system and dopamine (DA) depletion in the striatum. The most popular therapeutic medicine for treating PD, 3-(3,4-Dihydroxyphenyl)-L-alanine (L-DOPA), has adverse effects, such as dyskinesia and disease acceleration. As superoxide (·O(2)(-)) and hydroxyl radical (·OH) have been implicated in the pathogenesis of PD, free radical scavenging and antioxidants have attracted attention as agents to prevent disease progression. Rodents injected with 6-hydroxydopamine (6-OHDA) intracerebroventricularly are considered to be a good animal model of PD. Zingerone and eugenol, essential oils extracted from ginger and cloves, are known to have free radical scavenging and antioxidant effects. Therefore, we examined the effects of zingerone and eugenol on the behavioral problems in mouse model and on the DA concentration and antioxidant activities in the striatum after 6-OHDA administration and L-DOPA treatment. Daily oral administration of eugenol/zingerone and injection of L-DOPA intraperitoneally for 4 weeks following a single 6-OHDA injection did not improve abnormal behaviors induced by L-DOPA treatment. 6-OHDA reduced the DA level in the striatum; surprisingly, zingerone and eugenol enhanced the reduction of striatal DA and its metabolites. Zingerone decreased catalase activity, and increased glutathione peroxidase activity and the oxidized L-ascorbate level in the striatum. We previously reported that pre-treatment with zingerone or eugenol prevents 6-OHDA-induced DA depression by preventing lipid peroxidation. However, the present study shows that post-treatment with these substances enhanced the DA decrease. These substances had adverse effects dependent on the time of administration relative to model PD onset. These results suggest that we should be wary of ingesting these spice elements after the onset of PD symptoms.

  2. 4-Hydroperoxy-2-decenoic acid ethyl ester protects against 6-hydroxydopamine-induced cell death via activation of Nrf2-ARE and eIF2α-ATF4 pathways.

    PubMed

    Inoue, Yuki; Hara, Hirokazu; Mitsugi, Yukari; Yamaguchi, Eiji; Kamiya, Tetsuro; Itoh, Akichika; Adachi, Tetsuo

    2017-08-16

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra. Oxidative stress has been reported to be closely related to the pathogenesis and worsening of symptoms of PD. One therapeutic strategy is to alleviate neuronal injuries caused by oxidative stress. In this study, we investigated protective effects of royal jelly (RJ) fatty acids and their derivatives on oxidative stress-induced cell death using human neuroblastoma SH-SY5Y cells. 4-Hydroperoxy-2-decenoic acid ethyl ester (HPO-DAEE), a synthesized RJ fatty acid derivative, markedly induced antioxidant enzymes such as heme oxygenase-1 (HO-1). Pretreatment with HPO-DAEE protected against 6-hydroxydopamine (6-OHDA)-induced cell death. NF-E2-related factor 2 (Nrf2), a master regulator of antioxidative responses, plays a key role in the acquisition of resistance to oxidative stress. HPO-DAEE elicited nuclear accumulation of Nrf2 and activated antioxidant response element (ARE), a cis-activating regulatory element, indicating that HPO-DAEE induced expression of antioxidant genes through Nrf2-ARE signaling. Recently, the activating transcription factor-4 (ATF4) has been shown to cooperate with Nrf2 and modulate antioxidant gene expression. We also found that HPO-DAEE promoted phosphorylation of eukaryotic initiation factor 2α (eIF2α), which is an upstream effector of ATF4, and subsequent nuclear accumulation of ATF4. The eIF2α phosphatase inhibitor, salubrinal, augmented HPO-DAEE-induced HO-1 expression and protection against 6-OHDA-induced cell death. These results indicate that HPO-DAEE activates both the Nrf2-ARE and eIF2α-ATF4 pathways. Moreover, ROS generation occurred upon treatment of SH-SY5Y cells with HPO-DAEE, and the antioxidants N-acetylcysteine and glutathione suppressed HPO-DAEE-induced activation of the Nrf2-ARE and eIF2α-ATF4 pathways. Therefore, sublethal oxidative stress caused by HPO-DAEE is likely to activate both these pathways. Taken together, we conclude that HPO-DAEE elicits adaptive responses to oxidative stress through cooperative activation of the Nrf2-ARE and eIF2α-ATF4 pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Effect of Buspirone, Fluoxetine and 8-OH-DPAT on Striatal Expression of Bax, Caspase-3 and Bcl-2 Proteins in 6-Hydroxydopamine-Induced Hemi-Parkinsonian Rats.

    PubMed

    Sharifi, Hamdollah; Mohajjel Nayebi, Alireza; Farajnia, Safar; Haddadi, Rasool

    2015-11-01

    The exact pathogenesis of sporadic parkinson's disease (PD) is still unclear. Numerous evidences suggest involvement of apoptosis in the death of dopaminergic neurons. In this study we investigated the effect of sub-chronic administration of buspirone, fluoxetine and 8-hydroxy-2-[di-n-propylamino]tetralin (8-OH-DPAT) in 6-hydroxydopamine (6-OHDA)-lesioned rats and assayed striatal concentrations of apoptotic (Bax, Caspase3) and anti-apoptotic (Bcl-2) proteins. 6-OHDA (8μg/2μl/rat) was injected unilaterally into the central region of the substantia nigra pars copmacta (SNc) of male Wistar rats and then, after 21 days lesioned rats were treated with intraperitonel (i.p) 1 mg/kg injections of buspirone, fluoxetine and 8-OH-DPAT for 10 consecutive days. Striatum of rats was removed at tenth day of drugs administration and were analyzed by western blotting method to measure Bax, caspase3 and Bcl-2 expression. The results showed that the expression of Bax and caspase3 proteins was increased three weeks after 6-OHDA injection while they were decreased significantly in parkinsonian rats which were treated by buspirone, fluoxetine and 8-OH-DPAT. Bcl-2 was decreased and increased in parkinsonian rats and parkinsonian rats treated with buspirone, fluoxetine and 8-OH-DPAT, respectively. Our study indicates that sub-chronic administration of serotonergic drugs such as buspirone, fluoxetine and 8-OH-DPAT restores striatal concentration of apoptotic and anti-apoptotic factors to the basal levels of normal non-lesioned rats. We suggest that these drugs can be used as a potential adjunctive therapy in PD through attenuating neuronal apoptotic process.

  4. Relationships among rat ultrasonic vocalizations, behavioral measures of striatal dopamine loss, and striatal tyrosine hydroxylase immunoreactivity at acute and chronic time points following unilateral 6-hydroxydopamine-induced dopamine depletion

    PubMed Central

    Grant, Laura M; Barnett, David GS; Doll, Emerald J; Leverson, Glen; Ciucci, Michelle R

    2015-01-01

    Voice deficits in Parkinson disease (PD) emerge early in the disease process, but do not improve with standard treatments targeting dopamine. Experimental work in the rat shows that severe and chronic unilateral nigrostriatal dopamine depletion with 6-OHDA results in decreased intensity, bandwidth, and complexity of ultrasonic vocalizations. However, it is unclear if mild/acute dopamine depletion, paralleling earlier stages of PD, results in vocalization deficits, or to what degree vocalization parameters are correlated with other dopamine-dependent indicators of lesion severity or percent of tyrosine hydroxylase (%TH) loss. Here, we assayed ultrasonic vocalizations, forelimb asymmetry, and apomorphine rotations in rats with a range of unilateral dopamine loss resulting from 6-OHDA or vehicle control infusions to the medial forebrain bundle at acute (72 hours) and chronic (4 weeks) time points post-infusion. The %TH loss was evaluated at 4 weeks. At 72 hours, forelimb asymmetry and %TH loss were significantly correlated, while at 4 weeks, all measures of lesion severity were significantly correlated with each other. Call complexity was significantly correlated with all measures of lesion severity at 72 hours but only with %TH loss at 4 weeks. Bandwidth was correlated with forelimb asymmetry at both time points. Duration was significantly correlated with all dopamine depletion measures at 4 weeks. Notably, not all parameters were affected universally or equally across time. These results suggest that vocalization deficits may be a sensitive index of acute and catecholamine loss and further underscores the need to characterize the neural mechanisms underlying vocal deficits in PD. PMID:26026785

  5. Alterations of BDNF and trkB mRNA expression in the 6-hydroxydopamine-induced model of preclinical stages of Parkinson's disease: an influence of chronic pramipexole in rats.

    PubMed

    Berghauzen-Maciejewska, Klemencja; Wardas, Jadwiga; Kosmowska, Barbara; Głowacka, Urszula; Kuter, Katarzyna; Ossowska, Krystyna

    2015-01-01

    Our recent study has indicated that a moderate lesion of the mesostriatal and mesolimbic pathways in rats, modelling preclinical stages of Parkinson's disease, induces a depressive-like behaviour which is reversed by chronic treatment with pramipexole. The purpose of the present study was to examine the role of brain derived neurotrophic factor (BDNF) signalling in the aforementioned model of depression. Therefore, we investigated the influence of 6-hydoxydopamine (6-OHDA) administration into the ventral region of the caudate-putamen on mRNA levels of BDNF and tropomyosin-related kinase B (trkB) receptor. The BDNF and trkB mRNA levels were determined in the nigrostriatal and limbic structures by in situ hybridization 2 weeks after the operation. Pramipexole (1 mg/kg sc twice a day) and imipramine (10 mg/kg ip once a day) were injected for 2 weeks. The lesion lowered the BDNF and trkB mRNA levels in the hippocampus [CA1, CA3 and dentate gyrus (DG)] and amygdala (basolateral/lateral) as well as the BDNF mRNA content in the habenula (medial/lateral). The lesion did not influence BDNF and trkB expression in the caudate-putamen, substantia nigra, nucleus accumbens (shell and core) and ventral tegmental area (VTA). Chronic imipramine reversed the lesion-induced decreases in BDNF mRNA in the DG. Chronic pramipexole increased BDNF mRNA, but decreased trkB mRNA in the VTA in lesioned rats. Furthermore, it reduced BDNF and trkB mRNA expression in the shell and core of the nucleus accumbens, BDNF mRNA in the amygdala and trkB mRNA in the caudate-putamen in these animals. The present study indicates that both the 6-OHDA-induced dopaminergic lesion and chronic pramipexole influence BDNF signalling in limbic structures, which may be related to their pro-depressive and antidepressant activity in rats, respectively.

  6. Metallothionein-III protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a PI3K and ERK/Nrf2-dependent manner

    SciTech Connect

    Hwang, Yong Pil; Kim, Hyung Gyun; Han, Eun Hee; Jeong, Hye Gwang

    2008-09-15

    The zinc-binding protein metallothionein-III (MT-III) is associated with resistance to neuronal injury. However, the underlying mechanism for its effects is unclear. In this study, we demonstrate that MT-III prevents the accumulation of reactive oxygen species (ROS) in dopaminergic SH-SY5Y cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves phosphatidylinositol 3-kinase (PI3K) and ERK kinase/NF-E2-related factor 2 (Nrf2) dependent induction of the stress response protein heme oxygenase-1 (HO-1). Pretreatment of SH-SY5Y cells with MT-III significantly reduced 6-OHDA-induced generation of ROS, caspase-3 activation, and subsequent cell death. Also, MT-III up-regulates HO-1 expression and this expression confers neuroprotection against oxidative injury induced by 6-OHDA. Moreover, MT-III induces Nrf2 nuclear translocation, which is upstream of MT-III-induced HO-1 expression, and PI3K and ERK1/2 activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. Taken together, these results suggest that the PI3K and ERK/Nrf2 signaling pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.

  7. Cytochromes P450

    PubMed Central

    Werck-Reichhart, Danièle; Bak, Søren; Paquette, Suzanne

    2002-01-01

    There are 272 cytochrome P450 genes (including 26 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest families of proteins in higher plants. This explosion of the P450 family is thought to have occurred via gene duplication and conversion, and to result from the need of sessile plants to adapt to a harsh environment and to protect themselves from pathogens and predators. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions. Their biological functions range from the synthesis of structural macromolecules such as lignin, cutin or suberin, to the synthesis or catabolism of all types of hormone or signaling molecules, the synthesis of pigments and defense compounds, and to the metabolism of xenobiotics. In despite of a huge acceleration in our understanding of plant P450 functions in the recent years, the vast majority of these functions remain completely unknown. PMID:22303202

  8. Cytochromes P450

    PubMed Central

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  9. Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state.

    PubMed

    Moini, H; Arroyo, A; Vaya, J; Packer, L

    1999-01-01

    The polyphenolic structure common to flavonoids enables them to donate electrons and exert antioxidant activity. Since the mitochondrial electron transport chain consists of a series of redox intermediates, the effect of flavonoids in a complex mixture of polyphenols, as well as related pure flavonoids, was evaluated on the rat liver mitochondrial electron transport chain. A French maritime pine bark extract (PBE), a complex mixture of polyphenols and related pure flavonoids, was able to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group; the donated electrons can be utilized by cytochrome c oxidase. Among single flavonoids tested, (-)-epicatechin gallate had the greatest ability to reduce cytochrome c. In addition, PBE competitively inhibited electron chain activity in both whole mitochondria and submitochondrial particles. A 3.5-fold increase in the apparent Km value for succinate was calculated from reciprocal plots. Among the flavonoids tested, taxifolin and (-)-epicatechin gallate showed minor inhibitory effects, while (+/-)-catechin and (+)-epicatechin were ineffective. Activities of NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases were inhibited by low concentrations of PBE to a similar extent. However, inhibition of cytochrome c oxidase activity required 4-fold higher PBE concentrations. These results suggest that flavonoids reduce cytochrome c and that PBE inhibits electron transport chain activity mainly through NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases.

  10. In vitro effects of myricetin, morin, apigenin, (+)-taxifolin, (+)-catechin, (-)-epicatechin, naringenin and naringin on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase.

    PubMed

    Çelik, Haydar; Koşar, Müberra; Arinç, Emel

    2013-06-07

    The microsomal NADH-dependent electron transport system consisting of cytochrome b5 reductase and cytochrome b5 participates in a number of physiologically important processes including lipid metabolism as well as is involved in the metabolism of various drug and xenobiotics. In the present study, we assessed the inhibitory effects of eight dietary flavonoids representing five distinct chemical classes on cytochrome b5 reduction by purified cytochrome b5 reductase. From the flavonoids tested, myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC50 value of 0.35μM. Myricetin inhibited b5 reductase noncompetitively with a Ki of 0.21μM with respect to cofactor NADH, and exhibited a non-linear relationship indicating non-Michaelis-Menten kinetic binding with respect to cytochrome b5. In contrast to the potent inhibitory activity of myricetin, (+)-taxifolin was found to be a weak inhibitor (IC50=9.8μM). The remaining flavonoids were inactive within the concentration range tested (1-50μM). Analysis of structure-activity data suggested that simultaneous presence of three OH groups in ring B is a primary structural determinant for a potent enzyme inhibition. Our results suggest that inhibition of the activity of this system by myricetin or myricetin containing diets may influence the metabolism of therapeutic drugs as well as detoxification of xenobiotics. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  11. Cumene hydroperoxide supported demethylation of N,N-dimethylaniline by cytochrome P-450 from adrenal cortex mitochondria.

    PubMed

    Akhrem, A A; Khatyleva SYu; Shkumatov, V M; Chashchin, V L; Kiselev, P A

    1982-01-01

    The interaction of highly purified cytochrome P-450 from bovine adrenal cortex mitochondria (cytochrome P-450scc) with N,N-dimethylaniline (DMA), aniline, N-dimethylcyclohexylamine and cumene hydroperoxide (CHP) has been investigated. The formation of complexes between cytochrome P-450scc and the above listed compounds could be demonstrated. The reaction of oxidative demethylation of DMA by cumene hydroperoxide involving cytochrome P-450scc has been carried out at 37 degrees C; the mechanism of this process is discussed. Incubation of cytochrome P-450scc with negatively charged phospholipids, phosphatidylglycerol (PG), and phosphatidylinosite (PI) exerts an inhibiting effect on the reaction of oxidative demethylation. The interaction of cytochrome P-450scc with CHP is accompanied by hemoprotein destruction in a complex biphasic way. The process of oxidative demethylation of DMA in the system of cytochrome P-450scc-CHP has been concluded to have a predominantly radical character.

  12. Cytochrome P450 arachidonic acid metabolism in bovine corneal epithelium

    SciTech Connect

    Masferrer, J.; Schwartzman, M.L.; Abraham, N.G.; Dunn, M.W.; McGiff, J.C.

    1986-03-01

    The presence of the cytochrom P450 system and its involvement in the metabolism of AA was studied in the corneal epithelium. This tissue contains cytochrome P450 as assessed directly by measurement of the carbon monoxide reduced spectrum (specific activity of 161 pmol/10 mg protein) and indirectly by measuring the activity of aryl hydrocarbon hydroxylase (AHH) - a cytochrome P450-dependent enzyme (11-39 pmol 3-OH benzopyrene/mg protein/10 min). When corneal epithelial microsomes were incubated with /sup 14/C-arachidonic acid, 30-50% of the total radioactivity was converted to two peaks, I and II. Further separation using high performance liquid chromatography has shown that each peak contains two metabolites, A,B and C,D. Metabolite formation was dependent on the addition of NADPH (1 mM) and inhibited by carbon monoxide and SKF-525A (100 ..mu..M) suggesting a cytochrome P450-dependent mechanism. Compound C (5-10 ..mu..M) inhibited the activity of corneal epithelial Na-K-ATPase by 30-60%, being 100-fold more potent than ouabain. Compound D (10-100 ng) induced a dose dependent relaxation of the rat caudal artery. Compound D also inhibited corneal Na-K-ATPase activity but less potently than compound C. These compounds may be important to transport processes of ocular epithelia and participate in the control of the ocular circulation and aqueous humor dynamics.

  13. The cytochrome p450 homepage.

    PubMed

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  14. Cytochrome bd from Escherichia coli catalyzes peroxynitrite decomposition.

    PubMed

    Borisov, Vitaliy B; Forte, Elena; Siletsky, Sergey A; Sarti, Paolo; Giuffrè, Alessandro

    2015-02-01

    Cytochrome bd is a prokaryotic respiratory quinol oxidase phylogenetically unrelated to heme-copper oxidases, that was found to promote virulence in some bacterial pathogens. Cytochrome bd from Escherichia coli was previously reported to contribute not only to proton motive force generation, but also to bacterial resistance to nitric oxide (NO) and hydrogen peroxide (H2O2). Here, we investigated the interaction of the purified enzyme with peroxynitrite (ONOO(-)), another harmful reactive species produced by the host to kill invading microorganisms. We found that addition of ONOO(-) to cytochrome bd in turnover with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) causes the irreversible inhibition of a small (≤15%) protein fraction, due to the NO generated from ONOO(-) and not to ONOO(-) itself. Consistently, addition of ONOO(-) to cells of the E. coli strain GO105/pTK1, expressing cytochrome bd as the only terminal oxidase, caused only a minor (≤5%) irreversible inhibition of O2 consumption, without measurable release of NO. Furthermore, by directly monitoring the kinetics of ONOO(-) decomposition by stopped-flow absorption spectroscopy, it was found that the purified E. coli cytochrome bd in turnover with O2 is able to metabolize ONOO(-) with an apparent turnover rate as high as ~10 mol ONOO(-) (mol enzyme)(-1) s(-1) at 25°C. To the best of our knowledge, this is the first time that the kinetics of ONOO(-) decomposition by a terminal oxidase has been investigated. These results strongly suggest a protective role of cytochrome bd against ONOO(-) damage. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Enzyme kinetics of oxidative metabolism: cytochromes P450.

    PubMed

    Korzekwa, Ken

    2014-01-01

    The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 3 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapter 4 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

  16. Quinol-cytochrome c Oxidoreductase and Cytochrome c4 Mediate Electron Transfer during Selenate Respiration in Thauera selenatis*

    PubMed Central

    Lowe, Elisabeth C.; Bydder, Sarah; Hartshorne, Robert S.; Tape, Hannah L. U.; Dridge, Elizabeth J.; Debieux, Charles M.; Paszkiewicz, Konrad; Singleton, Ian; Lewis, Richard J.; Santini, Joanne M.; Richardson, David J.; Butler, Clive S.

    2010-01-01

    Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (Em + 234 ± 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified (∼24 and ∼6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c4 family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 ± 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c4 is a novel route for a member of the DMSO reductase family of molybdoenzymes. PMID:20388716

  17. Evidence for cytochrome b5 as an electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.).

    PubMed Central

    Smith, M A; Jonsson, L; Stymne, S; Stobart, K

    1992-01-01

    The major b-type cytochrome in microsomal membrane preparations from developing endosperm of castor bean (Ricinus communis) was cytochrome b5. Cytochrome P-450 was also present. The microsomal membranes had delta 12-hydroxylase activity and catalysed the NAD(P)H-dependent hydroxylation of oleate to yield ricinoleic acid. CO had no effect on the hydroxylase activity. Rabbit polyclonal antibodies were raised against the hydrophilic cytochrome b5 fragment purified from cauliflower (Brassica oleracea) floret microsomes. The anti-(cytochrome b5) IgG inhibited delta 12-hydroxylase, delta 12-desaturase and cytochrome c reductase activity in the microsomes. The results indicate that electrons from NAD(P)H were transferred to the site of hydroxylation via cytochrome b5 and that cytochrome P-450 was not involved. Images Fig. 1. PMID:1417766

  18. Human cytochrome c enters murine J774 cells and causes G{sub 1} and G{sub 2}/M cell cycle arrest and induction of apoptosis

    SciTech Connect

    Hiraoka, Yoshinori; Granja, Ana Teresa; Fialho, Arsenio M.; Schlarb-Ridley, Beatrix G.; Das Gupta, Tapas K.; Chakrabarty, Ananda M.; Yamada, Tohru . E-mail: tohru@uic.edu

    2005-12-16

    Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c {sub 551} from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c {sub 551}, the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G{sub 1} to S phase, as well as at the G{sub 2}/M phase at higher concentrations. Unlike P. aeruginosa cytochrome c {sub 551} which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G{sub 2}/M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 deg. C, suggesting energy requirement in the entry process.

  19. Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14 alpha-sterol demethylase purified from Candida albicans.

    PubMed Central

    Hitchcock, C A; Dickinson, K; Brown, S B; Evans, E G; Adams, D J

    1990-01-01

    The interaction of azole antifungal antibiotics with purified Candida albicans cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) was measured spectrophotometrically and by inhibition of enzyme activity. Ketoconazole and ICI 153066 (a triazole derivative) formed low-spin complexes with the ferric cytochrome and induced type II difference spectra. These spectra are indicative of an interaction between the azole moiety and the sixth co-ordination position of P-450DM haem. Both azoles inhibited the binding of CO to the sodium dithionite-reduced ferrous cytochrome, and inhibited reconstituted P-450DM activity by binding to the cytochrome with a one-to-one stoichiometry. Similarly, total inhibition of enzyme activity occurred when equimolar amounts of clotrimazole, miconazole or fluconazole were added to reconstituted P-450DM. These results correlated with the inhibition of P-450DM in broken cell preparations, confirming that all five azoles are potent inhibitors of ergosterol biosynthesis in C. albicans. PMID:2180400

  20. Cation binding site of cytochrome c oxidase: progress report.

    PubMed

    Vygodina, Tatiana V; Kirichenko, Anna; Konstantinov, Alexander A

    2014-07-01

    Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

  1. Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate▿

    PubMed Central

    Voordeckers, James W.; Kim, Byoung-Chan; Izallalen, Mounir; Lovley, Derek R.

    2010-01-01

    Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones. PMID:20154112

  2. Role of Geobacter sulfurreducens outer surface c-type cytochromes in reduction of soil humic acid and anthraquinone-2,6-disulfonate.

    PubMed

    Voordeckers, James W; Kim, Byoung-Chan; Izallalen, Mounir; Lovley, Derek R

    2010-04-01

    Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.

  3. Selective Targeting of Heme Protein in Cytochrome P450 and Nitric Oxide Synthase by Diphenyleneiodonium.

    PubMed

    Szilagyi, John T; Mishin, Vladimir; Heck, Diane E; Jan, Yi-Hua; Aleksunes, Lauren M; Richardson, Jason R; Heindel, Ned D; Laskin, Debra L; Laskin, Jeffrey D

    2016-05-01

    Cytochrome P450 (CYP) enzymes mediate mixed-function oxidation reactions important in drug metabolism. The aromatic heterocyclic cation, diphenyleneiodonium (DPI), binds flavin in cytochrome P450 reductase and inhibits CYP-mediated activity. DPI also inhibits CYP by directly interacting with heme. Herein, we report that DPI effectively inhibits a number of CYP-related monooxygenase reactions including NADPH oxidase, a microsomal enzyme activity that generates hydrogen peroxide in the absence of metabolizing substrates. Inhibition of monooxygenase by DPI was time and concentration dependent with IC50's ranging from 0.06 to 1.9 μM. Higher (4.6-23.9 μM), but not lower (0.06-1.9 μM), concentrations of DPI inhibited electron flow via cytochrome P450 reductase, as measured by its ability to reduce cytochrome c and mediate quinone redox cycling. Similar results were observed with inducible nitric oxide synthase (iNOS), an enzyme containing a C-terminal reductase domain homologous to cytochrome P450 reductase that mediates reduction of cytochrome c, and an N-terminal heme-thiolate oxygenase domain mediating nitric oxide production. Significantly greater concentrations of DPI were required to inhibit cytochrome c reduction by iNOS (IC50 = 3.5 µM) than nitric oxide production (IC50 = 0.16 µM). Difference spectra of liver microsomes, recombinant CYPs, and iNOS demonstrated that DPI altered heme-carbon monoxide interactions. In the presence of NADPH, DPI treatment of microsomes and iNOS yielded a type II spectral shift. These data indicate that DPI interacts with both flavin and heme in CYPs and iNOS. Increased sensitivity for inhibition of CYP-mediated metabolism and nitric oxide production by iNOS indicates that DPI targets heme moieties within the enzymes.

  4. Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

    PubMed Central

    Deming, Paula B.; Schafer, Zachary T.; Tashker, Jessica S.; Potts, Malia B.; Deshmukh, Mohanish; Kornbluth, Sally

    2004-01-01

    Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition. PMID:15542838

  5. Toxic dark effects of protoporphyrin on the cytochrome P-450 system in rat liver microsomes.

    PubMed Central

    Williams, M; Van der Zee, J; Van Steveninck, J

    1992-01-01

    In erythropoietic protoporphyria, accumulation of protoporphyrin has been found in various tissues and liver cirrhosis occurs frequently in this disease, probably due to toxic dark effects of protoporphyrin. We have studied the effect of porphyrins on various enzymic functions in rat liver microsomes. Incubation of microsomes with protoporphyrin resulted in a concentration-dependent inhibition of the oxidation of 7-ethoxycoumarin and aminopyrine by the cytochrome P-450 system. Kinetic analysis showed a decrease in Vmax., whereas the Km was not affected (non-competitive inhibition). Furthermore, reduction of cytochrome c by the NADPH-cytochrome P-450 reductase and by the NADH-cytochrome b5 reductase was inhibited. However, the activity of the reductases was only affected when the microsomes were pre-incubated with protoporphyrin, and it was found that the inhibition was dependent on the duration of the pre-incubation. Kinetic analysis again revealed non-competitive inhibition. When these experiments were repeated with uroporphyrin, no inhibition could be observed. With Stern-Volmer plots it was demonstrated that this was most likely caused by the localization of the porphyrins: protoporphyrin is localized in the membrane, whereas uroporphyrin remains in solution. From these results it is concluded that accumulation of protoporphyrin in the liver may markedly affect the cytochrome P-450 system and thus its detoxification function. PMID:1332695

  6. The role of cytochrome b5 in delta 12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.).

    PubMed

    Kearns, E V; Hugly, S; Somerville, C R

    1991-02-01

    The electron donors for the membrane-bound fatty acid desaturases of higher plants have not previously been identified. In order to assess the participation of cytochrome b5 in microsomal fatty acid desaturation, the cytoplasmic domain of microsomal cytochrome b5 was purified from Brassica oleracea, and murine polyclonal antibodies were prepared. The IgG fraction from ascites fluid inhibited 62% of NADH-dependent cytochrome c reduction in safflower (Carthamus tinctorius L.) microsomes. These antibodies also blocked desaturation of oleic acid to linoleic acid in lipids of C. tinctorius microsomes by 93%, suggesting that cytochrome b5 is the electron donor for the delta 12 desaturase.

  7. Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of "cytochrome a1" as cytochrome b595.

    PubMed

    Lorence, R M; Koland, J G; Gennis, R B

    1986-05-06

    Coulometric and spectroscopic analyses were performed on the three cytochrome components (cytochrome d, cytochrome b558, and the cytochrome previously described as cytochrome a1) of the purified cytochrome d complex, a terminal oxidase of the Escherichia coli aerobic respiratory chain. On the basis of heme extraction, spectroscopic, and coulometric data, the "cytochrome a1" component was identified as a b-type cytochrome: cytochrome b595. The pyridine hemochromogen technique revealed the presence of two molecules of protoheme IX per cytochrome d complex. This quantity of protoheme IX fully accounted for the sum of the cytochrome b558 and cytochrome b595 components as determined coulometrically. The renaming of cytochrome a1 as cytochrome b595 was further indicated by the lack of any heme a in the complex and by its resolved reduced-minus-oxidized spectrum. The latter was found to be similar to that of cytochrome c peroxidase, which contains protoheme IX. Coulometric titrations and carbon monoxide binding titrations revealed that there are two molecules of cytochrome d per complex. A convenient measurement of the amount of cytochrome b558 was found to be the beta-band at 531 nm since cytochrome b558 was observed to be the only component of the cytochrome d complex with a peak at this wavelength. By use of this method and the extinction coefficient for the purified cytochrome b558, it was estimated that there is one molecule of cytochrome b595 and one of cytochrome b558 per cytochrome complex.

  8. Cell respiration is controlled by ATP, an allosteric inhibitor of cytochrome-c oxidase.

    PubMed

    Arnold, S; Kadenbach, B

    1997-10-01

    The activity of cytochrome-c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is known to be regulated by the substrate pressure, i.e. the ferro-/ferricytochrome c ratio, by the oxygen concentration, and by the electrochemical proton gradient delta muH+ across the inner mitochondrial membrane. Here we describe a further mechanism of 'respiratory control' via allosteric inhibition of cytochrome-c oxidase by ATP, which binds to the matrix domain, of subunit IV. The cooperativity between cytochrome-c-binding sites in the dimeric enzyme complex is mediated by cardiolipin, which is essential for cooperativity of the enzyme within the lipid membrane.

  9. The Cytochrome P450 Homepage

    PubMed Central

    2009-01-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 (CYP) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described. PMID:19951895

  10. Cytochrome a620 in Tetrahymena pyriformis. Reactions with carbon monoxide and oxygen at subzero temperatures and photochemical action spectra.

    PubMed Central

    Lloyd, D; Scott, R I; Edwards, S W; Edwards, C; Chance, B

    1982-01-01

    1. Mitochondria-enriched fractions of the ciliate protozoan Tetrahymena pyriformis ST contained CO-reacting cytochromes b560 and a620. 2. A non-photodissociable oxygen-containing compound of cytochrome a620 was formed in whole cell suspensions at -114 degrees C after photolysis of CO in the presence of 200 microM-O2. 3. Electron transport, indicated by the oxidation of cytochrome a620 and cytochrome c, occurred at temperatures higher than -72 degrees C. 4. Photochemical action spectra for the relief of respiratory inhibition of whole cells by CO obtained by using a liquid dye laser indicate that the only CO-reacting terminal oxidase detectable was cytochrome a620. 5. It is concluded that the alternative electron transport chains in this organism utilize non-cytochrome terminal oxidases. PMID:6816222

  11. Cyanide-induced cytochrome a,a3 oxidation-reduction responses in rat brain in vivo.

    PubMed Central

    Piantadosi, C A; Sylvia, A L; Jöbsis, F F

    1983-01-01

    The sensitivity of the brain to cyanide-induced histotoxic hypoxia and the protective effects of known cyanide antagonists, have been assessed in vivo by reflectance spectrophotometry. Cyanide-related changes in cytochrome a,a3 (cytochrome c oxidase) oxidation-reduction (redox) state, tissue hemoglobin saturation, and local blood volume were continuously monitored in cerebral cortex of rats. Noncumulative, dose-dependent inhibition of the in situ mitochondrial respiratory chain was evaluated directly by measuring increases in reduction levels of the terminal oxidase. These transient cytochrome a,a3 reductions were accompanied by increases in regional cerebral hemoglobin saturation and blood volume. Cytochrome redox responses were not altered either in magnitude or kinetics by hyperoxia; however, the cyanide-cytochrome dose-response curve was greatly shifted to the right by pretreatment with sodium nitrite, and the recovery rate of cytochrome a,a3 from cyanide-induced reduction was enhanced fourfold by pretreatment with sodium thiosulfate. PMID:6313756

  12. Cytochrome b5 promotes the synthesis of delta 16-C19 steroids by homogeneous cytochrome P-450 C21 side-chain cleavage from pig testis.

    PubMed

    Nakajin, S; Takahashi, M; Shinoda, M; Hall, P F

    1985-10-30

    Conversion of progesterone to 17 alpha-hydroxyprogesterone plus androstenedione (17 alpha-hydroxylation) and to androstadienone (delta 16 synthetase activity) by microsomes from neonatal pig testis, were both inhibited by antibodies raised against homogeneous cytochrome P-450 C21 side-chain cleavage. Inhibition of the two activities showed the same relationship to the concentration of antibody added. Analogous results were obtained with pregnenolone as substrate. In a reconstituted enzyme system consisting of the homogeneous cytochrome P-450 C21 side-chain cleavage enzyme, P-450 reductase and NADPH, addition of cytochrome b5 resulted in the synthesis of the corresponding delta 16-C19-steroid from progesterone (androstadienone) and pregnenolone (androstadienol). The effect of cytochrome b5 was concentration-dependent and prevented by anti-cytochrome b5. It is concluded that the cytochrome P-450 C21 side-chain cleavage enzyme from pig testicular microsomes is also capable of synthesizing delta 16-C19-steroids and is, therefore, likely to be responsible for the large amounts of the pherormone androstadienone produced by male pigs.

  13. Primary structure determination of two cytochromes c2: close similarity to functionally unrelated mitochondrial cytochrome C.

    PubMed Central

    Ambler, R P; Meyer, T E; Kamen, M D

    1976-01-01

    The amino-acid sequences of the cytochromes c2 from the photosynthetic non-sulfur purple bacteria Rhodomicrobium vannielii and Rhodopseudomonas viridis have been determined. Only a single residue deletion (at position 11 in horse cytochrome c) is necessary to align the sequences with those of mitochondrial cytochromes c. The overall sequence similarity between these cytochromes c2 and mitochondrial cytochromes c is closer than that between mitochondrial cytochromes c and the other cytochromes c2 of known sequence, and in the latter multiple insertions and deletions must be postulated before a match can be obtained. Nevertheless, these two cytochromes c2 show no better reactivity with the mitochondrial cytochrome c oxidase than do the less well-matched cytochromes c2. The bearing of these findings on possible evolutionary relationship between mitochondria and prokaryotes is discussed. PMID:174109

  14. Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate.

    PubMed

    Stonehuerner, J; O'Brien, P; Kendrick, L; Hall, J; Millett, F

    1985-09-25

    Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.

  15. Inhibition of cytochrome P450 3A in rat liver by the Diorganotin (IV) compound di-n-Butyl-di-(4-chlorobenzo-hydroxamato)tin (IV) and Its Probable Mechanism.

    PubMed

    Zhang, Yunxia; Li, Yunlan; Li, Qingshan

    2012-09-12

    The specific aims of this study were to evaluate the inhibition effect on CYP3A of di-n-butyl-di-(4-chlorobenzohydroxamato)tin (IV) (DBDCT), a tin-based complex with high antitumor activity, and the probable mechanism(s) of this action. Adult male SD rats were treated separately with natural saline (NS), lipopolysaccharide (LPS, 5 mg/kg), DBDCT (1.25, 2.5 and 5.0 mg/kg) intraperitoneally for 2 days after induction of CYP3A with dexamethasone (DEX, 100 mg/kg) for 4 days. Western blot analysis and fluorescent quantitation PCR (FQ-PCR) were conducted to determine the changes in expression of CYP3A, PXR, CAR and RXR. The biological accumulation of DBDCT and total Sn were determined by high-performance liquid chromatography (HPLC) and atomic fluorescence spectrometry (AFS). CYP450 content and CYP3A activities were significantly inhibited (p < 0.05) in DBDCT-treated rats compared with the control group, as was the expression of CYP3A (p < 0.05) at both protein and mRNA levels. In DBDCT-treated groups, the expression of PXR protein and mRNA increased, while the expression of CAR decreased. The biological accumulation of DBDCT and Sn in rat livers treated with DBDCT was high. The accumulation of DBDCT and Sn due to the inhibition of CYP3A may be involved in the mechanism of toxicity of DBDCT in rat liver.

  16. Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease

    PubMed Central

    Khare, Shilpi; Roach, Steven L.; Barnes, S. Whitney; Hoepfner, Dominic; Walker, John R.; Chatterjee, Arnab K.; Neitz, R. Jeffrey; Arkin, Michelle R.; McNamara, Case W.; Ballard, Jaime; Lai, Yin; Fu, Yue; Molteni, Valentina; Yeh, Vince; McKerrow, James H.; Glynne, Richard J.; Supek, Frantisek

    2015-01-01

    Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease. PMID:26186534

  17. Cytochrome aa3 in Haloferax volcanii

    PubMed Central

    Tanaka, Mikiei; Ogawa, Naohide; Ihara, Kunio; Sugiyama, Yasuo; Mukohata, Yasuo

    2002-01-01

    A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN− complex spectrum that indicates the presence of heme a and heme a3. This cytochrome aa3 consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa3, providing physiological evidence for electron transfer from cytochrome c to cytochrome aa3 in archaea. PMID:11790755

  18. Spectral Modification and Catalytic Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2A6 and 2A13 by Four Chemopreventive Organoselenium Compounds

    PubMed Central

    Shimada, Tsutomu; Murayama, Norie; Tanaka, Katsuhiro; Takenaka, Shigeo; Guengerich, F. Peter; Yamazaki, Hiroshi; Komori, Masayuki

    2011-01-01

    Several organoselenium compounds including benzyl selenocyanate (BSC), 1,2-phenylenebis(methylene)selenocyanate (o-XSC), 1,3-phenylenebis(methylene)selenocyanate (m-XSC), and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) have been shown to prevent cancers caused by polycyclic aromatic hydrocarbons (PAHs) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in experimental animals; these chemical carcinogens are activated by human P450 1 and 2A family enzymes, respectively, to carcinogenic metabolites. In this study, we examined whether these selenium compounds interact with and inhibit human P450 1 and 2A enzymes in vitro. Four organoselenium compounds induced Reverse Type I binding spectra with P450 1A1, 1A2, and 1B1 and Type I binding spectra with P450 2A6 and 2A13. The spectral dissociation constants (Ks) for the interaction of P450 1B1 with these chemicals were 3.6–5.7 µM; the values were lower than those with seen with P450 1A1 (19–30 µM) or 1A2 (6.3–13 µM). The Ks values for Type I binding of P450 2A13 with m-XSC and BSC were both 0.20 µM; the values were very low compared to the interaction of P450 2A6 with m-XSC (5.7 µM) and BSC (2.0 µM). Four selenium compounds directly inhibited 7-ethoxyresorufin O-deethylation activities catalyzed by P450 1A1, 1A2, and 1B1 with IC50 values <1.0 µM, except for the inhibition of P450 1A2 by BSC (1.3 µM). Coumarin 7-hydroxylation activities of P450 2A13 were more inhibited by four selenium compounds than those of P450 2A6, with IC50 values of 0.22–1.4 µM for P450 2A13 and 2.4–6.2 µM for P450 2A6. Molecular docking studies of the interaction of four organoselenium compounds with human P450 enzymes suggest that these chemicals can be docked into the active sites of these human P450 enzymes and that the sites of the selenocyanate functional groups of these chemicals differ between the P450 1 and 2A family enzymes. PMID:21732699

  19. Cytochrome c Oxidation by the Electron Transport Fraction of Azotobacter vinelandii

    PubMed Central

    Jurtshuk, Peter; Old, Lynn

    1968-01-01

    The spectrophotometric oxidation of horse heart ferrocytochrome c was examined by use of the particulate electron transport fraction (R3) of Azotobacter vinelandii strain O. Unlike cytochrome c, purified preparations of native Azotobacter cytochromes c4 + c5 were oxidized only slowly by the electron transport fraction. The oxidation of mammalian cytochrome c proceeded at an appreciable rate and displayed “apparent” first-order kinetics at a pH optimum of 9.0 with tris(hydroxymethyl)aminomethane-chloride buffer. The calculated Vmax value was 0.22 μmole of cytochrome c oxidized per min per mg of protein (25 C) and a Km value for cytochrome c of 2.3 × 10−5m was obtained. Ferricytochrome c was a “strict” competitive inhibitor for this oxidation. Cytochrome c oxidation by the Azotobacter electron transport system was markedly sensitive to cyanide, azide, and hydroxylamine, although carbon monoxide inhibition could not be demonstrated. It was sensitive also to high concentrations of phosphate, ethylenediaminetetraacetate, and some metal cations. “Aging” or prolonged storage of the Azotobacter R3 fraction, at 4 C for 10 days, resulted in a threefold increase in specific activity. The cytochrome c peroxidase type of reaction did not occur with the R3 electron transport fraction. PMID:4967774

  20. Carbon tetrachloride changes the activity of cytochrome P450 system in the liver of male rats: role of antioxidants.

    PubMed

    Sheweita, S A; El-Gabar, M A; Bastawy, M

    2001-12-14

    The cytochrome P-450 enzymes are responsible for the oxidation of xenobiotic chemicals including drugs, pesticides, and carcinogens. These enzymes include cytochrome P450, cytochrome b(5), arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH), NADPH-cytochrome C reductase and dimethylnitrosamine N-demethylase I (DMN-dI). Changes in the activities of the above mentioned enzymes were studied in the liver microsomes of rats treated with antioxidants (ascorbic acid (AA), DL-a-tocopherol (vitamin E, VE), garlic) as single- and repeated doses prior to the administration of a single dose of CCl(4). Pretreatment of rats with single doses of AA, VE, or garlic prior to the administration of CCl(4) was found to decrease the hepatic content of cytochrome P450, and the activities of DMN-dI and AHH. On the other hand, these treatments induced the hepatic content of cytochrome b(5) and the activity of NADPH-cytochrome c reductase. Pretreatment of rats with repeated doses of AA, VE, or garlic for 12 consecutive days prior to the administration of CCl(4) as single dose was potentially decreased the activities of cytochrome P450, DMN-dI and NADPH-cytochrome c reductase. Also, the activity of AHH decreased after treatments of rats with repeated doses of garlic prior to the administration of CCl(4). It was noted that repeated doses of antioxidants are more effective than single dose in decreasing the activity of drug-metabolizing enzymes. It is concluded that repeated doses of antioxidants or garlic could reduce the toxic effects exerted by CCl(4) upon the liver, and probably other organs, through inhibition of cytochrome P450 system that activates CCl(4) into its active metabolite, trichloromethyl radical. Moreover, inhibition of cytochrome P450 system could also reduce the toxic and carcinogenic effects of chemical carcinogens such as benzo(a)pyrene and dimethylnitrosamine. The mechanisms of antioxidant protection were discussed in the text.

  1. The rabbit pulmonary cytochrome P450 arachidonic acid metabolic pathway: characterization and significance.

    PubMed Central

    Zeldin, D C; Plitman, J D; Kobayashi, J; Miller, R F; Snapper, J R; Falck, J R; Szarek, J L; Philpot, R M; Capdevila, J H

    1995-01-01

    Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19- and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung. Images PMID:7738183

  2. Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions.

    PubMed

    Unitt, David C; Hollis, Veronica S; Palacios-Callender, Miriam; Frakich, Nanci; Moncada, Salvador

    2010-03-01

    We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O(2)) conditions. The system measures the concentrations of O(2) and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O(2) concentration and electron turnover of the enzyme. At a high O(2) concentration (70 microM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O(2). At low O(2) (15 microM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O(2) consumption. At both high and low O(2) concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.

  3. Cytochromes P450 in Nanodiscs

    PubMed Central

    Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Nanodiscs have proven to be a versatile tool for the study all types of membrane proteins, including receptors, transporters, enzymes and viral antigens. The self-assembled Nanodisc system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native like bilayer environment that maintains functional activity. This system has thus provided a means for studying the extensive collection of membrane bound cytochromes P450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble P450s. These include a plethora of spectroscopic, kinetic and surface based methods. Significant improvements in homogeneity and stability of these preparations open new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of human cytochromes P450 involved in xenobiotic metabolism and in steroid biosynthesis. The experimental methods developed for physico-chemical and functional studies of membrane cytochromes P450 incorporated in Nanodiscs allow for more detailed understanding of the scientific questions along the lines pioneered by Professor Klaus Ruckpaul and his array of colleagues and collaborators. PMID:20685623

  4. Cytochrome c catalyses the formation of pentyl radical and octanoic acid radical from linoleic acid hydroperoxide.

    PubMed Central

    Iwahashi, Hideo; Nishizaki, Koji; Takagi, Ichiro

    2002-01-01

    A reaction of 13-hydroperoxide octadecadienoic acid (13-HPODE) with cytochrome c was analysed using ESR, HPLC-ESR and HPLC-ESR-MS by the combined use of the spin-trapping technique. The ESR, HPLC-ESR and HPLC-ESR-MS analyses showed that cytochrome c catalyses formation of pentyl and octanoic acid radicals from 13-HPODE. On the other hand, only the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone/octanoic acid radical adduct was detected in the elution profile of HPLC-ESR for a mixture of 13-HPODE with haematin, indicating that haematin catalyses the formation of octanoic acid radical. In addition, the reaction of 13-HPODE with cytochrome c was inhibited by chlorogenic acid, caffeic acid and ferulic acid via two possible mechanisms, i.e. reducing cytochrome c (chlorogenic acid and caffeic acid) and scavenging the radical intermediates (chlorogenic acid, caffeic acid and ferulic acid). PMID:11742529

  5. A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect.

    PubMed Central

    Abdullaev, Ziedulla Kh; Bodrova, Marina E; Chernyak, Boris V; Dolgikh, Dmitry A; Kluck, Ruth M; Pereverzev, Mikhail O; Arseniev, Alexander S; Efremov, Roman G; Kirpichnikov, Mikhail P; Mokhova, Elena N; Newmeyer, Donald D; Roder, Heinrich; Skulachev, Vladimir P

    2002-01-01

    A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other "horse" modifications in the N-terminal alpha-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12. PMID:11879204

  6. Zinc ions as cytochrome C oxidase inhibitors: two sites of action.

    PubMed

    Kuznetsova, S S; Azarkina, N V; Vygodina, T V; Siletsky, S A; Konstantinov, A A

    2005-02-01

    Zinc ions are shown to be an efficient inhibitor of mitochondrial cytochrome c oxidase activity, both in the solubilized and the liposome-reconstituted enzyme. The effect of zinc is biphasic. First there occurs rapid interaction of zinc with the enzyme at a site exposed to the aqueous phase corresponding to the mitochondrial matrix. This interaction is fully reversed by EDTA and results in a partial inhibition of the enzyme activity (50-90%, depending on preparation) with an effective K(i) of approximately 10 microM. The rapid effect of zinc is observed with the solubilized enzyme, it vanishes upon incorporation of cytochrome oxidase in liposomes, and it re-appears when proteoliposomes are supplied with alamethicin that makes the membrane permeable to low molecular weight substances. Zinc presumably blocks the entrance of the D-protonic channel opening into the inner aqueous phase. Second, zinc interacts slowly (tens of minutes, hours) with a site of cytochrome oxidase accessible from the outer aqueous phase bringing about complete inhibition of the enzymatic activity. The slow phase is characterized by high affinity of the inhibitor for the enzyme: full inhibition can be achieved upon incubation of the solubilized oxidase for 24 h with zinc concentration as low as 2 microM. The rate of zinc inhibitory action in the slow phase is proportional to Zn(2+) concentration. The slow interaction of zinc with the outer surface of liposome-reconstituted cytochrome oxidase is observed only with the enzyme turning over or in the presence of weak reductants, whereas incubation of zinc with the fully oxidized proteoliposomes does not induce the inhibition. It is shown that zinc ions added to cytochrome oxidase proteoliposomes from the outside inhibit specifically the slow electrogenic phase of proton transfer, coupled to a transition of cytochrome oxidase from the oxo-ferryl to the oxidized state (the F --> O step corresponding to transfer of the 4th electron in the catalytic

  7. Cytochrome Electron Transfer and Biomolecular Electronics.

    DTIC Science & Technology

    1988-06-22

    polarograms of cytochrome c3 "a) DvM: Desulfovibrio vulgaris Miyazaki F (solid line,rmeasured; dots, si ulated) .. b) DvH: Desulfovibrio vulgaris ...Miyazaki); 2. D. vulgaris (H ldenborough); 3. D. sulfurlcans (Norway) and % 4. D. gigas. The macroscopic redox potentials for each of the hemes in the...Structure of Cytochrome C 3 Four cytochromes C have been selected for study: 1. D. vulgaris (Miyazaki) DvM) ; 2. D. vulgaris (Hildenborough) (DvH); 3. D

  8. Molecular interaction between cyclophilin D and adenine nucleotide translocase in cytochrome c release: does it determine whether cytochrome c release is dependent on permeability transition or not?

    PubMed

    Machida, Kiyotaka; Osada, Hiroyuki

    2003-12-01

    We propose a model for the mechanism of permeability transition (PT) related cytochrome c release. It is likely that the Ca(2+) requirement for the induction of the mitochondrial permeability transition pore (MPTP) opening might be due to the Ca(2+)-dependent interaction between cyclophilin D and ANT. We show here that the modification of adenine nucleotide translocase (ANT), which is one of the components of MPTP, can induce two different types of the cytochrome c release. One is dependent on classical PT, resulting in mitochondrial swelling, and is inhibited by cyclosporin A. The other is PT-independent, without swelling, and is insensitive to cyclosporin A.

  9. Organization of the electron transfer chain to oxygen in the obligate human pathogen Neisseria gonorrhoeae: roles for cytochromes c4 and c5, but not cytochrome c2, in oxygen reduction.

    PubMed

    Li, Ying; Hopper, Amanda; Overton, Tim; Squire, Derrick J P; Cole, Jeffrey; Tovell, Nicholas

    2010-05-01

    Although Neisseria gonorrhoeae is a prolific source of eight c-type cytochromes, little is known about how its electron transfer pathways to oxygen are organized. In this study, the roles in the respiratory chain to oxygen of cytochromes c(2), c(4), and c(5), encoded by the genes cccA, cycA, and cycB, respectively, have been investigated. Single mutations in genes for either cytochrome c(4) or c(5) resulted in an increased sensitivity to growth inhibition by excess oxygen and small decreases in the respiratory capacity of the parent, which were complemented by the chromosomal integration of an ectopic, isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible copy of the cycA or cycB gene. In contrast, a cccA mutant reduced oxygen slightly more rapidly than the parent, suggesting that cccA is expressed but cytochrome c(2) is not involved in electron transfer to cytochrome oxidase. The deletion of cccA increased the sensitivity of the cycB mutant to excess oxygen but decreased the sensitivity of the cycA mutant. Despite many attempts, a double mutant defective in both cytochromes c(4) and c(5) could not be isolated. However, a strain with the ectopically encoded, IPTG-inducible cycB gene with deletions in both cycA and cycB was constructed: the growth and survival of this strain were dependent upon the addition of IPTG, so gonococcal survival is dependent upon the synthesis of either cytochrome c(4) or c(5). These results define the gonococcal electron transfer chain to oxygen in which cytochromes c(4) and c(5), but not cytochrome c(2), provide alternative pathways for electron transfer from the cytochrome bc(1) complex to the terminal oxidase cytochrome cbb(3).

  10. Cytochrome P450-activated prodrugs

    PubMed Central

    Ortiz de Montellano, Paul R

    2013-01-01

    A prodrug is a compound that has negligible, or lower, activity against a specified pharmacological target than one of its major metabolites. Prodrugs can be used to improve drug delivery or pharmacokinetics, to decrease toxicity, or to target the drug to specific cells or tissues. Ester and phosphate hydrolysis are widely used in prodrug design because of their simplicity, but such approaches are relatively ineffective for targeting drugs to specific sites. The activation of prodrugs by the cytochrome P450 system provides a highly versatile approach to prodrug design that is particularly adaptable for targeting drug activation to the liver, to tumors or to hypoxic tissues. PMID:23360144

  11. Cytochrome C oxidase activity in germinating Phaseolus vulgaris l. seeds: Effects of carbon monoxide

    SciTech Connect

    Caughey, W.S. ); Sowa, S.; Roos, E.E.

    1989-04-01

    Cytochrome c oxidase is a key bioenergetic enzyme required for seed germination. The enzyme was isolated from 2-day germinating beans and biochemically compared to its bovine heart counterpart. Carbon monoxide, which binds to the heme a{sub 3} site of cytochrome c oxidase, we used to probe O{sub 2} utilization activity in isolated enzyme, mitochondrial particles, and whole seeds. Bean seeds under 80% CO/20% O{sub 2} exhibited 46% growth inhibition as determined by root length. Reversible, dose-dependent partial inhibition of bean seed mitochondrial respiration was observed in the presence of CO; heart mitochondria had a more sensitive, less reversible response. Effects of CO on bean and bovine heart enzyme were similar. The close correlation of CO effects observed on seedling growth, mitochondrial respiration and cytochrome oxidase activity indicate an important role for this enzyme during the early stages of seed germination.

  12. Interaction of new sulfaphenazole derivatives with human liver cytochrome p450 2Cs: structural determinants required for selective recognition by CYP 2C9 and for inhibition of human CYP 2Cs.

    PubMed

    Ha-Duong, N T; Marques-Soares, C; Dijols, S; Sari, M A; Dansette, P M; Mansuy, D

    2001-10-15

    A series of new derivatives of sulfaphenazole (SPA), in which the NH(2) and phenyl substituents of SPA are replaced by various groups or in which the sulfonamide function of SPA is N-alkylated, were synthesized in order to further explore CYP 2C9 active site and to determine the structural factors explaining the selectivity of SPA for CYP 2C9 within the human P450 2C subfamily. Compounds in which the NH(2) group of SPA was replaced with R(1) = CH(3), Br, CH = CH(2), CH(2)CH = CH(2), and CH(2)CH(2)OH exhibited a high affinity for CYP 2C9, as shown by the dissociation constant of their CYP 2C9 complexes, K(s), which was determined by difference visible spectroscopy (K(s) between 0.1 and 0.4 microM) and their constant of CYP 2C9 inhibition (K(i) between 0.3 and 0.6 microM). This indicates that the CYP 2C9-iron(III)-NH(2)R bond previously described to exist in the CYP 2C9-SPA complex does not play a key role in the high affinity of SPA for CYP 2C9. Compounds in which the phenyl group of SPA was replaced with various aryl or alkyl R(2) substituents only exhibited a high affinity for CYP 2C9 if R(2) is a freely rotating and sufficiently electron-rich aryl substituent. Finally, compounds resulting from a N-alkylation of the SPA sulfonamide function (R(3) = CH(3), C(2)H(5), or C(3)H(7)) did not retain the selective inhibitory properties of SPA toward CYP 2C9. However, they are reasonably good inhibitors of CYP 2C8 and CYP 2C18 (IC(50) approximately 20 microM). These data allow one to better understand the structural factors that are important for selective binding in the CYP 2C9 active site. They also provide us with clues towards new selective inhibitors of CYP 2C8 and CYP 2C18.

  13. Cytochrome c Adducts with PCB Quinoid Metabolites

    PubMed Central

    Li, Miao; Teesch, Lynn M.; Murry, Daryl J.; Pope, R. Marshal; Li, Yalan; Robertson, Larry W.; Ludewig, Gabriele

    2015-01-01

    PCBs are a group of 209 individual congeners widely used as industrial chemicals. PCBs are found as by-products in dye and paint manufacture and are legacy, ubiquitous and persistent as human and environmental contaminants. PCBs with fewer chlorine atoms may be metabolized to hydroxy- and dihydroxy- metabolites and further oxidized to quinoid metabolites both in vitro and in vivo. Specifically, quinoid metabolites may form adducts on nucleophilic sites within cells. We hypothesized that the PCB-quinones covalently bind to cytochrome c and thereby cause defects in the function of cytochrome c. In this study synthetic PCB quinones (2-(4’-chlorophenyl)-1,4-benzoquinone, 2-(3’, 5’-dichlorophenyl)-1,4-benzoquinone, 2-(3’,4’, 5’-trichlorophenyl)-1,4-benzoquinone, and 2-(4’-chlorophenyl)-3,6-dichloro-1,4-benzoquinone) were incubated with cytochrome c, and adducts were detected by LC-MS and MALDI TOF. SDS PAGE gel electrophoresis was employed to separate the adducted proteins, while trypsin digestion and LC-MS/MS were applied to identify the amino acid binding sites on cytochrome c. Conformation change of cytochrome c after binding with PCB3-para-quinone was investigated by SYBYL-X simulation and cytochrome c function was examined. We found that more than one molecule of PCB-quinone may bind to one molecule of cytochrome c. Lysine and glutamic acid were identified as the predominant binding sites. Software simulation showed conformation changes of adducted cytochrome c. Additionally, cross-linking of cytochrome c was observed on the SDS PAGE gel. Cytochrome c was found to be in the reduced form after incubation with PCB quinones. These data provide evidence that the covalent binding of PCB quinone metabolites to cytochrome c may be included among the toxic effects of PCBs. PMID:26062463

  14. Early Decrease in Respiration and Uncoupling Event Independent of Cytochrome c Release in PC12 Cells Undergoing Apoptosis

    PubMed Central

    Berghella, Libera; Ferraro, Elisabetta

    2012-01-01

    Cytochrome c is a key molecule in mitochondria-mediated apoptosis. It also plays a pivotal role in cell respiration. The switch between these two functions occurs at the moment of its release from mitochondria. This process is therefore extremely relevant for the fate of the cell. Since cytochrome c mediates respiration, we studied the changes in respiratory chain activity during the early stages of apoptosis in order to contribute to unravel the mechanisms of cytochrome c release. We found that, during staurosporine (STS)- induced apoptosis in PC12 cells, respiration is affected before the release of cytochrome c, as shown by a decrease in the endogenous uncoupled respiration and an uncoupling event, both occurring independently of cytochrome c release. The decline in the uncoupled respiration occurs also upon Bcl-2 overexpression (which inhibits cytochrome c release), while the uncoupling event is inhibited by Bcl-2. We also observed that the first stage of nuclear condensation during STS-induced apoptosis does not depend on the release of cytochrome c into the cytosol and is a reversibile event. These findings may contribute to understand the mechanisms affecting mitochondria during the early stages of apoptosis and priming them for the release of apoptogenic factors. PMID:22666257

  15. Cytochrome P450 structure, function and clinical significance: A review.

    PubMed

    Palrasu, Manikandan; Nagini, Siddavaram

    2017-01-25

    The cytochrome P450 (CYP) enzymes are membrane-bound hemoproteins that play a pivotal role in the detoxification of xenobiotics, cellular metabolism and homeostasis. Induction or inhibition of CYP enzymes is a major mechanism that underlies drug-drug interactions. CYP enzymes can be transcriptionally activated by various xenobiotics and endogenous substrates through receptor-dependent mechanisms. CYP enzyme inhibition is a principal mechanism for metabolism-based drug-drug interactions. Many chemotherapeutic drugs can cause drug interactions due to their ability to either inhibit or induce the CYP enzyme system. Predictions based on in silico analyses followed by validation have identified several microRNAs that regulate CYPs. Genetic polymorphisms and epigenetic changes in CYP genes may be responsible for inter-individual and inter-ethnic variations in disease susceptibility and the therapeutic efficacy of drugs. Knowledge about the substrates, inducers, inhibitors of CYP isoforms, and the polymorphisms of CYP enzymes may be used as an aid by clinicians to determine therapeutic strategy, and treatment doses for drugs that are metabolized by CYP gene products. The present review is a comprehensive compilation of cytochrome P450 structure, function, pharmacogenetics, and pharmacoepigenetics and clinical significance.

  16. Mitochondrial cytochrome c oxidase deficiency.

    PubMed

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-03-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance of studying different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy. © 2016 Authors; published by Portland Press Limited.

  17. Mitochondrial Cytochrome c Oxidase Deficiency

    PubMed Central

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-01-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance to study different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy. PMID:26846578

  18. [Localization of the redox-center of cytochrome b562 on the internal (M-) side of the mitochondrial membrane].

    PubMed

    Konstantinov, A A; Kunts, V S

    1984-06-01

    In the inside-out submitochondrial particles, cytochrome b-562 is readily reduced by a hydrophilic redox mediator Ru(NH3)6(2)+; this reaction is not inhibited by antimycin and myxothiazol. In mitochondria, cytochromes b do not virtually interact with Ru(NH3)6(2)+. The accessibility of cytochrome b-562 to Ru(NH3)6(2)+ in submitochondrial particles and its inaccessibility in mitochondria suggest the localization of the hemoprotein redox center on the inner surface of the mitochondrial membrane.

  19. Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ.

    PubMed

    Krasnikov, Boris F; Melik-Nubarov, Nickolay S; Zorova, Lubava D; Kuzminova, Alevtina E; Isaev, Nickolay K; Cooper, Arthur J L; Zorov, Dmitry B

    2011-05-01

    A synthetic polyanion composed of styrene, maleic anhydride, and methacrylic acid (molar ratio 56:37:7) significantly inhibited the respiration of isolated rat liver mitochondria in a time-dependent fashion that correlated with 1) collapse of the mitochondrial membrane potential and 2) high amplitude mitochondrial swelling. The process is apparently Ca(2+) dependent. Since it is blocked by cyclosporin A, the process is ascribed to induction of the mitochondrial permeability transition. In mitoplasts, i.e., mitochondria lacking their outer membranes, the polyanion rapidly blocked respiration. After incubation of rat liver mitochondria with the polyanion, cytochrome c was released into the incubation medium. In solution, the polyanion modified by conjugation with fluorescein formed a complex with cytochrome c. Addition of the polyanion to cytochrome c-loaded phosphatidylcholine/cardiolipin liposomes induced the release of the protein from liposomal membrane evidently due to coordinated interplay of Coulomb and hydrophobic interactions of the polymer with cytochrome c. We conclude that binding of the polyanion to cytochrome c renders it inactive in the respiratory chain due to exclusion from its native binding sites. Apparently, the polyanion interacts with cytochrome c in mitochondria and releases it to the medium through breakage of the outer membrane as a result of severe swelling. Similar properties were demonstrated for the natural polyanion, tobacco mosaic virus RNA. An electron microscopy study confirmed that both polyanions caused mitochondrial swelling. Exposure of cerebellar astroglial cells in culture to the synthetic polyanion resulted in cell death, which was associated with nuclear fragmentation.

  20. Cumene hydroperoxide effected hydroperoxidation by cytochrome P-450.

    PubMed

    Chen, C; Gurka, D P

    1985-04-01

    9-Methylfluorene was found to be oxygenated to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene by cytochrome P-450 in the presence of cumene hydroperoxide. Molecular oxygen is required and carbon monoxide is inhibitory. The reaction is inhibited by SKF-525A and metyrapone. Metyrapone and cumene hydroperoxide also retard the conversion of 9-hydroperoxy-9-methylfluorene to 9-hydroxy-9-methylfluorene. The reaction is different from hydroperoxide-supported oxygenation, since the cumene hydroperoxide appears to act as an effector of the enzyme rather than oxygen donor. It is suggested that substrates with stable radicals can be dioxygenated in this manner.

  1. Cytochrome c Negatively Regulates NLRP3 Inflammasomes

    PubMed Central

    Shi, Chong-Shan; Kehrl, John H.

    2016-01-01

    The release of cytochrome c from the inner mitochondrial membrane, where it is anchored by caridolipin, triggers the formation of the Apaf-1 apoptosome. Cardiolipin also interacts with NLRP3 recruiting NLRP3 to mitochondria and facilitating inflammasome assembly. In this study we investigated whether cytosolic cytochrome c impacts NLRP3 inflammasome activation in macrophages. We report that cytochrome c binds to the LRR domain of NLRP3 and that cytochrome c reduces the interactions between NLRP3 and cardiolipin and between NLRP3 and NEK7, a recently recognized component of the NLRP3 inflammasome needed for NLRP3 oligomerization. Protein transduction of cytochrome c impairs NLRP3 inflammasome activation, while partially silencing cytochrome c expression enhances it. The addition of cytochrome c to an in vitro inflammasome assay severely limited caspase-1 activation. We propose that there is a crosstalk between the NLRP3 inflammasome and apoptosome pathways mediated by cytochrome c, whose release during apoptosis acts to limit NLRP3 inflammasome activation. PMID:28030552

  2. PROPICONAZOLE-INDUCED CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RAT AND MOUSE LIVER

    EPA Science Inventory

    Conazoles are N-substituted azole antifungal agents used as both pesticides and drugs. Some of these compounds are hepatocarcinogenic in mice and some can induce thyroid tumors in rats. Many of these compounds are able to induce and/or inhibit mammalian hepatic cytochrome P450s t...

  3. PROPICONAZOLE-INDUCED CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RAT AND MOUSE LIVER

    EPA Science Inventory

    Conazoles are N-substituted azole antifungal agents used as both pesticides and drugs. Some of these compounds are hepatocarcinogenic in mice and some can induce thyroid tumors in rats. Many of these compounds are able to induce and/or inhibit mammalian hepatic cytochrome P450s t...

  4. Amino acid sequences of bacterial cytochromes c' and c-556.

    PubMed Central

    Ambler, R P; Bartsch, R G; Daniel, M; Kamen, M D; McLellan, L; Meyer, T E; Van Beeumen, J

    1981-01-01

    The cytochrome c' are electron transport proteins widely distributed in photosynthetic and aerobic bacteria. We report the amino acid sequences of the proteins from 12 different bacterial species, and we show by sequences that the cytochromes c-556 from 2 different bacteria are structurally related to the cytochromes c'. Unlike the mitochondrial cytochromes c, the heme binding site in the cytochromes c' and c-556 is near the COOH terminus. The cytochromes c-556 probably have a methionine sixth heme ligand located near the NH2 terminus, whereas the cytochromes c' may be pentacoordinate. Quantitative comparison of cytochrome c' and c-556 sequences indicates a relatively low 28% average identity. PMID:6273892

  5. Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase.

    PubMed

    Lehninger, A L; Reynafarje, B; Costa, L

    1985-01-01

    The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.

  6. Contribution of cytochrome P-450 omega-hydroxylase to altered arteriolar reactivity with high-salt diet and hypertension.

    PubMed

    Frisbee, J C; Falck, J R; Lombard, J H

    2000-05-01

    The present study evaluated the contribution of cytochrome P-450 omega-hydroxylase in modulating the reactivity of cremaster muscle arterioles in normotensive rats on high-salt (HS) and low-salt (LS) diet and in rats with reduced renal mass hypertension (RRM-HT). Changes in arteriolar diameter in response to ACh, sodium nitroprusside (SNP), ANG II, and elevated O(2) were measured via television microscopy under control conditions and following cytochrome P-450 omega-hydroxylase inhibition with 17-octadecynoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In normotensive rats on either LS or HS diet, resting tone was unaffected and arteriolar reactivity to ACh or SNP was minimally affected by cytochrome P-450 omega-hydroxylase inhibition. In RRM-HT rats, cytochrome P-450 omega-hydroxylase inhibition reduced resting tone and significantly enhanced arteriolar dilation to ACh and SNP. Treatment with 17-ODYA or DDMS inhibited arteriolar constriction to ANG II and O(2) in all the groups, although the degree of inhibition was greater in RRM-HT than in normotensive animals. These results suggest that metabolites of cytochrome P-450 omega-hydroxylase contribute to the altered reactivity of skeletal muscle arterioles to vasoconstrictor and vasodilator stimuli in RRM-HT.

  7. Maturation of Plastid c-type Cytochromes.

    PubMed

    Gabilly, Stéphane T; Hamel, Patrice P

    2017-01-01

    Cytochromes c are hemoproteins, with the prosthetic group covalently linked to the apoprotein, which function as electron carriers. A class of cytochromes c is defined by a CXXCH heme-binding motif where the cysteines form thioether bonds with the vinyl groups of heme. Plastids are known to contain up to three cytochromes c. The membrane-bound cytochrome f and soluble cytochrome c6 operate in photosynthesis while the activity of soluble cytochrome c6A remains unknown. Conversion of apo- to holocytochrome c occurs in the thylakoid lumen and requires the independent transport of apocytochrome and heme across the thylakoid membrane followed by the stereospecific attachment of ferroheme via thioether linkages. Attachment of heme to apoforms of plastid cytochromes c is dependent upon the products of the CCS (for cytochrome csynthesis) genes, first uncovered via genetic analysis of photosynthetic deficient mutants in the green alga Chlamydomonas reinhardtii. The CCS pathway also occurs in cyanobacteria and several bacteria. CcsA and CCS1, the signature components of the CCS pathway are polytopic membrane proteins proposed to operate in the delivery of heme from the stroma to the lumen, and also in the catalysis of the heme ligation reaction. CCDA, CCS4, and CCS5 are components of trans-thylakoid pathways that deliver reducing equivalents in order to maintain the heme-binding cysteines in a reduced form prior to thioether bond formation. While only four CCS components are needed in bacteria, at least eight components are required for plastid cytochrome c assembly, suggesting the biochemistry of thioether formation is more nuanced in the plastid system.

  8. A world of cytochrome P450s.

    PubMed

    Nelson, David R

    2013-02-19

    The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450.

  9. Aryl Hydroxylation of the Herbicide Diclofop by a Wheat Cytochrome P-450 Monooxygenase 1

    PubMed Central

    Zimmerlin, Alfred; Durst, Francis

    1992-01-01

    Wheat (Triticum aestivum L. cv Etoile de Choisy) microsomes catalyzed the cytochrome P-450-dependent oxidation of the herbicide diclofop to three hydroxy-diclofop isomers. Hydroxylation was predominant at carbon 4, with migration of chlorine to carbon 5 (67%) and carbon 3 (25%). The 2,4-dichloro-5-hydroxy isomer was identified as a minor reaction product (8%). Substrate-specificity studies showed that the activity was not inhibited or was weakly inhibited by a range of xenobiotic or physiological cytochrome P-450 substrates, with the exception of lauric acid. Wheat microsomes also catalyze the metabolism of the herbicides chlorsulfuron, chlortoluron, and 2,4-dichlorophenoxyacetic acid and of the model substrate ethoxycoumarin, as well as the hydroxylation of the endogenous substrates cinnamic and lauric acids. Treatments of wheat seedlings with phenobarbital or the safener naphthalic acid anhydride enhanced the cytochrome P-450 content of the microsomes and all related activities except that of cinnamic acid 4-hydroxylase, which was reduced. The stimulation patterns of diclofop aryl hydroxylase and lauric acid hydroxylase were similar, in contrast with the other activities tested. Lauric acid inhibited competitively (Ki = 9 μm) the oxidation of diclofop and reciprocally. The similarity of diclofop aryl hydroxylase and lauric acid hydroxylase was further investigated by alternative substrate kinetics, autocatalytic inactivation, and computer-aided molecular modelisation studies, and the results suggest that both reactions are catalyzed by the same cytochrome P-450 isozyme. PMID:16653070

  10. Comparative kinetics of Qi site inhibitors of cytochrome bc1 complex: picomolar antimycin and micromolar cyazofamid.

    PubMed

    Li, Hui; Zhu, Xiao-Lei; Yang, Wen-Chao; Yang, Guang-Fu

    2014-01-01

    Antimycin and cyazofamid are specific inhibitors of the mitochondrial respiratory chain and bind to the Qi site of the cytochrome bc1 complex. With the aim to understand the detailed molecular inhibition mechanism of Qi inhibitors, we performed a comparative investigation of the inhibitory kinetics of them against the porcine bc1 complex. The results showed that antimycin is a slow tight-binding inhibitor of succinate-cytochrome c reductase (SCR) with Ki  = 0.033 ± 0.00027 nm and non-competitive inhibition with respect to cytochrome c. Cyazofamid is a classical inhibitor of SCR with Ki  = 12.90 ± 0.91 μm and a non-competitive inhibitor with respect to cytochrome c. Both of them show competitive inhibition with respect to substrate DBH2 . Further molecular docking and quantum mechanics calculations were performed. The results showed that antimycin underwent significant conformational change upon the binding. The energy barrier between the conformations in the crystal and in the binding pocket is ~13.63 kcal/mol. Antimycin formed an H-bond with Asp228 and two water-bridged H-bonds with Lys227 and His201, whereas cyazofamid formed only one H-bond with Asp228. The conformational change and the different hydrogen bonding network might account for why antimycin is a slow tight-binding inhibitor, whereas cyazofamid is a classic inhibitor.

  11. Role of cytochrome P450 in drug interactions.

    PubMed

    Bibi, Zakia

    2008-10-18

    Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events.

  12. Role of cytochrome P450 in drug interactions

    PubMed Central

    Bibi, Zakia

    2008-01-01

    Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events. PMID:18928560

  13. Anthocyanins block ischemia-induced apoptosis in the perfused heart and support mitochondrial respiration potentially by reducing cytosolic cytochrome c.

    PubMed

    Skemiene, Kristina; Rakauskaite, Gintare; Trumbeckaite, Sonata; Liobikas, Julius; Brown, Guy C; Borutaite, Vilmante

    2013-01-01

    Anthocynanins, found in fruits and vegetables, have a variety of protective properties, which have generally been attributed to their antioxidant capacity. However, antioxidants are generally strong reductants, and some reductants have been found to block apoptosis by reducing cytosolic cytochrome c, which prevents caspase activation. We tested the ability of various anthocyanins: to reduce cytochrome c, to support cytochrome c-induced mitochondrial respiration and to inhibit apoptosis induced by heart ischemia. Anthocyanins such as delphinidin-3-glucoside (Dp3G) and cyanidin-3-glucoside (Cy3G) were able to reduce cytochrome c directly and rapidly, whereas pelargonidin-3-glucoside (Pg3G), malvinidin-3-glucoside (Mv3G) and peonidin-3-glucoside (Pn3G) had relatively low cytochrome c reducing activities. Dp3G and Cy3G but not Pg3G supported mitochondrial state 4 respiration in the presence of exogenous cytochrome c. Pre-perfusion of hearts with 20 μM Cy3G but not Pg3G prevented ischemia-induced caspase activation. This suggests that the ability of anthocyanins to block caspase activation may be due to their ability to reduce cytosolic cytochrome c. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Cyclosporin A-induced free radical generation is not mediated by cytochrome P-450

    PubMed Central

    Krauskopf, Alexandra; Buetler, Timo M; Nguyen, Nathalie S D; Macé, Katherine; Ruegg, Urs T

    2002-01-01

    Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA). The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS. We show that CsA (1 – 10 μM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7-dichlorofluorescin diacetate. Using cytochrome c as substrate, we show that CsA (10 μM) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. CsA (10 μM) did not uncouple the electron flow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. CsA (10 μM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes. PMID:11861326

  15. Canine cytochrome P450 (CYP) pharmacogenetics

    PubMed Central

    Court, Michael H.

    2013-01-01

    Synopsis The cytochrome P450 (CYP) drug metabolizing enzymes are essential for the efficient elimination of many clinically used drugs. These enzymes typically display high interindividual variability in expression and function resulting from enzyme induction, inhibition, and genetic polymorphism thereby predisposing patients to adverse drug reactions or therapeutic failure. There are also substantial species differences in CYP substrate specificity and expression that complicate direct extrapolation of information from humans to veterinary species. This article reviews the available published data regarding the presence and impact of genetic polymorphisms on CYP-dependent drug metabolism in dogs in the context of known human-dog CYP differences. Canine CYP1A2, which metabolizes phenacetin, caffeine, and theophylline, is the most widely studied polymorphic canine CYP. A single nucleotide polymorphism resulting in a CYP1A2 premature stop codon (c.1117C>T; R383X) with a complete lack of enzyme is highly prevalent in certain dog breeds including Beagle and Irish wolfhound. This polymorphism was shown to substantially affect the pharmacokinetics of several experimental compounds in Beagles during preclinical drug development. However, the impact on the pharmacokinetics of phenacetin (a substrate specific for human CYP1A2) was quite modest probably because other canine CYPs are capable of metabolizing phenacetin. Other canine CYPs with known genetic polymorphisms include CYP2C41 (gene deletion), as well as CYP2D15, CYP2E1, and CYP3A12 (coding SNPs). However the impact of these variants on drug metabolism in vitro or on drug pharmacokinetics is unknown. Future systematic investigations are needed to comprehensively identify CYP genetic polymorphisms that are predictive of drug effects in canine patients. PMID:23890236

  16. Cannabinoids and Cytochrome P450 Interactions.

    PubMed

    Zendulka, Ondřej; Dovrtělová, Gabriela; Nosková, Kristýna; Turjap, Miroslav; Šulcová, Alexandra; Hanuš, Lumír; Juřica, Jan

    2016-01-01

    This review consists of three parts, representing three different possibilities of interactions between cannabinoid receptor ligands of both exogenous and endogenous origin and cytochrome P450 enzymes (CYPs). The first part deals with cannabinoids as CYP substrates, the second summarizes current knowledge on the influence of various cannabinoids on the metabolic activity of CYP, and the third outline a possible involvement of the endocannabinoid system and cannabinoid ligands in the regulation of CYP liver activity. We performed a structured search of bibliographic and drug databases for peer-reviewed literature using focused review questions. Biotransformation via a hydrolytic pathway is the major route of endocannabinoid metabolism and the deactivation of substrates is characteristic, in contrast to the minor oxidative pathway via CYP involved in the bioactivation reactions. Phytocannabinoids are extensively metabolized by CYPs. The enzymes CYP2C9, CYP2C19, and CYP3A4 catalyze most of their hydroxylations. Similarly, CYP represents a major metabolic pathway for both synthetic cannabinoids used therapeutically and drugs that are abused. In vitro experiments document the mostly CYP inhibitory activity of the major phytocannabinoids, with cannabidiol as the most potent inhibitor of many CYPs. The drug-drug interactions between cannabinoids and various drugs at the CYP level are reported, but their clinical relevance remains unclear. The direct activation/inhibition of nuclear receptors in the liver cells by cannabinoids may result in a change of CYP expression and activity. Finally, we hypothesize the interplay of central cannabinoid receptors with numerous nervous systems, resulting in a hormone-mediated signal towards nuclear receptors in hepatocytes.

  17. Role of cytochrome P sub 450 in the control of the production of erythropoietin

    SciTech Connect

    Fandrey, J.; Seydel, F.P.; Siegers, C.P.; Jelkmann, W. )

    1990-01-01

    Effects of agents affecting cytochrome P{sub 450} were studied on the production of erythropoietin (Epo) in cultures of the human hepatoma cell line HepG2. Epo was measured by radioimmunoassay of the culture media after 24 h of incubation. The addition of phenobarbital or 3-methylcholanthrene, which induce cytochrome P{sub 450}, significantly enhanced the formation of Epo. Likewise, the thyroid hormones T{sub 3} and T{sub 4} stimulated the rate of the production of Epo. On the other hand, the formation of Epo was lowered following the addition of diethyl-dithiocarbamate or cysteamine chloride, which inhibit cytochrome P{sub 450}. These findings support the idea that O{sub 2} sensitive hemoproteins of the microsomal mixed-functional oxidases play a role in the control of the synthesis of Epo.

  18. Cytochrome bd oxidase from Escherichia coli displays high catalase activity: an additional defense against oxidative stress.

    PubMed

    Borisov, Vitaliy B; Forte, Elena; Davletshin, Albert; Mastronicola, Daniela; Sarti, Paolo; Giuffrè, Alessandro

    2013-07-11

    Cytochrome bd oxygen reductase from Escherichia coli has three hemes, b558, b595 and d. We found that the enzyme, as-prepared or in turnover with O2, rapidly decomposes H2O2 with formation of approximately half a mole of O2 per mole of H2O2. Such catalase activity vanishes upon cytochrome bd reduction, does not compete with the oxygen-reductase activity, is insensitive to NO, CO, antimycin-A and N-ethylmaleimide (NEM), but is inhibited by cyanide (Ki ~2.5μM) and azide. The activity, possibly associated with heme-b595, was also observed in catalase-deficient E. coli cells following cytochrome bd over-expression suggesting a protective role against oxidative stress in vivo. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Molecular surveillance of mutations in the cytochrome b gene of Plasmodium falciparum in Gabon and Ethiopia

    PubMed Central

    Gebru, Tamirat; Hailu, Asrat; Kremsner, Peter G; Kun, Jürgen FJ; Grobusch, Martin P

    2006-01-01

    Background Atovaquone is part of the antimalarial drug combination atovaquone-proguanil (Malarone®) and inhibits the cytochrome bc1 complex of the electron transport chain in Plasmodium spp. Molecular modelling showed that amino acid mutations are clustered around a putative atovaquone-binding site resulting in a reduced binding affinity of atovaquone for plasmodial cytochrome b, thus resulting in drug resistance. Methods The prevalence of cytochrome b point mutations possibly conferring atovaquone resistance in Plasmodium falciparum isolates in atovaquone treatment-naïve patient cohorts from Lambaréné, Gabon and from South Western Ethiopia was assessed. Results Four/40 (10%) mutant types (four different single polymorphisms, one leading to an amino acid change from M to I in a single case) in Gabonese isolates, but all 141/141 isolates were wild type in Ethiopia were found. Conclusion In the absence of drug pressure, spontaneous and possibly resistance-conferring mutations are rare. PMID:17118179

  20. Cytochromes P460 and c'-beta; a new family of high-spin cytochromes c.

    PubMed

    Elmore, Bradley O; Bergmann, David J; Klotz, Martin G; Hooper, Alan B

    2007-03-06

    Cytochromes-P460 of Nitrosomonas europaea and Methylococcus capsulatus (Bath), and the cytochrome c' of M. capsulatus, believed to be involved in binding or transformation of N-oxides, are shown to represent an evolutionarily related new family of monoheme, approximately 17kDa, cytochromes c found in the genomes of diverse Proteobacteria. All members of this family have a predicted secondary structure predominantly of beta-sheets in contrast to the predominantly alpha-helical cytochromes c' found in photoheterotrophic and denitrifying Proteobacteria.

  1. A Mycobacterium tuberculosis cytochrome bd oxidase mutant is hypersensitive to bedaquiline.

    PubMed

    Berney, Michael; Hartman, Travis E; Jacobs, William R

    2014-07-15

    The new medicinal compound bedaquiline (BDQ) kills Mycobacterium tuberculosis by inhibiting F1Fo-ATP synthase. BDQ is bacteriostatic for 4 to 7 days and kills relatively slowly compared to other frontline tuberculosis (TB) drugs. Here we show that killing with BDQ can be improved significantly by inhibiting cytochrome bd oxidase, a non-proton-pumping terminal oxidase. BDQ was instantly bactericidal against a cytochrome bd oxidase null mutant of M. tuberculosis, and the rate of killing was increased by more than 50%. We propose that this exclusively bacterial enzyme should be a high-priority target for new drug discovery. Importance: A major drawback of current TB chemotherapy is its long duration. New drug regimens with rapid killing kinetics are desperately needed. Our study demonstrates that inhibition of a nonessential bacterial enzyme greatly improves the efficacy of the latest TB drug bedaquiline and emphasizes that screening for compounds with synergistic killing mechanisms is a promising strategy.

  2. Reaction of ozone with protein tryptophans: band III, serum albumin, and cytochrome C.

    PubMed

    Mudd, J B; Dawson, P J; Tseng, S; Liu, F P

    1997-02-15

    Treatment of red cell ghosts with ozone inhibited both AChE (marking the outside of the membrane) and G3PDH (marking the inside of the membrane). There was no change in tryptophan fluorescence of the ghosts after the ozone treatment. Band 3 protein was isolated from the ozone-treated ghosts. The protein was digested with trypsin to obtain water soluble peptides from the cytoplasmic N-terminal tail and the interhelical loops. Fluorescent peptides included GWVIHPLGLR from the outer loop between helices 7 and 8, and peptide WMEAAR from the N-terminal cytoplasmic tail. Neither one of these peptides was oxidized by ozone. This was true whether or not the ghosts were sealed. We conclude that the position of these tryptophans either in the membrane structure, or because of binding to other proteins in the cytoplasmic tail, protects them from oxidation by ozone. Treatment of horse heart cytochrome c with ozone did not change the absorbance spectrum in the heme region or the tryptophan absorbing region. HPLC of the ozone-treated cytochrome c showed that cytochrome c was being modified, indicated by a change in the elution time. Treatment of cytochrome c with ozone did not change the activity in the NADH-cytochrome c reductase assay. Digestion of the ozone-treated cytochrome c with trypsin gave peptides which demonstrated normal fluorescence. (Cytochrome c has abnormally low fluorescence, which is not changed by ozone exposure.) The peptides were separated by HPLC. The fluorescence of the tryptophan-containing peptide (GITWK) was not decreased by treatment of the cytochrome c by ozone. Amino acid analysis of the ozone-treated cytochrome c indicated that methionine was oxidized. We conclude that tryptophan in cytochrome c is protected from oxidation by ozone because of the interaction with the porphyrin ring. Bovine serum albumin and human serum albumin were treated with ozone. There was a monotonic decrease in tryptophan fluorescence in both cases. Digestion of BSA with

  3. Inhibition of Hageman factor activation

    PubMed Central

    Nossel, H. L.; Rubin, H.; Drillings, M.; Hsieh, R.

    1968-01-01

    A method for studying inhibitors of the contact stages of blood coagulation is described. A number of positively charged substances were shown to inhibit the contact stages. The inhibitory substances include spermine, cytochrome c, ribonuclease, and lysozyme. The inhibitory effect of these substances was neutralized by the addition of an activated plasma thromboplastin antecedent, factor XI, (PTA) fraction. Other positively charged substances including protamine, hexadimethrine, polylysine, polyornithine, methylene blue, and ortho-toluidine blue also inhibited the contact stages of coagulation, but the inhibitory effect on coagulation was not neutralized by the activated PTA fraction. Negatively charged substances such as heparin and insulin did not inhibit the contact stages of coagulation. Cytochrome c inhibited Celite adsorption of a partially purified Hageman factor fraction, and cytochrome, ribonuclease, spermine, and lysozome inhibited the adsorption of Hageman factor from PTA-deficient plasma. Very much smaller quantities of Celite completely adsorbed Hageman factor from the fraction rather than from whole plasma, which suggested the possibility that plasma contains an inhibitor or inhibitors of Hageman factor adsorption. Furthermore cytochrome c, spermine, ribonuclease, and lysozyme inhibited the coagulant activity of the following activators of the Hageman and PTA factors: Celite, kaolin, sodium stearate, ellagic acid, and skin. It is suggested that negatively charged sites on these activators are critical for adsorption and activation and that inhibition results from neutralization of the negatively charged sites by the adsorbed inhibtor. Tests with polylysine polymers indicate that inhibitory activity is directly related to molecular size over the molecular weight range of 4000 to 100,000. PMID:5645860

  4. Interaction of rocuronium with human liver cytochromes P450.

    PubMed

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-02-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  5. Metazoan cytochrome P450 evolution.

    PubMed

    Nelson, D R

    1998-11-01

    There are 37 cytochrome P450 families currently identified in animals. The concept of higher order groupings of P450 families called P450 CLANS is introduced. The mammalian CYP3 and CYP5 families belong to the same clan as insect CYP6 and CYP9. All mitochondrial P450s seem to belong to the same clan. Lack of mitochondrial P450s in C. elegans suggests that mitochondrial P450s probably arose from the mistargeting of a microsomal P450 after the coelomates diverged from acoelomates and pseudocoelomates. Different taxonomic groups appear to have recruited different ancestral P450s for expansion as they evolved, since each major taxon seems to have one large cluster of P450s. In insects, this cluster derives from the ancestor to the CYP4 family. Vertebrates and C. elegans may have used the same ancestor independently to generate the CYP1, 2, 17, and 21 families in vertebrates and a large distinctive clan with 45 genes in C. elegans.

  6. Cytochrome bd Displays Significant Quinol Peroxidase Activity

    PubMed Central

    Al-Attar, Sinan; Yu, Yuanjie; Pinkse, Martijn; Hoeser, Jo; Friedrich, Thorsten; Bald, Dirk; de Vries, Simon

    2016-01-01

    Cytochrome bd is a prokaryotic terminal oxidase that catalyses the electrogenic reduction of oxygen to water using ubiquinol as electron donor. Cytochrome bd is a tri-haem integral membrane enzyme carrying a low-spin haem b558, and two high-spin haems: b595 and d. Here we show that besides its oxidase activity, cytochrome bd from Escherichia coli is a genuine quinol peroxidase (QPO) that reduces hydrogen peroxide to water. The highly active and pure enzyme preparation used in this study did not display the catalase activity recently reported for E. coli cytochrome bd. To our knowledge, cytochrome bd is the first membrane-bound quinol peroxidase detected in E. coli. The observation that cytochrome bd is a quinol peroxidase, can provide a biochemical basis for its role in detoxification of hydrogen peroxide and may explain the frequent findings reported in the literature that indicate increased sensitivity to hydrogen peroxide and decreased virulence in mutants that lack the enzyme. PMID:27279363

  7. Chemoreceptor discharges and cytochrome redox changes of the rat carotid body: Role of heme ligands

    PubMed Central

    Lahiri, Sukhamay; Ehleben, Wilhelm; Acker, Helmut

    1999-01-01

    In superfused in vitro rat carotid body, we recorded chemoreceptor discharges and the redox state of cytochromes simultaneously to identify the primary oxygen-sensing protein controlling transmitter release and electrical activity of the carotid sinus nerve. These parameters were tested under the influence of heme ligands such as oxygen, cyanide, 4-(2-aminoethyl)-benzenesulfonyl fluoride, and CO. During stimulation, there was an initial increase in discharge frequency followed by a decline or suppression of activity. Photometric changes lagged and were maintained as nerve activity decreased. Reducing mitochondrial cytochromes by cyanide or prolonged severe hypoxia, suppressed the chemoreceptor discharge. 4-(2-Aminoethyl)-benzenesulfonyl fluoride, a specific inhibitor of the phagocytic cytochrome b558, also silenced the chemoreceptors after an initial excitation. CO increased the chemoreceptor discharge under normoxia, an effect inhibited by light, when the cytochromes were not reduced. When the discharges were depressed by severe hypoxia, exposure to light excited the chemoreceptors and the cytochromes were reduced. The rapidity of the chemosensory responses to light and lack of effect on dopamine release from type I cells led us to hypothesize that carotid body type I cells and the apposed nerve endings use different mechanisms for oxygen sensing: the nerve endings generate action potentials in association with membrane heme proteins whereas cytosolic heme proteins signal the redox state, releasing modulators or transmitters from type I cells. PMID:10430959

  8. Kinetics of flavin semiquinone reduction of the components of the cytochrome c-cytochrome b5 complex.

    PubMed

    Eltis, L; Mauk, A G; Hazzard, J T; Cusanovich, M A; Tollin, G

    1988-07-26

    The kinetics of flavin semiquinone reduction of the components of the 1:1 complex formed by cytochrome c with either cytochrome b5 or a derivative of cytochrome b5 in which the heme propionates are esterified (DME-cytochrome b5) have been studied. The rate constant for the reduction of horse heart cytochrome c by the electrostatically neutral lumiflavin semiquinone (LfH) is unaffected by complexation with native cytochrome b5 at pH 7. However, complex formation with DME-cytochrome b5 (pH 7) decreases by 35% the rate constant for cytochrome c reduction by LfH. At pH 8, complex formation with native cytochrome b5 decreases the rate constant for cytochrome c reduction by LfH markedly, whereas the rate constant for cytochrome c reduction, either unbound or in the complex formed with DME-cytochrome b5, is increased 2-fold relative to pH 7. These results indicate that the accessibility of the cytochrome c heme is not the same in the complexes formed with the two cytochrome b5 derivatives and that the docking geometry of the complex formed by the two native cytochromes is pH dependent. Binding of horse heart and tuna cytochromes c to native and DME-cytochromes b5 decreases the rate constants for reduction of cytochrome c by the negatively charged flavin mononucleotide semiquinone (FMNH) by approximately 30% and approximately 40%, respectively. This finding is attributed to substantial neutralization of the positive electrostatic potential surface of cytochrome c that occurs when it binds to either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Pharmacological profile of the abeorphine 201-678, a potent orally active and long lasting dopamine agonist

    SciTech Connect

    Jaton, A.L.; Giger, R.K.A.; Vigouret, J.M.; Enz, A.; Frick, W.; Closse, A.; Markstein, R.

    1986-01-13

    The central dopaminergic effects of an abeorphine derivative 201-678 were compared to those of apomorphine and bromocriptine in different model systems. After oral administration, this compound induced contralateral turning in rats with 6-hydroxydopamine induced nigral lesions and exhibited strong anti-akinetic properties in rats with 6-hydroxydopamine induced hypothalamic lesions. It decreased dopamine metabolism in striatum and cortex, but did not modify noradrenaline and serotonin metabolism in the rat brain. 201-678 counteracted the in vivo increase of tyrosine hydroxylase activity induced by ..gamma..-butyrolactone. In vitro it stimulated DA-sensitive adenylate cyclase and inhibited acetylcholine release from rat striatal slices. This compound had high affinity for /sup 3/H-dopamine and /sup 3/H-clonidine binding sites. These results indicate that 201-678 is a potent, orally active dopamine agonist with a long duration of action. Furthermore it appears more selective than other dopaminergic drugs. 29 references, 5 figures, 3 tables.

  10. Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

    PubMed

    Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

    2013-01-01

    Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems.

  11. Control of proteoliposomal cytochrome c oxidase: the overall reaction.

    PubMed

    Nicholls, P; Cooper, C E; Wrigglesworth, J M

    1990-09-01

    The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (delta psi) and pH gradient (delta pH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse delta psi and increase delta pH; the respiration rate decreases. High levels of valinomycin, however, decrease delta pH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases delta psi and collapses delta pH; the respiration rate increases. On a millivolt equivalent basis delta pH is a more effective inhibitor of activity than is delta psi. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both delta pH and delta psi components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing delta pH but in a nonlinear way to delta psi ("non-ohmically"). High levels of both delta psi and delta pH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both delta pH and delta psi components of the proton-motive force, but is more sensitive to delta pH than to delta psi at an equivalent delta mu H+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella.

  12. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    PubMed

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two bin