Quantification of tamoxifen and three of its phase-I metabolites in human plasma by liquid chromatography/triple-quadrupole mass spectrometry.

PubMed

Binkhorst, Lisette; Mathijssen, Ron H J; Ghobadi Moghaddam-Helmantel, Inge M; de Bruijn, Peter; van Gelder, Teun; Wiemer, Erik A C; Loos, Walter J

2011-12-15

In view of future pharmacokinetic studies, a highly sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous quantification of tamoxifen and three of its main phase I metabolites in human lithium heparinized plasma. The analytical method has been thoroughly validated in agreement with FDA recommendations. Plasma samples of 200 μl were purified by liquid-liquid extraction with 1 ml n-hexane/isopropanol, after deproteination through addition of 50 μl acetone and 50 μl deuterated internal standards in acetonitrile. Tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and endoxifen were chromatographically separated on an Acquity UPLC(®) BEH C18 1.7 μm 2.1 mm×100 mm column eluted at a flow-rate of 0.300 ml/min on a gradient of 0.2mM ammonium formate and acetonitrile, both acidified with 0.1% formic acid. The overall run time of the method was 10 min, with elution times of 2.9, 3.0, 4.1 and 4.2 min for endoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen and tamoxifen, respectively. Tamoxifen and its metabolites were quantified by triple-quadrupole mass spectrometry in the positive ion electrospray ionization mode. The multiple reaction monitoring transitions were set at 372>72 (m/z) for tamoxifen, 358>58 (m/z) for N-desmethyl-tamoxifen, 388>72 (m/z) for 4-hydroxy-tamoxifen and 374>58 (m/z) for endoxifen. The analytical method was highly sensitive with the lower limit of quantification validated at 5.00 nM for tamoxifen and N-desmethyl-tamoxifen and 0.500 nM for 4-hydroxy-tamoxifen and endoxifen, which is equivalent to 1.86, 1.78, 0.194 and 0.187 ng/ml for tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and endoxifen, respectively. The method was also precise and accurate, with within-run and between-run precisions within 12.0% and accuracy ranging from 89.5 to 105.3%. The method has been applied to samples from a clinical study and cross-validated with a validated LC

4-Hydroxylated metabolites of the antiestrogens tamoxifen and toremifene are metabolized to unusually stable quinone methides.

PubMed

Fan, P W; Zhang, F; Bolton, J L

2000-01-01

Tamoxifen is widely prescribed for the treatment of hormone-dependent breast cancer, and it has recently been approved by the Food and Drug Administration for the chemoprevention of this disease. However, long-term usage of tamoxifen has been linked to increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. The resulting quinone methide has the potential to alkylate DNA and may initiate the carcinogenic process. To further probe the chemical reactivity and toxicity of such an electrophilic species, we have prepared the 4-hydroxytamoxifen quinone methide chemically and enzymatically, examined its reactivity under physiological conditions, and quantified its reactivity with GSH. Interestingly, this quinone methide is unusually stable; its half-life under physiological conditions is approximately 3 h, and its half-life in the presence of GSH is approximately 4 min. The reaction between 4-hydroxytamoxifen quinone methide and GSH appears to be a reversible process because the quinone methide GSH conjugates slowly decompose over time, regenerating the quinone methide as indicated by LC/MS/MS data. The tamoxifen GSH conjugates were detected in microsomal incubations with 4-hydroxytamoxifen; however, none were observed in breast cancer cell lines (MCF-7) perhaps because very little quinone methides is formed. Toremifene, which is a chlorinated analogue of tamoxifen, undergoes similar oxidative metabolism to give 4-hydroxytoremifene, which is further oxidized to the corresponding quinone methide. The toremifene quinone methide has a half-life of approximately 1 h under physiological conditions, and its rate of reaction in the presence of excess GSH is approximately 6 min. More detailed analyses have indicated that the 4-hydroxytoremifene quinone methide reacts with two

Formulation of Anti-miR-21 and 4-Hydroxytamoxifen Co-loaded Biodegradable Polymer Nanoparticles and Their Antiproliferative Effect on Breast Cancer Cells

PubMed Central

2015-01-01

Breast cancer is the second leading cause of cancer-related death in women. The majority of breast tumors are estrogen receptor-positive (ER+) and hormone-dependent. Neoadjuvant anti-estrogen therapy has been widely employed to reduce tumor mass prior to surgery. Tamoxifen is a broadly used anti-estrogen for early and advanced ER+ breast cancers in women and the most common hormone treatment for male breast cancer. 4-Hydroxytamoxifen (4-OHT) is an active metabolite of tamoxifen that functions as an estrogen receptor antagonist and displays higher affinity for estrogen receptors than that of tamoxifen and its other metabolites. MicroRNA-21 (miR-21) is a small noncoding RNA of 23 nucleotides that regulates several apoptotic and tumor suppressor genes and contributes to chemoresistance in numerous cancers, including breast cancer. The present study investigated the therapeutic potential of 4-OHT and anti-miR-21 coadministration in an attempt to combat tamoxifen resistance, a common problem often encountered in anti-estrogen therapy. A biodegradable poly(d,l-lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) copolymer was utilized as a carrier to codeliver 4-OHT and anti-miR-21 to ER+ breast cancer cells. 4-OHT and anti-miR-21 co-loaded PLGA-b-PEG nanoparticles (NPs) were developed using emulsion-diffusion evaporation (EDE) and water-in-oil-in-water (w/o/w) double emulsion methods. The EDE method was found to be best method for 4-OHT loading, and the w/o/w method proved to be more effective for coloading NPs with anti-miR-21 and 4-OHT. The optimal NPs, which were prepared using the double emulsion method, were evaluated for their antiproliferative and apoptotic effects against MCF7, ZR-75-1, and BT-474 human breast cancer cells as well as against 4T1 mouse mammary carcinoma cells. We demonstrated that PLGA-b-PEG NP encapsulation significantly extended 4-OHT’s stability and biological activity compared to that of free 4-OHT. MTT assays indicated that

Mass spectrometric characterization of tamoxifene metabolites in human urine utilizing different scan parameters on liquid chromatography/tandem mass spectrometry.

PubMed

Mazzarino, Monica; de la Torre, Xavier; Di Santo, Roberto; Fiacco, Ilaria; Rosi, Federica; Botrè, Francesco

2010-03-01

Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were considered for the characterization of tamoxifene metabolites in human urine for anti-doping purpose. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 166, 152 and 129) and neutral loss scan (neutral loss of 72 Da and 58 Da) in positive ion mode were assessed to recognize common ions or common losses of tamoxifene metabolites. The applicability of these methods was checked first by infusion and then by the injection of solution of a mixture of reference standards of four tamoxifene metabolites available in our laboratory. The data obtained by the analyses of the mixture of the reference standards showed that the five methods used exhibited satisfactory results for all tamoxifene metabolites considered at a concentration level of 100 ng/mL, whereas the analysis of blank urine samples spiked with the same tamoxifene metabolites at the same concentration showed that the neutral loss scan of 58 Da lacked sufficient specificity and sensitivity. The limit of detection in urine of the compounds studied was in the concentration range 10-100 ng/mL, depending on the compound structure and on the selected product ion. The suitability of these approaches was checked by the analysis of urine samples collected after the administration of a single dose of 20 mg of tamoxifene. Six metabolites were detected: 4-hydroxytamoxifene, 3,4-dihydroxytamoxifene, 3-hydroxy-4-methoxytamoxifene, N-demethyl-4-hydroxytamoxifene, tamoxifene-N-oxide and N-demethyl-3-hydroxy-4-methoxytamoxifene, which is in conformity to our previous work using a time-of-flight (TOF) mass spectrometer in full scan acquisition mode. Copyright (c) 2010 John Wiley & Sons, Ltd.

Tamoxifen dose and serum concentrations of tamoxifen and six of its metabolites in routine clinical outpatient care.

PubMed

Jager, N G L; Rosing, H; Schellens, J H M; Linn, S C; Beijnen, J H

2014-02-01

A sensitive and selective HPLC-MS/MS assay was used to analyze steady-state serum concentrations of tamoxifen, N-desmethyltamoxifen (E)-endoxifen, (Z)-endoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen, and 4'-hydroxytamoxifen to support therapeutic drug monitoring (TDM) in patients treated with tamoxifen according to standard of care. When the (Z)-endoxifen serum concentration was below the predefined therapeutic threshold concentration of 5.9 ng/mL, the clinician was advised to increase the tamoxifen dose and to collect another serum sample. Paired serum samples from patients at one dose level at different time points during the tamoxifen treatment were used to assess the intra-patient variability. A total of 251 serum samples were analyzed, obtained from 205 patients. Of these patients, 197 used 20 mg tamoxifen per day and 8 patients used 10 mg/day. There was wide variability in tamoxifen and metabolite concentrations within the dosing groups. The threshold concentration for (Z)-endoxifen was reached in one patient (12 %) in the 10 mg group, in 153 patients (78 %) in the 20 mg group, and in 26 (96 %) of the patients who received a dose increase to 30 or 40 mg/day. Dose increase from 20 to 30 or 40 mg per day resulted in a significant increase in the mean serum concentrations of all analytes (p < 0.001). The mean intra-patient variability was between 10 and 20 % for all analytes. These results support the suitability of TDM for optimizing the tamoxifen treatment. It is shown that tamoxifen dose is related to (Z)-endoxifen exposure and increasing this dose leads to a higher serum concentration of tamoxifen and its metabolites. The low intra-patient variability suggests that only one serum sample is needed for TDM, making this a relatively noninvasive way to optimize the patient's treatment.

Role of human pregnane X receptor in tamoxifen- and 4-hydroxytamoxifen-mediated CYP3A4 induction in primary human hepatocytes and LS174T cells.

PubMed

Sane, Rucha S; Buckley, Donna J; Buckley, Arthur R; Nallani, Srikanth C; Desai, Pankaj B

2008-05-01

Previously we observed that the antiestrogens tamoxifen and 4-hydroxytamoxifen (4OHT) induce CYP3A4 in primary human hepatocytes and activate human pregnane X receptor (PXR) in cell-based reporter assays. Given the complex cross-talk between nuclear receptors, tissue-specific expression of CYP3A4, and the potential for tamoxifen and 4OHT to interact with a myriad of receptors, this study was undertaken to gain mechanistic insights into the inductive effects of tamoxifen and 4OHT. First, we observed that transfection of the primary cultures of human hepatocytes with PXR-specific small interfering RNA reduced the PXR mRNA expression and the extent of CYP3A4 induction by tamoxifen and 4OHT by 50%. Second, in LS174T colon carcinoma cells, which were observed to have significantly lower PXR expression relative to human hepatocytes, neither tamoxifen nor 4OHT induced CYP3A4. Third, N-desmethyltamoxifen, which did not induce CYP3A4 in human hepatocytes, also did not activate PXR in LS174T cells. We then used cell-based reporter assay to evaluate the effects of other receptors such as glucocorticoid receptor GR alpha and estrogen receptor ER alpha on the transcriptional activation of PXR. The cotransfection of GR alpha in LS174T cells augmented PXR activation by tamoxifen and 4OHT. On the other hand, the presence of ER alpha inhibited PXR-mediated basal activation of CYP3A4 promoter, possibly via competing for common cofactors such as steroid receptor coactivator 1 and glucocorticoid receptor interacting protein 1. Collectively, our findings suggest that the CYP3A4 induction by tamoxifen and 4OHT is primarily mediated by PXR but the overall stoichiometry of other nuclear receptors and transcription cofactors also contributes to the extent of the inductive effect.

Developing analytical approaches to explore the connection between endocrine-active pharmaceuticals in water to effects in fish

USGS Publications Warehouse

Jones-Lepp, Tammy L.; Taniguchi-Fu, Randi L.; Morgan, Jade; Nance Jr., Trevor; Ward, Matthew; Alvarez, David A.; Mills, Lesley

2015-01-01

The emphasis of this research project was to develop and optimize a solid-phase extraction method and highperformance liquid chromatography-electrospray ionizationmass spectrometry method, such that a linkage between the detection of endocrine-active pharmaceuticals (EAPs) in the aquatic environment and subsequent effects on fish populations could eventually be studied. Four EAPs were studied: tamoxifen (TAM), exemestane (EXE), letrozole (LET), anastrozole (ANA); and three TAM metabolites: 4- hydroxytamoxifen, e/z endoxifen, and n-desmethyl tamoxifen. In aqueous matrices, the use of isotopically labeled standards for the EAPs allowed for the generation of good recoveries, greater than 80 %, and low relative standard deviations (% RSDs) (3 to 27 %). TAM metabolites had lower recoveries in the spiked water matrices: 35 to 93 % in waste/source water compared to 58 to 110 % in DI water. The precision in DI water was acceptable ranging from 8 to 38 % RSD. However, the precision in real environmental wastewaters could be poor, ranging from 15 to 120 % RSD, dependent upon unique matrix effects. In plasma, the overall recoveries of the EAPs were acceptable: 88 to 110 %, with %RSDs of 6 to 18 % (Table 3). The spiked recoveries of the TAM metabolites from plasma were good, ranging from 77 to 120 %, with %RSDs ranging from 27 to 32 %. Two of the TAM metabolites, 4- hydroxytamoxifen and n-desmethyl tamoxifen, were confirmed in most of the environmental aqueous samples. The discovery of TAM metabolites demonstrates that the source of the TAM metabolites, TAM, is constant, introducing a pseudo-persistence of this chemical into the environment.

Green Tea Catechin Metabolites Exert Immunoregulatory Effects on CD4(+) T Cell and Natural Killer Cell Activities.

PubMed

Kim, Yoon Hee; Won, Yeong-Seon; Yang, Xue; Kumazoe, Motofumi; Yamashita, Shuya; Hara, Aya; Takagaki, Akiko; Goto, Keiichi; Nanjo, Fumio; Tachibana, Hirofumi

2016-05-11

Tea catechins, such as (-)-epigallocatechin-3-O-gallate (EGCG), have been shown to effectively enhance immune activity and prevent cancer, although the underlying mechanism is unclear. Green tea catechins are instead converted to catechin metabolites in the intestine. Here, we show that these green tea catechin metabolites enhance CD4(+) T cell activity as well as natural killer (NK) cell activity. Our data suggest that the absence of a 4'-hydroxyl on this phenyl group (B ring) is important for the effect on immune activity. In particular, 5-(3',5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5), a major metabolite of EGCG, not only increased the activity of CD4(+) T cells but also enhanced the cytotoxic activity of NK cells in vivo. These data suggest that EGC-M5 might show immunostimulatory activity.

Approaches for predicting effects of unintended environmental ...

EPA Pesticide Factsheets

Tamoxifen is an endocrine-active pharmaceutical (EAP) that is used world-wide. Because tamoxifen is a ubiquitous pharmaceutical and interacts with estrogen receptors, a case study was conducted with this compound to (1) determine effects on reproductive endpoints in a nontarget species (i.e., a fish), (2) compare biologically-active metabolites across species, (3) assess whether in vitro assays predict in vivo results, and (4) investigate metabolomic profiles in tamoxifen-treated fish to better understand the biological mechanisms of tamoxifen toxicity. In reproductive assays, tamoxifen exposure caused a significant reduction in egg production and significantly increased ovarian aromatase activity in spawning adult cunner fish (Tautogolabrus adspersus). In plasma from tamoxifen-exposed cunner, the predominant metabolite was 4-hydroxytamoxifen, while in rats it was N-desmethyltamoxifen. Because 4-hydroxytamoxifen is a more biologically active metabolite than N-desmethyltamoxifen, this difference could result in a different level of risk for the two species. The results of in vitro assays with fish hepatic microsomes to assess tamoxifen metabolism did not match in vivo results, indicating probable differences in excretion of tamoxifen metabolites in fish compared with rats. For the first time, a complete in vitro characterization of the metabolism of tamoxifen using fish microsomes is presented. Furthermore, a metabolomic investigation of cunner gonad extracts dem

Differences in metabolite-mediated toxicity of tamoxifen in rodents versus humans elucidated with DNA/microsome electro-optical arrays and nanoreactors.

PubMed

Zhao, Linlin; Krishnan, Sadagopan; Zhang, Yun; Schenkman, John B; Rusling, James F

2009-02-01

Tamoxifen, a therapeutic and chemopreventive breast cancer drug, was chosen as a model compound because of acknowledged species specific toxicity differences. Emerging approaches utilizing electro-optical arrays and nanoreactors based on DNA/microsome films were used to compare metabolite-mediated toxicity differences of tamoxifen in rodents versus humans. Hits triggered by liver enzyme metabolism were first provided by arrays utilizing a DNA damage end point. The arrays feature thin-film spots containing an electrochemiluminescent (ECL) ruthenium polymer ([Ru(bpy)(2)PVP(10)](2+); PVP, polyvinylpyridine), DNA, and liver microsomes. When DNA damage resulted from reactions with tamoxifen metabolites, it was detected by an increase in light from the oxidation of the damaged DNA by the ECL metallopolymer. The slope of ECL generation versus enzyme reaction time correlated with the rate of DNA damage. An approximate 2-fold greater ECL turnover rate was observed for spots with rat liver microsomes compared to that with human liver microsomes. These results were supported by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of reaction products using nanoreactors featuring analogous films on silica nanoparticles, allowing the direct measurement of the relative formation rate for alpha-(N(2)-deoxyguanosinyl)tamoxifen. We observed 2-5-fold more rapid formation rates for three major metabolites, i.e., alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and tamoxifen N-oxide, catalyzed by rat liver microsomes compared to human liver microsomes. Comparable formation rates were observed for N-desmethyl tamoxifen with rat and human liver microsomes. A better detoxifying capacity for human liver microsomes than rat liver microsomes was confirmed utilizing glucuronyltransferase in microsomes together with UDP-glucuronic acid. Taken together, lower genotoxicity and higher detoxication rates presented by human liver microsomes correlate with the lower risk of tamoxifen in

Endoxifen, 4-Hydroxytamoxifen and an Estrogenic Derivative Modulate Estrogen Receptor Complex Mediated Apoptosis in Breast Cancer.

PubMed

Maximov, Philipp Y; Abderrahman, Balkees; Fanning, Sean W; Sengupta, Surojeet; Fan, Ping; Curpan, Ramona F; Quintana Rincon, Daniela Maria; Greenland, Jeffery A; Rajan, Shyamala S; Greene, Geoffrey L; Jordan, V Craig

2018-05-08

Estrogen therapy was used to treat advanced breast cancer in postmenopausal women for decades until the introduction of tamoxifen. Resistance to long-term estrogen deprivation (LTED) with tamoxifen and aromatase inhibitors used as a treatment for breast cancer inevitably occurs, but unexpectedly low dose estrogen can cause regression of breast cancer and increase disease free survival in some patients. This therapeutic effect is attributed to estrogen-induced apoptosis in LTED breast cancer. Here we describe modulation of the estrogen receptor liganded with antiestrogens (endoxifen, 4-hydroxytamoxifen) and an estrogenic triphenylethylene (TPE) EthoxyTPE (EtOXTPE) on estrogen-induced apoptosis in LTED breast cancer cells. Our results show that the angular TPE estrogen (EtOXTPE) is able to induce the ER-mediated apoptosis only at a later time compared to planar estradiol in these cells. Using RT-PCR, ChIP, Western blotting, molecular modelling and X-ray crystallography techniques we report novel conformations of the ER complex with an angular estrogen EtOXTPE and endoxifen. We propose that alteration of the conformation of the ER complexes, with changes in coactivator binding, governs estrogen-induced apoptosis through the PERK sensor system to trigger an Unfolded Protein Response (UPR). The American Society for Pharmacology and Experimental Therapeutics.

Hot flashes are not predictive for serum concentrations of tamoxifen and its metabolites

PubMed Central

2013-01-01

Background Tamoxifen has dramatically reduced the recurrence and mortality rate of estrogen receptor positive breast cancer. However, the efficacy of tamoxifen varies between individuals and 40% of patients will have a recurrence despite adjuvant tamoxifen treatment. Factors that predict tamoxifen efficacy would be helpful for optimizing treatment. Serum concentrations of the active metabolite, endoxifen, may be positively related to treatment outcome. In addition, hot flashes are suggested to be positively associated with tamoxifen treatment outcome. Methods We investigated in a series of 109 patients whether the frequency and severity of hot flashes were related to concentrations of tamoxifen and its metabolites. A serum sample of all patients was analyzed for the concentration of tamoxifen, N-desmethyltamoxifen, endoxifen and 4-hydroxytamoxifen, as well as for estradiol concentrations and several single nucleotide polymorphisms in CYP2D6. Additionally, these patients completed a questionnaire concerning biometric data and treatment side effects. Results We found no evidence supporting an association between concentrations of tamoxifen or metabolites and either the frequency or severity of hot flashes in the covariate unadjusted analyses. However, including interactions with menopausal status and pre-treatment hot flash (PTHF) history indicated that post-menopausal women with PTHF experienced an increasing frequency of hot flashes with increasing serum concentrations of tamoxifen and its metabolites. This finding was not altered when adjusting for potential confounding factors (duration of tamoxifen treatment, CYP2D6 phenotype, estradiol serum concentration, age and body mass index). In addition we observed a positive association between body mass index and both hot flash frequency (p = 0.04) and severity (p < 0.0001). We also observed that patients with lower estradiol levels reported more severe hot flashes (p = 0.02). Conclusions No univariate

Evaluation of tamoxifen and metabolites by LC-MS/MS and HPLC methods.

PubMed

Heath, D D; Flat, S W; Wu, A H B; Pruitt, M A; Rock, C L

2014-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and its metabolites, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxytamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam), is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and its metabolites. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. This study analysed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high-performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentrations for the LC-MS/MS method (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC method (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108 [55] ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL) did not show any significant differences. The results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites.

Tamoxifen inhibits macrophage FABP4 expression through the combined effects of the GR and PPARγ pathways.

PubMed

Jiang, Meixiu; Zhang, Ling; Ma, Xingzhe; Hu, Wenquan; Chen, Yuanli; Yu, Miao; Wang, Qixue; Li, Xiaoju; Yin, Zhinan; Zhu, Yan; Gao, Xiumei; Hajjar, David P; Duan, Yajun; Han, Jihong

2013-09-15

Macrophage adipocyte fatty acid-binding protein (FABP4) plays an important role in foam cell formation and development of atherosclerosis. Tamoxifen inhibits this disease process. In the present study, we determined whether the anti-atherogenic property of tamoxifen was related to its inhibition of macrophage FABP4 expression. We initially observed that tamoxifen inhibited macrophage/foam cell formation, but the inhibition was attenuated when FABP4 expression was selectively inhibited by siRNA.We then observed that tamoxifen and 4-hydroxytamoxifen inhibited FABP4 protein expression in primary macrophages isolated from both the male and female wild-type mice, suggesting that the inhibition is sex-independent. Tamoxifen and 4-hydroxytamoxifen inhibited macrophage FABP4 protein expression induced either by activation of GR (glucocorticoid receptor) or PPARγ (peroxisome-proliferator-activated receptor γ). Associated with the decreased protein expression, Fabp4 mRNA expression and promoter activity were also inhibited by tamoxifen and 4-hydroxytamoxifen, indicating transcriptional regulation. Analysis of promoter activity and EMSA/ChIP assays indicated that tamoxifen and 4-hydroxytamoxifen activated the nGRE (negative glucocorticoid regulatory element), but inhibited the PPRE (PPARγ regulatory element) in the Fabp4 gene. In vivo, administration of tamoxifen to ApoE (apolipoprotein E)-deficient (apoE-/-) mice on a high-fat diet decreased FABP4 expression in macrophages and adipose tissues as well as circulating FABP4 levels. Tamoxifen also inhibited FABP4 protein expression by human blood monocyte-derived macrophages. Taken together, the results of the present study show that tamoxifen inhibited FABP4 expression through the combined effects of GR and PPARγ signalling pathways. Our findings suggest that the inhibition of macrophage FABP4 expression can be attributed to the antiatherogenic properties of tamoxifen.

Fast method for simultaneous quantification of tamoxifen and metabolites in dried blood spots using an entry level LC-MS/MS system.

PubMed

Tré-Hardy, Marie; Capron, Arnaud; Antunes, Marina Venzon; Linden, Rafael; Wallemacq, Pierre

2016-11-01

The purpose of this study was to develop and validate a new liquid chromatography-tandem mass spectrometric (LC-MSMS) assay for the simultaneous quantification of tamoxifen (TAM) and its main therapeutically active metabolites, N-desmethyltamoxifen (NDT), 4-hydroxytamoxifen (4HT) and endoxifen (END) in dried blood spots. Ultrasound assisted methanolic extraction was used for TAM and metabolites extraction from dried blood spot. After evaporation and methanol reconstitution, the extract was injected into a LC-MSMS system. Reversed phase chromatography was performed on a C18 grafted column in gradient mode. TAM, metabolites, and internal standard (diazepam-d 5 ; IS) were identified in positive electrospray ionization mode using m/z transition of 372.5>72.1 (TAM); 374.23>58.10 (END); 358.27>58.10 (NDT); 388.23>44.80 (4HT) and 290.00>198.00 (IS). Total analytical run time was 6.5min. Assay was linear from 1 to 500ng/mL for all substances and presented intra and inter-assay precision and accuracy <15%. TAM, NDT, 4HT and END limits of quantification and detection were of 1 and 0.5ng/mL; 1 and 3ng/mL; 1.7 and 3ng/mL; 0.6 and 2ng/mL, respectively. Recovery ranged from 83.8 to 96.3% with matrix effect ranged from 4.3 to 29.8% for TAM and its metabolites. Hematocrit value ≤40% appeared to negatively influence accuracy of the method. In conclusion, the method described here is somewhat accessible, relatively fast, sensitive and selective with no interference. This assay might be used to investigate the level of TAM and its metabolites in DBS for therapeutic drug monitoring purposes. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the g protein-coupled receptor GPR30.

PubMed

Vivacqua, Adele; Bonofiglio, Daniela; Albanito, Lidia; Madeo, Antonio; Rago, Vittoria; Carpino, Amalia; Musti, Anna Maria; Picard, Didier; Andò, Sebastiano; Maggiolini, Marcello

2006-10-01

The higher incidence of thyroid carcinoma (TC) in women during reproductive years compared with men and the increased risk associated with the therapeutic use of estrogens have suggested a pathogenetic role exerted by these steroids in the development of TC. In the present study, we evaluated the potential of 17beta-estradiol (E2), genistein (G), and 4-hydroxyta-moxifen (OHT) to regulate the expression of diverse estrogen target genes and the proliferation of human WRO, FRO, and ARO thyroid carcinoma cells, which were used as a model system. We have ascertained that ARO cells are devoid of estrogen receptors (ERs), whereas both WRO and FRO cells express a single variant of ERalpha that was neither transactivated, modulated, nor translocated into the nucleus upon treatment with ligands. However, E2, G, and OHT were able either to induce the transcriptional activity of c-fos promoter constructs, including those lacking the estrogen-responsive elements, or to increase c-fos, cyclin A, and D1 expression. It is noteworthy that we have demonstrated that the G protein-coupled receptor 30 (GPR30) and the mitogen-activated protein kinase (MAPK) pathway mediate both the up-regulation of c-fos and the growth response to E2, G, and OHT in TC cells studied, because these stimulatory effects were prevented by silencing GPR30 and using the MEK inhibitor 2'-amino-3'-methoxyflavone (PD 98059). Our findings provide new insight into the molecular mechanisms through which estrogens may induce the progression of TC.

Evaluation of Tamoxifen and metabolites by LC-MS/MS and HPLC Methods

PubMed Central

Heath, D.D.; Flatt, S.W.; Wu, A.H.B.; Pruitt, M.A.; Rock, C.L.

2015-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and the metabolites of tamoxifen, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxy-tamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam) is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and the metabolites of tamoxifen. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. We analyzed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentration for the LC-MS/MS (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108[55]ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL), the methods did not show any significant differences. Our results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites. PMID:24693573

Endoxifen and Other Metabolites of Tamoxifen Inhibit Human Hydroxysteroid Sulfotransferase 2A1 (hSULT2A1)

PubMed Central

Squirewell, Edwin J.; Qin, Xiaoyan

2014-01-01

Although tamoxifen is a successful agent for treatment and prevention of estrogen-dependent breast cancer, its use has been limited by the low incidence of endometrial cancer. Human hydroxysteroid sulfotransferase 2A1 (hSULT2A1) catalyzes the formation of an α-sulfooxy metabolite of tamoxifen that is reactive toward DNA, and this has been implicated in its carcinogenicity. Also, hSULT2A1 functions in the metabolism of steroid hormones such as dehydroepiandrosterone (DHEA) and pregnenolone (PREG). These roles of hSULT2A1 in steroid hormone metabolism and in generating a reactive metabolite of tamoxifen led us to examine its interactions with tamoxifen and several of its major metabolites. We hypothesized that metabolites of tamoxifen may regulate the catalytic activity of hSULT2A1, either through direct inhibition or through serving as alternate substrates for the enzyme. We found that 4-hydroxy-N-desmethyltamoxifen (endoxifen) is a potent inhibitor of hSULT2A1-catalyzed sulfation of PREG and DHEA, with Ki values of 3.5 and 2.8 μM, respectively. In the hSULT2A1-catalyzed sulfation of PREG, 4-hydroxytamoxifen (4-OHTAM) and N-desmethyltamoxifen (N-desTAM) exhibited Ki values of 12.7 and 9.8 μM, respectively, whereas corresponding Ki values of 19.4 and 17.2 μM were observed with DHEA as substrate. A Ki value of 9.1 μM was observed for tamoxifen-N-oxide with DHEA as substrate, and this increased to 16.9 μM for the hSULT2A1-catalyzed sulfation of PREG. Three metabolites were substrates for hSULT2A1, with relative sulfation rates of 4-OHTAM > N-desTAM > > endoxifen. These results may be useful in interpreting ongoing clinical trials of endoxifen and in improving the design of related molecules. PMID:25157097

Formononetin, a phyto-oestrogen, and its metabolites up-regulate interleukin-4 production in activated T cells via increased AP-1 DNA binding activity

PubMed Central

Park, Jin; Kim, Seung H; Cho, Daeho; Kim, Tae S

2005-01-01

Phyto-oestrogens are polyphenolic non-steroidal plant compounds with oestrogen-like biological activity. Phyto-oestrogens have many biological effects including oestrogen agonist/antagonist properties. However, the effect of phyto-oestrogens on allergic responses remains unclear. In this study we investigated whether formononetin, a phyto-oestrogen, and its metabolites, daidzein and equol, affect production of interleukin-4 (IL-4), a pro-inflammatory cytokine closely associated with allergic immune response, in primary CD4+ T cells and EL4 T lymphoma cells. Formononetin, daidzein and equol significantly enhanced IL-4 production from both CD4+ T cells and EL4 cells in a dose-dependent manner. Formononetin, daidzein and equol also enhanced IL-4 gene promoter activity in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this effect was impaired in EL4 cells transfected with an IL-4 promoter construct deleted of P4 site carrying nuclear factor of activated T cells (NF-AT) and activator protein-1 (AP-1) binding sites. In addition, formononetin, daidzein and equol increased AP-1 DNA binding activities while did not affect NF-AT DNA binding activities. The enhancing effects on IL-4 production and AP-1 DNA binding activities were abrogated by specific inhibitors for phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC) and p38 mitogen-activated protein kinase (MAPK), indicating that formononetin, daidzein and equol might enhance IL-4 production by increased activation of AP-1 through the PI3-K/PKC/p38 MAPK signalling pathway. These results suggest that phyto-oestrogens and some of their metabolites may increase allergic responses via the enhancement of IL-4 production in T cells. PMID:16108819

UPLC-MS/MS method for the determination of tobacco-specific biomarker NNAL, tamoxifen and its main metabolites in rat plasma.

PubMed

Xiong, Wei; Zhao, Jiajia; Wang, Ling; Jiang, Xuehua

2017-06-01

Cigarette smoke is known to interact with tamoxifen-metabolizing enzymes and transporters and potentially affect its treatment outcome. 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important metabolite of 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) because it is frequently used as a biomarker to assess human smoke exposure. In order to study the potential pharmacokinetic interaction between cigarette smoke and tamoxifen in rats a UPLC-MS/MS method for the simultaneous determination of NNAL and tamoxifen along with its metabolites in rat plasma has been developed and validated. Analytes were extracted with methanol and separated on a HSS T3 column by a gradient elution with the mobile phase consisting of acetonitrile and water. The lower limits of quantitation ranged from 0.05 to 0.62 ng/mL. Precisions showed RSD <15.8% and accuracy in the range 80.6-116.0%. Mean analyte recoveries ranged from 76.9 to 108.4%. The method was successfully applied to study the effects of cigarette smoke condensate (CSC), NNK and benzo(a)pyrene pre-treatment on the pharmacokinetics of tamoxifen and its metabolites in rats. Significant effects of CSC, NNK, benzo(a)pyrene were observed on pharmacokinetics of tamoxifen and its metabolites. We also found that plasma NNAL levels are statistically significant correlated with plasma 4-hydroxy-tamoxifen and endoxifen. Copyright © 2016 John Wiley & Sons, Ltd.

Activation of cryptic metabolite production through gene disruption: Dimethyl furan-2,4-dicarboxylate produced by Streptomyces sahachiroi.

PubMed

Simkhada, Dinesh; Zhang, Huitu; Mori, Shogo; Williams, Howard; Watanabe, Coran M H

2013-01-01

At least 65% of all small molecule drugs on the market today are natural products, however, re-isolation of previously identified and characterized compounds has become a serious impediment to the discovery of new bioactive natural products. Here, genetic knockout of an unusual non-ribosomal peptide synthetase (NRPS) C-PCP-C module, aziA2, is performed resulting in the accumulation of the secondary metabolite, dimethyl furan-2,4-dicarboxylate. The cryptic metabolite represents the first non-azinomycin related compound to be isolated and characterized from the soil bacterium, S. sahachiroi. The results from this study suggest that abolishing production of otherwise predominant natural products through genetic knockout may constitute a means to "activate" the production of novel secondary metabolites that would otherwise lay dormant within microbial genome sequences.

Pathway Activity Profiling (PAPi): from the metabolite profile to the metabolic pathway activity.

PubMed

Aggio, Raphael B M; Ruggiero, Katya; Villas-Bôas, Silas Granato

2010-12-01

Metabolomics is one of the most recent omics-technologies and uses robust analytical techniques to screen low molecular mass metabolites in biological samples. It has evolved very quickly during the last decade. However, metabolomics datasets are considered highly complex when used to relate metabolite levels to metabolic pathway activity. Despite recent developments in bioinformatics, which have improved the quality of metabolomics data, there is still no straightforward method capable of correlating metabolite level to the activity of different metabolic pathways operating within the cells. Thus, this kind of analysis still depends on extremely laborious and time-consuming processes. Here, we present a new algorithm Pathway Activity Profiling (PAPi) with which we are able to compare metabolic pathway activities from metabolite profiles. The applicability and potential of PAPi was demonstrated using a previously published data from the yeast Saccharomyces cerevisiae. PAPi was able to support the biological interpretations of the previously published observations and, in addition, generated new hypotheses in a straightforward manner. However, PAPi is time consuming to perform manually. Thus, we also present here a new R-software package (PAPi) which implements the PAPi algorithm and facilitates its usage to quickly compare metabolic pathways activities between different experimental conditions. Using the identified metabolites and their respective abundances as input, the PAPi package calculates pathways' Activity Scores, which represents the potential metabolic pathways activities and allows their comparison between conditions. PAPi also performs principal components analysis and analysis of variance or t-test to investigate differences in activity level between experimental conditions. In addition, PAPi generates comparative graphs highlighting up- and down-regulated pathway activity. These datasets are available in http://www.4shared

Development and validation of an UPLC-MS/MS method for the quantification of tamoxifen and its main metabolites in human scalp hair.

PubMed

Drooger, Jan C; Jager, Agnes; Lam, Mei-Ho; den Boer, Mathilda D; Sleijfer, Stefan; Mathijssen, Ron H J; de Bruijn, Peter

2015-10-10

The aim of this study was to validate an earlier developed high-performance highly sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for quantification of tamoxifen and its three main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen) in scalp hair. This non-invasive method might, by segmental analysis of hair, be useful in the determination of the concentration of drugs and its metabolites over time, which can be used to study a wide variety of clinical relevant questions. Hair samples (150-300 hair strands, cut as close to the scalp as possible from the posterior vertex region of the head) were collected from female patients taking tamoxifen 20mg daily (n=19). The analytes were extracted using a liquid-liquid extraction procedure with carbonate buffer at pH 8.8 and a mixture of n-hexane/isopropranol method, followed by UPLC-MS/MS chromatography, based on an earlier validated method. The calibration curves were linear in the range of 1.00-200 pmol for tamoxifen and N-desmethyl-tamoxifen, with lower limit of quantitation of 1.00 pmol and 0.100-20.0 pmol with lower limit of quantitation of 0.100 pmol for endoxifen and 4-hydroxy-tamoxifen. Assay performance was fair with a within-run and between-run variability less than 9.24 at the three quality control samples and less than 15.7 for the lower limit of quantitation. Importantly, a steep linear decline was observed from distal to proximal hair segments. Probably, this is due to UV exposure as we showed degradation of tamoxifen and its metabolites after exposure to UV-light. Furthermore, higher concentrations of tamoxifen were found in black hair samples compared to blond and brown hair samples. We conclude that measurement of the concentration of tamoxifen and its main metabolites in hair is possible, with the selective, sensitive, accurate and precise UPLC-MS/MS method. However, for tamoxifen, it seems not possible to determine

Effects of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites on cardiovascular function in conscious rats.

PubMed

Schindler, Charles W; Thorndike, Eric B; Blough, Bruce E; Tella, Srihari R; Goldberg, Steven R; Baumann, Michael H

2014-01-01

The cardiovascular effects produced by 3,4-methylenedioxymethamphetamine (MDMA; 'Ecstasy') contribute to its acute toxicity, but the potential role of its metabolites in these cardiovascular effects is not known. Here we examined the effects of MDMA metabolites on cardiovascular function in rats. Radiotelemetry was employed to evaluate the effects of s.c. administration of racemic MDMA and its phase I metabolites on BP, heart rate (HR) and locomotor activity in conscious male rats. MDMA (1-20 mg·kg(-1)) produced dose-related increases in BP, HR and activity. The peak effects on HR occurred at a lower dose than peak effects on BP or activity. The N-demethylated metabolite, 3,4-methylenedioxyamphetamine (MDA), produced effects that mimicked those of MDMA. The metabolite 3,4-dihydroxymethamphetamine (HHMA; 1-10 mg·kg(-1)) increased HR more potently and to a greater extent than MDMA, whereas 3,4-dihydroxyamphetamine (HHA) increased HR, but to a lesser extent than HHMA. Neither dihydroxy metabolite altered motor activity. The metabolites 4-hydroxy-3-methoxymethamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) did not affect any of the parameters measured. The tachycardia produced by MDMA and HHMA was blocked by the β-adrenoceptor antagonist propranolol. Our results demonstrate that HHMA may contribute significantly to the cardiovascular effects of MDMA in vivo. As such, determining the molecular mechanism of action of HHMA and the other hydroxyl metabolites of MDMA warrants further study. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Effects of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites on cardiovascular function in conscious rats

PubMed Central

Schindler, Charles W; Thorndike, Eric B; Blough, Bruce E; Tella, Srihari R; Goldberg, Steven R; Baumann, Michael H

2014-01-01

BACKGROUND AND PURPOSE The cardiovascular effects produced by 3,4-methylenedioxymethamphetamine (MDMA; ‘Ecstasy’) contribute to its acute toxicity, but the potential role of its metabolites in these cardiovascular effects is not known. Here we examined the effects of MDMA metabolites on cardiovascular function in rats. EXPERIMENTAL APPROACH Radiotelemetry was employed to evaluate the effects of s.c. administration of racemic MDMA and its phase I metabolites on BP, heart rate (HR) and locomotor activity in conscious male rats. KEY RESULTS MDMA (1–20 mg·kg−1) produced dose-related increases in BP, HR and activity. The peak effects on HR occurred at a lower dose than peak effects on BP or activity. The N-demethylated metabolite, 3,4-methylenedioxyamphetamine (MDA), produced effects that mimicked those of MDMA. The metabolite 3,4-dihydroxymethamphetamine (HHMA; 1–10 mg·kg−1) increased HR more potently and to a greater extent than MDMA, whereas 3,4-dihydroxyamphetamine (HHA) increased HR, but to a lesser extent than HHMA. Neither dihydroxy metabolite altered motor activity. The metabolites 4-hydroxy-3-methoxymethamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) did not affect any of the parameters measured. The tachycardia produced by MDMA and HHMA was blocked by the β-adrenoceptor antagonist propranolol. CONCLUSIONS AND IMPLICATIONS Our results demonstrate that HHMA may contribute significantly to the cardiovascular effects of MDMA in vivo. As such, determining the molecular mechanism of action of HHMA and the other hydroxyl metabolites of MDMA warrants further study. PMID:24328722

Estrogenic activities of diuron metabolites in female Nile tilapia (Oreochromis niloticus).

PubMed

Pereira, Thiago Scremin Boscolo; Boscolo, Camila Nomura Pereira; Felício, Andreia Arantes; Batlouni, Sergio Ricardo; Schlenk, Daniel; de Almeida, Eduardo Alves

2016-03-01

Some endocrine disrupting chemicals (EDCs) can alter the estrogenic activities of the organism by directly interacting with estrogen receptors (ER) or indirectly through the hypothalamus-pituitary-gonadal axis. Recent studies in male Nile tilapia (Oreochromis niloticus) indicated that diuron may have anti-androgenic activity augmented by biotransformation. In this study, the effects of diuron and three of its metabolites were evaluated in female tilapia. Sexually mature female fish were exposed for 25 days to diuron, as well as to its metabolites 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 3,4-dichlorophenyl-N-methylurea (DCPMU), at concentrations of 100 ng/L. Diuron metabolites caused increases in E2 plasma levels, gonadosomatic indices and in the percentage of final vitellogenic oocytes. Moreover, diuron and its metabolites caused a decrease in germinative cells. Significant differences in plasma concentrations of the estrogen precursor and gonadal regulator17α-hydroxyprogesterone (17α-OHP) were not observed. These results show that diuron metabolites had estrogenic effects potentially mediated through enhanced estradiol biosynthesis and accelerated the ovarian development of O. niloticus females. Copyright © 2015 Elsevier Ltd. All rights reserved.

Targeted conjugation of breast anticancer drug tamoxifen and its metabolites with synthetic polymers.

PubMed

Sanyakamdhorn, S; Agudelo, D; Bekale, L; Tajmir-Riahi, H A

2016-09-01

Conjugation of antitumor drug tamoxifen and its metabolites, 4-hydroxytamxifen and ednoxifen with synthetic polymers poly(ethylene glycol) (PEG), methoxypoly (ethylene glycol) polyamidoamine (mPEG-PAMAM-G3) and polyamidoamine (PAMAM-G4) dendrimers was studied in aqueous solution at pH 7.4. Multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling were used to characterize the drug binding process to synthetic polymers. Structural analysis showed that drug-polymer binding occurs via both H-bonding and hydrophobic contacts. The order of binding is PAMAM-G4>mPEG-PAMAM-G3>PEG-6000 with 4-hydroxttamoxifen forming more stable conjugate than tamoxifen and endoxifen. Transmission electron microscopy showed significant changes in carrier morphology with major changes in the shape of the polymer aggregate as drug encapsulation occurred. Modeling also showed that drug is located in the surface and in the internal cavities of PAMAM with the free binding energy of -3.79 for tamoxifen, -3.70 for 4-hydroxytamoxifen and -3.69kcal/mol for endoxifen, indicating of spontaneous drug-polymer interaction at room temperature. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation, synthesis, and pharmacology of metabolites of 1-(3,4-dichlorobenzyl)-3,4,5,6-tetrahydro-2(1H)-pyrimidone.

PubMed

Schwan, T J; Ellis, K O; Wessels, F L; Pugh, D; Bell, R

1980-10-01

The metabolism of 1-(3,4-dichlorobenzyl)-3,4,5,6-tetrahydro-2(1H)-pyrimidone, an antianxiety/antidepressant agent, in dogs is reported. Two metabolites, 3-[1-(3,4-dichlorobenzyl)-1-ureido]propanoic acid and 1-(3,4-dichlorobenzyl)uracil, were isolated, characterized, and synthesized. Neither metabolite was acutely toxic, and they did not exhibit antidepressant or antianxiety/anticonvulsant activity.

New brominated flame retardants and their metabolites as activators of the pregnane X receptor.

PubMed

Gramec Skledar, Darja; Tomašič, Tihomir; Carino, Adriana; Distrutti, Eleonora; Fiorucci, Stefano; Peterlin Mašič, Lucija

2016-09-30

The present study investigated the activities on different nuclear receptors of the new brominated flame retardants 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) and bis(2-ethylhexyl) 2,3,4,5-tetrabromophthalate (TBPH), and their main carboxylic acid metabolites 2,3,4,5-tetrabromobenzoic acid (TBBA) and mono(2-ethylhexyl) tetrabromophthalate (TBMEPH). None of selected chemicals exhibited marked activity towards PPARα and PPARγ by the use of transactivation assays in HepG2 cells transfected with peroxisome proliferator-activated receptors. In contrast, selected flame retardants all exhibited potent agonist activity on pregnane X receptor (PXR), with EC50 values of 5.5μM for TBPH and 2.0μM for its metabolite TBMEPH. Molecular docking of TBPH and TBMEPH to the PXR ligand binding site revealed similar interactions, with differences only for conformation and orientation of the alkyl chains. Additionally, TBPH showed antagonist activity on PXR (IC50, 13.9μM). Moreover, there was significant up-regulation of CYP3A4 expression via PXR activation for TBB and TBPH and their metabolites. Induction of CYP3A4 might cause undesired drug-drug interactions, lower bioavailability of pharmaceutical drugs, higher formation of reactive toxic metabolites, or enhanced elimination of endogenous hormones, such as T3/T4, to lead to endocrine disruption. These data provide new and important insights into the toxicity of these new polybrominated flame retardants, TBB and TBPH, and their metabolites. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Glucuronic acid and the ethanol metabolite ethyl-glucuronide cause Toll-like receptor 4 activation and enhanced pain

PubMed Central

Lewis, Susannah S.; Hutchinson, Mark R.; Zhang, Yingning; Hund, Dana K.; Maier, Steven F.; Rice, Kenner C.; Watkins, Linda R.

2013-01-01

We have previously observed that the non-opioid morphine metabolite, morphine-3-glucuronide, enhances pain via a toll-like receptor 4 (TLR4) dependent mechanism. The present studies were undertaken to determine whether TLR4-dependent pain enhancement generalizes to other classes of glucuronide metabolites. In silico modeling predicted that glucuronic acid alone and ethyl glucuronide, a minor but long-lasting ethanol metabolite, would dock to the same MD-2 portion of the TLR4 receptor complex previously characterized as the docking site for morphine-3-glucuronide. Glucuronic acid, ethyl glucuronide and ethanol all caused an increase in TLR4-dependent reporter protein expression in a cell line transfected with TLR4 and associated co-signaling molecules. Glucuronic acid-, ethyl glucuronide-, and ethanol-induced increases in TLR4 signaling were blocked by the TLR4 antagonists LPS-RS and (+)-naloxone. Glucuronic acid and ethyl glucuronide both caused allodynia following intrathecal injection in rats, which was blocked by intrathecal co-administration of the TLR4 antagonist LPS-RS. The finding that ethyl glucuronide can cause TLR4-dependent pain could have implications for human conditions such as hangover headache and alcohol withdrawal hyperalgesia, as well as suggesting that other classes of glucuronide metabolites could have similar effects. PMID:23348028

Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.

PubMed Central

Kitareewan, S; Burka, L T; Tomer, K B; Parker, C E; Deterding, L J; Stevens, R D; Forman, B M; Mais, D E; Heyman, R A; McMorris, T; Weinberger, C

1996-01-01

RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways. PMID:8856661

Relationship between Genotypes Sult1a2 and Cyp2d6 and Tamoxifen Metabolism in Breast Cancer Patients

PubMed Central

Fernández-Santander, Ana; Gaibar, María; Novillo, Apolonia; Romero-Lorca, Alicia; Rubio, Margarita; Chicharro, Luis Miguel; Tejerina, Armando; Bandrés, Fernando

2013-01-01

Tamoxifen is a pro-drug widely used in breast cancer patients to prevent tumor recurrence. Prior work has revealed a role of cytochrome and sulfotransferase enzymes in tamoxifen metabolism. In this descriptive study, correlations were examined between concentrations of tamoxifen metabolites and genotypes for CYP2D6, CYP3A4, CYP3A5, SULT1A1, SULT1A2 and SULT1E1 in 135 patients with estrogen receptor-positive breast cancer. Patients were genotyped using the Roche-AmpliChip® CYP450 Test, and Real-Time and conventional PCR-RFLP. Plasma tamoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen, endoxifen and tamoxifen-N-oxide were isolated and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Significantly higher endoxifen levels were detected in patients with the wt/wt CYP2D6 compared to the v/v CYP2D6 genotype (p<0.001). No differences were detected in the remaining tamoxifen metabolites among CYP2D6 genotypes. Patients featuring the SULT1A2*2 and SULT1A2*3 alleles showed significantly higher plasma levels of 4-hydroxy-tamoxifen and endoxifen (p = 0.025 and p = 0.006, respectively), as likely substrates of the SULT1A2 enzyme. Our observations indicate that besides the CYP2D6 genotype leading to tamoxifen conversion to potent hydroxylated metabolites in a manner consistent with a gene-dose effect, SULT1A2 also seems to play a role in maintaining optimal levels of both 4-hydroxy-tamoxifen and endoxifen. PMID:23922954

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

Pharmaceutically active secondary metabolites of marine actinobacteria.

PubMed

Manivasagan, Panchanathan; Venkatesan, Jayachandran; Sivakumar, Kannan; Kim, Se-Kwon

2014-04-01

Marine actinobacteria are one of the most efficient groups of secondary metabolite producers and are very important from an industrial point of view. Many representatives of the order Actinomycetales are prolific producers of thousands of biologically active secondary metabolites. Actinobacteria from terrestrial sources have been studied and screened since the 1950s, for many important antibiotics, anticancer, antitumor and immunosuppressive agents. However, frequent rediscovery of the same compounds from the terrestrial actinobacteria has made them less attractive for screening programs in the recent years. At the same time, actinobacteria isolated from the marine environment have currently received considerable attention due to the structural diversity and unique biological activities of their secondary metabolites. They are efficient producers of new secondary metabolites that show a range of biological activities including antibacterial, antifungal, anticancer, antitumor, cytotoxic, cytostatic, anti-inflammatory, anti-parasitic, anti-malaria, antiviral, antioxidant, anti-angiogenesis, etc. In this review, an evaluation is made on the current status of research on marine actinobacteria yielding pharmaceutically active secondary metabolites. Bioactive compounds from marine actinobacteria possess distinct chemical structures that may form the basis for synthesis of new drugs that could be used to combat resistant pathogens. With the increasing advancement in science and technology, there would be a greater demand for new bioactive compounds synthesized by actinobacteria from various marine sources in future. Copyright © 2013 Elsevier GmbH. All rights reserved.

Biologically Active Secondary Metabolites from the Fungi.

PubMed

Bills, Gerald F; Gloer, James B

2016-11-01

Many Fungi have a well-developed secondary metabolism. The diversity of fungal species and the diversification of biosynthetic gene clusters underscores a nearly limitless potential for metabolic variation and an untapped resource for drug discovery and synthetic biology. Much of the ecological success of the filamentous fungi in colonizing the planet is owed to their ability to deploy their secondary metabolites in concert with their penetrative and absorptive mode of life. Fungal secondary metabolites exhibit biological activities that have been developed into life-saving medicines and agrochemicals. Toxic metabolites, known as mycotoxins, contaminate human and livestock food and indoor environments. Secondary metabolites are determinants of fungal diseases of humans, animals, and plants. Secondary metabolites exhibit a staggering variation in chemical structures and biological activities, yet their biosynthetic pathways share a number of key characteristics. The genes encoding cooperative steps of a biosynthetic pathway tend to be located contiguously on the chromosome in coregulated gene clusters. Advances in genome sequencing, computational tools, and analytical chemistry are enabling the rapid connection of gene clusters with their metabolic products. At least three fungal drug precursors, penicillin K and V, mycophenolic acid, and pleuromutilin, have been produced by synthetic reconstruction and expression of respective gene clusters in heterologous hosts. This review summarizes general aspects of fungal secondary metabolism and recent developments in our understanding of how and why fungi make secondary metabolites, how these molecules are produced, and how their biosynthetic genes are distributed across the Fungi. The breadth of fungal secondary metabolite diversity is highlighted by recent information on the biosynthesis of important fungus-derived metabolites that have contributed to human health and agriculture and that have negatively impacted crops

4-Hydroxytamoxifen-stimulated processing of cyclin E is mediated via G protein-coupled receptor 30 (GPR30) and accompanied by enhanced migration in MCF-7 breast cancer cells.

PubMed

Li, Yang; Chen, Yan; Zhu, Zhu-Xia; Liu, Xiao-Hong; Yang, Li; Wan, Lei; Lei, Ting-Wen; Wang, Xu-Dong

2013-07-05

Over-expression of cleaved cyclin E in breast tumors is closely associated with tumor progression and resistance to antiestrogens. 17β-Estradiol (E2) has been recently shown to induce cyclin E processing in breast cancer cells. Tamoxifen has been used in patients with estrogen-sensitive breast cancer, yet resistance to antiestrogens and recurrence will appear in some of the patients after its continued use. We therefore addressed possible effects of tamoxifen on the generation of cleaved cyclin E and its signal mechanism(s) in estrogen-responsive MCF-7 breast cancer cells that express both G protein-coupled protein (GPR) 30 and estrogen receptor α (ERα). 4-Hydroxytamoxifen (OHT, tamoxifen's active form) failed to prevent E2-induced proteolysis of cyclin E and migration, but rather triggered cyclin E cleavage coincident with augmented migration. OHT-induced cyclin E truncation also occurred in SK-BR-3 cells that express GPR30 and lack ERα, but not in MDA-MB-231 cells that express neither GPR30 nor ERα. G1, a specific GPR 30 agonist, caused dramatic proteolysis of cyclin E and enhanced migration. Furthermore, OHT-stimulated cleavage of cyclin E and migration were tremendously attenuated by G15, a GPR30 antagonist, or siRNA against GPR30. In addition, inhibitors for EGFR or ERK1/2 remarkably suppressed OHT-induced truncation of cyclin E, suggesting involvement of EGFR signaling. Collectively, our data indicate that OHT contributes to the production of proteolyzed cyclin E via GPR30 with augmented migration in MCF-7 cells. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Secondary metabolites and insecticidal activity of Anemone pavonina.

PubMed

Varitimidis, Christos; Petrakis, Panos V; Vagias, Constantinos; Roussis, Vassilios

2006-01-01

The insecticidal properties of the crude extracts of the leaves and flowers of Anemone pavonina were evaluated on Pheidole pallidula ants and showed significant levels of activity. Bioassay-guided fractionations led to the isolation of the butenolide ranunculin (1) as the active principle. Chemical investigations of the extracts showed them to contain as major components the sitosterol glycopyranoside lipids 2-5 and the glycerides 6-8. The structures of the metabolites were elucidated, following acetylation and hydrolysis of the natural products, by interpretation of their NMR and mass spectral data. The uncommon lipid metabolites 2-8 were isolated for the first time from the genus Anemone and this is the first report of insecticidal activity of the Anemone metabolite ranunculin against ants.

Buprenorphine metabolites, buprenorphine-3-glucuronide and norbuprenorphine-3-glucuronide, are biologically active

PubMed Central

Brown, Sarah M.; Holtzman, Michael; Kim, Thomas; Kharasch, Evan D.

2012-01-01

Background The long-lasting high affinity opioid buprenorphine has complex pharmacology including ceiling effects with respect to analgesia and respiratory depression. Plasma concentrations of the major buprenorphine metabolites norbuprenorphine, buprenorphine-3-glucuronide, and norbuprenorphine-3-glucuronide approximate or exceed those of the parent drug. Buprenorphine glucuronide metabolites pharmacology is undefined. This investigation determined binding and pharmacological activity of the two glucuronide metabolites, and in comparison with buprenorphine and norbuprenorphine. Methods Competitive inhibition of radioligand binding to human mu, kappa, delta opioid and nociceptin receptors was used to determine glucuronide binding affinities for these receptors. Common opiate effects were assessed in vivo in Swiss Webster mice. Antinociception was assessed using a tail-flick assay, respiratory effects were measured using unrestrained whole-body plethysmography, and sedation was assessed by inhibition of locomotion measured by open-field testing. Results Buprenorphine-3-glucuronide had high affinity for human mu (Ki = 4.9±2.7 pM), delta (Ki = 270±0.4 nM), and nociceptin (Ki = 36±0.3 μM) but not kappa receptors. Norbuprenorphine-3-glucuronide had affinity for human kappa (Ki = 300±0.5 nM) and nociceptin (Ki= 18±0.2 μM) but not mu or delta receptors. At the dose tested, buprenorphine-3-glucuronide had a small antinociceptive effect. Neither glucuronide had significant effects on respiratory rate, but norbuprenorphine-3-glucuronide decreased tidal volume. Norbuprenorphine-3-glucuronide also caused sedation. Conclusions Both glucuronide metabolites of buprenorphine are biologically active at doses relevant to metabolite exposures which occur after buprenorphine. Activity of the glucuronides may contribute to the overall pharmacology of buprenorphine. PMID:22037640

Microbial transformation of (+)-nootkatone and the antiproliferative activity of its metabolites.

PubMed

Gliszczyńska, Anna; Łysek, Agnieszka; Janeczko, Tomasz; Świtalska, Marta; Wietrzyk, Joanna; Wawrzeńczyk, Czesław

2011-04-01

Six metabolites were obtained as a result of microbial transformation of (+)-nootkatone (1) by the fungal strains: Botrytis, Didymosphaeria, Aspergillus, Chaetomium and Fusarium. Their structure were established as (+)-(4R,5S,7R,9R)-9α-hydroxynootkatone (2), (+)-(4R,5S,7R)-13-hydroxynootkatone (3) and (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9α-hydroxynootkatone (4), (+)-(4R,5S,7R,11S)-11,12-epoksynootkatone (5), (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) and (+)-(4R,5S,7R)-7,11,12-trihydroxynootkatone (7) on the basis of their spectral data. Two products: (4) and (7) were not previously reported in the literature. The antiproliferative activity of (+)-nootkatone (1) and isolated metabolites (2-7) of its biotransformation has been evaluated. Copyright © 2011 Elsevier Ltd. All rights reserved.

Microbiome-Derived Tryptophan Metabolites and Their Aryl Hydrocarbon Receptor-Dependent Agonist and Antagonist Activities

PubMed Central

Jin, Un-Ho; Lee, Syng-Ook; Sridharan, Gautham; Lee, Kyongbum; Davidson, Laurie A.; Jayaraman, Arul; Chapkin, Robert S.; Alaniz, Robert

2014-01-01

The tryptophan metabolites indole, indole-3-acetate, and tryptamine were identified in mouse cecal extracts and fecal pellets by mass spectrometry. The aryl hydrocarbon receptor (AHR) agonist and antagonist activities of these microbiota-derived compounds were investigated in CaCo-2 intestinal cells as a model for understanding their interactions with colonic tissue, which is highly aryl hydrocarbon (Ah)–responsive. Activation of Ah-responsive genes demonstrated that tryptamine and indole 3-acetate were AHR agonists, whereas indole was an AHR antagonist that inhibited TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)–induced CYP1A1 expression. In contrast, the tryptophan metabolites exhibited minimal anti-inflammatory activities, whereas TCDD decreased phorbol ester-induced CXCR4 [chemokine (C-X-C motif) receptor 4] gene expression, and this response was AHR dependent. These results demonstrate that the tryptophan metabolites indole, tryptamine, and indole-3-acetate modulate AHR-mediated responses in CaCo-2 cells, and concentrations of indole that exhibit AHR antagonist activity (100–250 μM) are detected in the intestinal microbiome. PMID:24563545

[Influence of corynebacteria metabolites on antagonistic activity of H2O2 producing lactobacilli].

PubMed

Bukharin, O V; Sgibnev, A V

2012-01-01

Study combined influence of Corynebacterium genus bacteria metabolites and H2O2 producing lactobacilli on survival rate of Staphylococcus aureus, Escherichia coli and Lactobacillus acidophilus. The ability to inhibit catalase of the test strains used and to reduce bactericidal effect of hydroxyl radical were determined in corynebacteria. H2O2 containing metabolites were obtained by cultivating lactobacilli in mineral medium, the amount of H2O2 was determined by oxidation of TMB by peroxidase. Bactericidal effect of lactobacilli metabolites for test strains treated by corynebacteria metabolites was evaluated by seeding results. Results. Inhibitio by corynebacteria metabolites of S. aureus catalase activity by 30-40% and E. coli catalase activ ity by 40-70% was shown. A reduction of bactericidal effect of hydroxyl radicals by corynebacteria metabolites by 30-35% for S. aureus, 38-42% for E. coli and 70-73% for L. acidophilus was noted. The enchantment of bactericidal effect of lactobacilli after treatment of the test strain by corynebacteria metabolites against S. aureus and E. coli manifested by reduction of the numbe of viable cells by 2-3 lg CFU. For L. acidophilus the bactericidal effect oflactobacilli metabolite in the same conditions reduced, and that led to the increase ofviability by 2-4 lg PFU. A conclusion on the possibility of regulation by associative bacteria the manifestations of antagonistic activity of H2O2 producing dominant microorganisms is made based on the data obtained.

Resveratrol glucuronides as the metabolites of resveratrol in humans: characterization, synthesis, and anti-HIV activity.

PubMed

Wang, Lai-Xi; Heredia, Alonso; Song, Haijing; Zhang, Zhaojun; Yu, Biao; Davis, Charles; Redfield, Robert

2004-10-01

Resveratrol is a natural product with diverse biological activities. We have previously reported that resveratrol possesses potent synergistic inhibitory activity against human immunodeficiency virus (HIV)-1 infection in combination with nucleoside analogs (Heredia et al. 2000. J Acquir Immune Defic Syndr 25:246-255). As a part of our program in developing resveratrol as a component for anti-HIV chemotherapy, we describe in this article the characterization, chemical synthesis, and biological effects of the human metabolites of resveratrol. We found that resveratrol was metabolized in humans into two metabolites, which were characterized as resveratrol-3-O- and 4'-O-glucuronides. For further biological studies, we reported two simple, alternative methods for the synthesis of the metabolites. The cytotoxic and antiviral activities of resveratrol and its metabolites were compared in cell culture experiments using human peripheral blood mononuclear cells. Whereas resveratrol was cytotoxic at > or =30 microM, no cytotoxicity was observed for the metabolites at concentrations as high as 300 microM. However, resveratrol showed strong synergistic anti-HIV activity with didanosine at 10 microM, but no synergistic effects were observed for either of the metabolites at up to 300 microM. Nevertheless, the in vitro activity of the metabolites (resveratrol glucuronides) may not necessarily reflect their in vivo function, given the fact that the ubiquitously existing human beta-glucuronidase could convert the metabolites back to resveratrol locally or systematically in vivo. The present studies have implications for future development of resveratrol and/or its derivatives as a chemotherapeutic agent. Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association

Clustering of 3D-Structure Similarity Based Network of Secondary Metabolites Reveals Their Relationships with Biological Activities.

PubMed

Ohtana, Yuki; Abdullah, Azian Azamimi; Altaf-Ul-Amin, Md; Huang, Ming; Ono, Naoaki; Sato, Tetsuo; Sugiura, Tadao; Horai, Hisayuki; Nakamura, Yukiko; Morita Hirai, Aki; Lange, Klaus W; Kibinge, Nelson K; Katsuragi, Tetsuo; Shirai, Tsuyoshi; Kanaya, Shigehiko

2014-12-01

Developing database systems connecting diverse species based on omics is the most important theme in big data biology. To attain this purpose, we have developed KNApSAcK Family Databases, which are utilized in a number of researches in metabolomics. In the present study, we have developed a network-based approach to analyze relationships between 3D structure and biological activity of metabolites consisting of four steps as follows: construction of a network of metabolites based on structural similarity (Step 1), classification of metabolites into structure groups (Step 2), assessment of statistically significant relations between structure groups and biological activities (Step 3), and 2-dimensional clustering of the constructed data matrix based on statistically significant relations between structure groups and biological activities (Step 4). Applying this method to a data set consisting of 2072 secondary metabolites and 140 biological activities reported in KNApSAcK Metabolite Activity DB, we obtained 983 statistically significant structure group-biological activity pairs. As a whole, we systematically analyzed the relationship between 3D-chemical structures of metabolites and biological activities. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of drug metabolizing cytochrome P450s by the aromatase inhibitor drug letrozole and its major oxidative metabolite 4,4′-methanol-bisbenzonitrile in vitro

PubMed Central

Jeong, Seongwook; Woo, Margaret M.; Flockhart, David A.

2009-01-01

Purpose To determine the inhibitory potency of letrozole and its main human metabolite, 4,4′-methanol-bisbenzonitrilee, on the activities of eight cytochrome P450 (CYP) enzymes. Methods Letrozole and its metabolite were incubated with human liver microsomes (HLMs) (or expressed CYP isoforms) and NADPH in the absence (control) and presence of the test inhibitor. Results Letrozole was a potent competitive inhibitor of CYP2A6 (Ki 4.6 ± 0.05 μM and 5.0 ± 2.4 μM in HLMs and CYP2A6, respectively) and a weak inhibitor of CYP2C19 (Ki 42.2 μM in HLMs and 33.3 μM in CYP2C19), while its metabolite showed moderate inhibition of CYP2C19 and CYP2B6. Letrozole or its metabolite had negligible effect on other CYPs. Conclusions Based on the in vitro Ki values, letrozole is predicted to be a weak inhibitor of CYP2A6 in vivo. Letrozole and its major human metabolite show inhibitory activity towards other CYPs, but clinically relevant drug interactions seem less likely as the Ki values are above the therapeutic plasma concentrations of letrozole. PMID:19198839

Physical activity, sedentary behavior, and vitamin D metabolites.

PubMed

Hibler, Elizabeth A; Sardo Molmenti, Christine L; Dai, Qi; Kohler, Lindsay N; Warren Anderson, Shaneda; Jurutka, Peter W; Jacobs, Elizabeth T

2016-02-01

Physical activity is associated with circulating 25-hydroxyvitamin D (25(OH)D). However, the influence of activity and/or sedentary behavior on the biologically active, seco-steroid hormone 1α,25-dihydroxyvitamin D (1,25(OH)2D) is unknown. We conducted a cross-sectional analysis among ursodeoxycholic acid (UDCA) randomized trial participants (n=876) to evaluate associations between physical activity, sedentary behavior, and circulating vitamin D metabolite concentrations. Continuous vitamin D metabolite measurements and clinical thresholds were evaluated using multiple linear and logistic regression models, mutually adjusted for either 1,25(OH)2D or 25(OH)D and additional confounding factors. A statistically significant linear association between 1,25(OH)2D and moderate-vigorous physical activity per week was strongest among women (β (95% CI): 3.10 (1.51-6.35)) versus men (β (95% CI): 1.35 (0.79-2.29)) in the highest tertile of activity compared to the lowest (p-interaction=0.003). Furthermore, 25(OH)D was 1.54ng/ml (95% CI 1.09-1.98) higher per hour increase in moderate-vigorous activity (p=0.001) and odds of sufficient 25(OH)D status was higher among physically active participants (p=0.001). Sedentary behavior was not significantly associated with either metabolite in linear regression models, nor was a statistically significant interaction by sex identified. The current study identified novel associations between physical activity and serum 1,25(OH)2D levels, adjusted for 25(OH)D concentrations. These results identify the biologically active form of vitamin D as a potential physiologic mechanism related to observed population-level associations between moderate-vigorous physical activity with bone health and chronic disease risk. However, future longitudinal studies are needed to further evaluate the role of physical activity and vitamin D metabolites in chronic disease prevention. Copyright © 2015 Elsevier Inc. All rights reserved.

A Potential Biofilm Metabolite Signature for Caries Activity - A Pilot Clinical Study

PubMed Central

Zandona, F; Soini, HA; Novotny, MV; Santiago, E; Eckert, GJ; Preisser, JS; Benecha, HK; Arthur, RA; Zero, DT

2016-01-01

Background This study's aim was to compare the dental biofilm metabolite-profile of caries-active (N=11) or caries-free (N=4) children by gas chromatography-mass spectrometry (GC/MS) analyses. Methods Samples collected after overnight fasting, with or without a previous glucose rinse, were combined for each child based on the caries status of the site, re-suspended in ethanol and analyzed by GC/MS. Results Biofilm from caries-active sites exhibited a different chromatographic profile compared to caries-free sites. Qualitative and quantitative analysis suggested a special cluster of branched alcohols and esters present at substantially higher intensity in biofilms of caries-active sites. Conclusions This pilot study indicates that there are metabolites present in the biofilm which have the potential to provide a characteristic metabolomics signature for caries activity. PMID:27885354

Diadenosine tetraphosphate (Ap4A) - an E. coli alarmone or a damage metabolite?

PubMed

Despotović, Dragana; Brandis, Alexander; Savidor, Alon; Levin, Yishai; Fumagalli, Laura; Tawfik, Dan S

2017-07-01

Under stress, metabolism is changing: specific up- or down-regulation of proteins and metabolites occurs as well as side effects. Distinguishing specific stress-signaling metabolites (alarmones) from side products (damage metabolites) is not trivial. One example is diadenosine tetraphosphate (Ap4A) - a side product of aminoacyl-tRNA synthetases found in all domains of life. The earliest observations suggested that Ap4A serves as an alarmone for heat stress in Escherichia coli. However, despite 50 years of research, the signaling mechanisms associated with Ap4A remain unknown. We defined a set of criteria for distinguishing alarmones from damage metabolites to systematically classify Ap4A. In a nutshell, no indications for a signaling cascade that is triggered by Ap4A were found; rather, we found that Ap4A is efficiently removed in a constitutive, nonregulated manner. Several fold perturbations in Ap4A concentrations have no effect, yet accumulation at very high levels is toxic due to disturbance of zinc homeostasis, and also because Ap4A's structural overlap with ATP can result in spurious binding and inactivation of ATP-binding proteins. Overall, Ap4A met all criteria for a damage metabolite. While we do not exclude any role in signaling, our results indicate that the damage metabolite option should be considered as the null hypothesis when examining Ap4A and other metabolites whose levels change upon stress. © 2017 Federation of European Biochemical Societies.

Biologically Active Metabolites Synthesized by Microalgae

PubMed Central

Costa, Jorge Alberto Vieira

2015-01-01

Microalgae are microorganisms that have different morphological, physiological, and genetic traits that confer the ability to produce different biologically active metabolites. Microalgal biotechnology has become a subject of study for various fields, due to the varied bioproducts that can be obtained from these microorganisms. When microalgal cultivation processes are better understood, microalgae can become an environmentally friendly and economically viable source of compounds of interest, because production can be optimized in a controlled culture. The bioactive compounds derived from microalgae have anti-inflammatory, antimicrobial, and antioxidant activities, among others. Furthermore, these microorganisms have the ability to promote health and reduce the risk of the development of degenerative diseases. In this context, the aim of this review is to discuss bioactive metabolites produced by microalgae for possible applications in the life sciences. PMID:26339647

In-stream attenuation of neuro-active pharmaceuticals and their metabolites

USGS Publications Warehouse

Writer, Jeffrey; Antweiler, Ronald C.; Ferrar, Imma; Ryan, Joseph N.; Thurman, Michael

2013-01-01

In-stream attenuation was determined for 14 neuro-active pharmaceuticals and associated metabolites. Lagrangian sampling, which follows a parcel of water as it moves downstream, was used to link hydrological and chemical transformation processes. Wastewater loading of neuro-active compounds varied considerably over a span of several hours, and thus a sampling regime was used to verify that the Lagrangian parcel was being sampled and a mechanism was developed to correct measured concentrations if it was not. In-stream attenuation over the 5.4-km evaluated reach could be modeled as pseudo-first-order decay for 11 of the 14 evaluated neuro-active pharmaceutical compounds, illustrating the capacity of streams to reduce conveyance of neuro-active compounds downstream. Fluoxetine and N-desmethyl citalopram were the most rapidly attenuated compounds (t1/2 = 3.6 ± 0.3 h, 4.0 ± 0.2 h, respectively). Lamotrigine, 10,11,-dihydro-10,11,-dihydroxy-carbamazepine, and carbamazepine were the most persistent (t1/2 = 12 ± 2.0 h, 12 ± 2.6 h, 21 ± 4.5 h, respectively). Parent compounds (e.g., buproprion, carbamazepine, lamotrigine) generally were more persistent relative to their metabolites. Several compounds (citalopram, venlafaxine, O-desmethyl-venlafaxine) were not attenuated. It was postulated that the primary mechanism of removal for these compounds was interaction with bed sediments and stream biofilms, based on measured concentrations in stream biofilms and a column experiment using stream sediments.

Evaluation of the in vitro activity of levornidazole, its metabolites and comparators against clinical anaerobic bacteria.

PubMed

Hu, Jiali; Zhang, Jing; Wu, Shi; Zhu, Demei; Huang, Haihui; Chen, Yuancheng; Yang, Yang; Zhang, Yingyuan

2014-12-01

This study evaluated the in vitro anti-anaerobic activity and spectrum of levornidazole, its metabolites and comparators against 375 clinical isolates of anaerobic bacteria, including Gram-negative bacilli (181 strains), Gram-negative cocci (11 strains), Gram-positive bacilli (139 strains) and Gram-positive cocci (44 strains), covering 34 species. Minimum inhibitory concentrations (MICs) of levornidazole, its five metabolites and three comparators against these anaerobic isolates were determined by the agar dilution method. Minimum bactericidal concentrations (MBCs) of levornidazole and metronidazole were measured against 22 strains of Bacteroides fragilis. Levornidazole showed good activity against B. fragilis, other Bacteroides spp., Clostridium difficile, Clostridium perfringens and Peptostreptococcus magnus, evidenced by MIC90 values of 0.5, 1, 0.25, 2 and 1mg/L, respectively. The activity of levornidazole and the comparators was poor for Veillonella spp. Generally, levornidazole displayed activity similar to or slightly higher than that of metronidazole, ornidazole and dextrornidazole against anaerobic Gram-negative bacilli, Gram-positive bacilli and Gram-positive cocci, especially B. fragilis. Favourable anti-anaerobic activity was also seen with levornidazole metabolites M1 and M4 but not M2, M3 or M5. For the 22 clinical B. fragilis strains, MBC50 and MBC90 values of levornidazole were 2mg/L and 4mg/L, respectively. Both MBC50/MIC50 and MBC90/MIC90 ratios of levornidazole were 4, similar to those of metronidazole. Levornidazole is an important anti-anaerobic option in clinical settings in terms of its potent and broad-spectrum in vitro activity, bactericidal property, and the anti-anaerobic activity of its metabolites M1 and M4. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Identification of the urinary metabolites of 4-bromoaniline and 4-bromo-[carbonyl-13C]-acetanilide in rat.

PubMed

Scarfe, G B; Nicholson, J K; Lindon, J C; Wilson, I D; Taylor, S; Clayton, E; Wright, B

2002-04-01

1. The urinary excretion of 4-bromoaniline and its [carbonyl-(13)C]-labelled N-acetanilide, together with their corresponding metabolites, have been investigated in the rat following i.p. administration at 50 mg kg(-1). 2. Metabolite profiling was performed by reversed-phase HPLC with UV detection, whilst identification was performed using a combination of enzymic hydrolysis and directly coupled HPLC-NMR-MS analysis. The urinary metabolite profile was quantitatively and qualitatively similar for both compounds with little of either excreted unchanged. 3. The major metabolite present in urine was 2-amino-5-bromophenylsulphate, but, in addition, a number of metabolites with modification of the N-acetyl moiety were identified (from both the [(13)C]-acetanilide or produced following acetylation of the free bromoaniline). 4. For 4-bromoacetanilide, N-deacetylation was a major route of metabolism, but despite the detection of the acetanilide following the administration of the free aniline, there was no evidence of reacetylation (futile deacetylation). 5. Metabolites resulting from the oxidation of the acetyl group included a novel glucuronide of an N-glycolanilide, an unusual N-oxanilic acid and a novel N-acetyl cysteine conjugate.

Neuropharmacology of 3,4-Methylenedioxypyrovalerone (MDPV), Its Metabolites, and Related Analogs

PubMed Central

Baumann, Michael H.; Bukhari, Mohammad O.; Lehner, Kurt R.; Anizan, Sebastien; Rice, Kenner C.; Concheiro, Marta; Huestis, Marilyn A.

2017-01-01

3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive component of so-called bath salts products that has caused serious medical consequences in humans. In this chapter, we review the neuropharmacology of MDPV and related analogs, and supplement the discussion with new results from our preclinical experiments. MDPV acts as a potent uptake inhibitor at plasma membrane transporters for dopamine (DAT) and norepinephrine (NET) in nervous tissue. The MDPV formulation in bath salts is a racemic mixture, and the S isomer is much more potent than the R isomer at blocking DAT and producing abuse-related effects. Elevations in brain extracellular dopamine produced by MDPV are likely to underlie its locomotor stimulant and addictive properties. MDPV displays rapid pharmacokinetics when injected into rats (0.5–2.0 mg/kg), with peak plasma concentrations achieved by 10–20 min and declining quickly thereafter. MDPV is metabolized to 3,4-dihydroxypyrovalerone (3,4-catechol-PV) and 4-hydroxy-3-methoxypyrovalerone (4-OH-3-MeO-PV) in vivo, but motor activation produced by the drug is positively correlated with plasma concentrations of parent drug and not its metabolites. 3,4-Catechol-PV is a potent uptake blocker at DAT in vitro but has little activity after administration in vivo. 4-OH-3-MeO-PV is the main MDPV metabolite but is weak at DAT and NET. MDPV analogs, such as α-pyrrolidinovalerophenone (α-PVP), display similar ability to inhibit DAT and increase extracellular dopamine concentrations. Taken together, these findings demonstrate that MDPV and its analogs represent a unique class of transporter inhibitors with a high propensity for abuse and addiction. PMID:27830575

Secondary Metabolites from Three Florida Sponges with Antidepressant Activity

PubMed Central

Kochanowska, Anna J.; Rao, Karumanchi V.; Childress, Suzanne; El-Alfy, Abir; Matsumoto, Rae R.; Kelly, Michelle; Stewart, Gina S.; Sufka, Kenneth J.; Hamann, Mark T.

2016-01-01

Brominated indole alkaloids are a common class of metabolites reported from sponges of the order Verongida. Herein we report the isolation, structure determination, and activity of metabolites from three Florida sponges, namely, Verongula rigida (order Verongida, family Aplysinidae), Smenospongia aurea, and S. cerebriformis (order Dictyoceratida, family Thorectidae). All three species were investigated chemically, revealing similarities in secondary metabolites. Brominated compounds, as well as sesquiterpene quinones and hydroquinones, were identified from both V. rigida and S. aurea despite their apparent taxonomic differences at the ordinal level. Similar metabolites found in these distinct sponge species of two different genera provide evidence for a microbial origin of the metabolites. Isolated compounds were evaluated in the Porsolt forced swim test (FST) and the chick anxiety–depression continuum model. Among the isolated compounds, 5,6-dibromo-N,N-dimethyltryptamine (1) exhibited significant antidepressant-like action in the rodent FST model, while 5-bromo-N,N-dimethyltryptamine (2) caused significant reduction of locomotor activity indicative of a potential sedative action. The current study provides ample evidence that marine natural products with the diversity of brominated marine alkaloids will provide potential leads for antidepressant and anxiolytic drugs. PMID:18217716

Secondary metabolites from three Florida sponges with antidepressant activity.

PubMed

Kochanowska, Anna J; Rao, Karumanchi V; Childress, Suzanne; El-Alfy, Abir; Matsumoto, Rae R; Kelly, Michelle; Stewart, Gina S; Sufka, Kenneth J; Hamann, Mark T

2008-02-01

Brominated indole alkaloids are a common class of metabolites reported from sponges of the order Verongida. Herein we report the isolation, structure determination, and activity of metabolites from three Florida sponges, namely, Verongula rigida (order Verongida, family Aplysinidae), Smenospongia aurea, and S. cerebriformis (order Dictyoceratida, family Thorectidae). All three species were investigated chemically, revealing similarities in secondary metabolites. Brominated compounds, as well as sesquiterpene quinones and hydroquinones, were identified from both V. rigida and S. aurea despite their apparent taxonomic differences at the ordinal level. Similar metabolites found in these distinct sponge species of two different genera provide evidence for a microbial origin of the metabolites. Isolated compounds were evaluated in the Porsolt forced swim test (FST) and the chick anxiety-depression continuum model. Among the isolated compounds, 5,6-dibromo- N,N-dimethyltryptamine ( 1) exhibited significant antidepressant-like action in the rodent FST model, while 5-bromo- N,N-dimethyltryptamine ( 2) caused significant reduction of locomotor activity indicative of a potential sedative action. The current study provides ample evidence that marine natural products with the diversity of brominated marine alkaloids will provide potential leads for antidepressant and anxiolytic drugs.

Sulfate Metabolites of 4-Monochlorobiphenyl in Whole Poplar Plants

PubMed Central

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

2013-01-01

4-Monochlorobiphenyl (PCB3) has been proven to be transformed into hydroxylated metabolites of PCB3 (OH-PCB3s) in whole poplar plants in our previous work. However, hydroxylated metabolites of PCBs, including OH-PCB3s, as the substrates of sulfotransferases have not been studied in many organisms including plants in vivo. Poplar (Populus deltoides × nigra, DN34) was used to investigate the further metabolism from OH-PCB3s to PCB3 sulfates because it is a model plant and one that is frequently utilized in phytoremediation. Results showed poplar plants could metabolize PCB3 into PCB3 sulfates during 25 day exposures. Three sulfate metabolites, including 2′-PCB3 sulfate, 3′-PCB3 sulfate and 4′-PCB3 sulfate, were identified in poplar roots and their concentrations increased in the roots from day 10 to day 25. The major products were 2′-PCB3 sulfate and 4′-PCB3 sulfate. However, the concentrations of PCB3 sulfates were much lower than those of OH-PCB3s in the roots, suggesting the sequential transformation of these hydroxylated PCB3 metabolites into PCB3 sulfates in whole poplars. In addition, 2′-PCB3 sulfate or 4′-PCB3 sulfate was also found in the bottom wood samples indicating some translocation or metabolism in woody tissue. Results suggested that OH-PCB3s were the substrates of sulfotransferases which catalyzed the formation of PCB3 sulfates in the metabolic pathway of PCB3. PMID:23215248

Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer

PubMed Central

Dachineni, Rakesh; Kumar, D. Ramesh; Callegari, Eduardo; Kesharwani, Siddharth S.; Sankaranarayanan, Ranjini; Seefeldt, Teresa; Tummala, Hemachand; Bhat, G. Jayarama

2017-01-01

Aspirin's potential as a drug continues to be evaluated for the prevention of colorectal cancer (CRC). Although multiple targets for aspirin and its metabolite, salicylic acid, have been identified, no unifying mechanism has been proposed to clearly explain its chemopreventive effects. Our goal here was to investigate the ability of salicylic acid metabolites, known to be generated through cytochrome P450 (CYP450) enzymes, and its derivatives as cyclin dependent kinase (CDK) inhibitors to gain new insights into aspirin's chemopreventive actions. Using in vitro kinase assays, for the first time, we demonstrate that salicylic acid metabolites, 2,3-dihydroxy-benzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), as well as derivatives 2,4-dihydroxybenzoic acid (2,4-DHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, another derivative, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking studies showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and pre-incubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supported the data obtained from molecular docking studies. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells, or the salicylic acid metabolites generated by gut microflora may significantly contribute to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin's chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of novel CDK inhibitors in cancer prevention and

Cytochrome P450 Genetic Variation Associated with Tamoxifen Biotransformation in American Indian and Alaska Native People

PubMed Central

Khan, Burhan A.; Robinson, Renee; Fohner, Alison E.; Muzquiz, LeeAnna I.; Schilling, Brian D.; Beans, Julie A.; Olnes, Matthew J.; Trawicki, Laura; Frydenlund, Holly; Laukes, Cindi; Beatty, Patrick; Phillips, Brian; Nickerson, Deborah; Howlett, Kevin; Dillard, Denise A.; Thornton, Timothy A.; Thummel, Kenneth E.

2018-01-01

Abstract Despite evidence that pharmacogenetics can improve tamoxifen pharmacotherapy, there are few studies with American Indian and Alaska Native (AIAN) people. We examined variation in cytochrome P450 (CYP) genes (CYP2D6, CYP3A4, CYP3A5, and CYP2C9) and tamoxifen biotransformation in AIAN patients with breast cancer (n = 42) from the Southcentral Foundation in Alaska and the Confederated Salish and Kootenai Tribes in Montana. We tested for associations between CYP diplotypes and plasma concentrations of tamoxifen and metabolites. Only the CYP2D6 variation was significantly associated with concentrations of endoxifen (P = 0.0008) and 4‐hydroxytamoxifen (P = 0.0074), tamoxifen's principal active metabolites, as well as key metabolic ratios. The CYP2D6 was also the most significant predictor of active metabolites and metabolic ratios in a multivariate regression model, including all four genes as predictors, with minor roles for other CYP genes. In AIAN populations, CYP2D6 is the largest contributor to tamoxifen bioactivation, illustrating the importance of validating pharmacogenetic testing for therapy optimization in an understudied population. PMID:29436156

Stereoselective synthesis of an active metabolite of the potent PI3 kinase inhibitor PKI-179.

PubMed

Chen, Zecheng; Venkatesan, Aranapakam M; Dos Santos, Osvaldo; Delos Santos, Efren; Dehnhardt, Christoph M; Ayral-Kaloustian, Semiramis; Ashcroft, Joseph; McDonald, Leonard A; Mansour, Tarek S

2010-03-05

The synthesis and stereochemical determination of 1-(4-(4-((1R,5R,6R)-6-hydroxy-3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-yl)phenyl)-3-(pyridin-4-yl)urea (2), an active metabolite of the potent PI3 kinase inhibitor PKI-179 (1), is described. Stereospecific hydroboration of the double bond of 2,5-dihydro-1H-pyrrole 8 gave the 2,3-trans alcohol 9 exclusively. The configuration of the 3-hydroxyl group in 9 was inverted by an oxidation and stereoselective reduction sequence to give the corresponding 2,3-cis isomer 23. Both exo (21) and endo (27) isomers of the metabolite 2 were prepared via a practical synthetic route from 9 and 23, respectively, and the stereochemistry of 2 was determined to be endo. The endo isomer (27) was separated into two enantiomers 28 and 29 by chiral HPLC. Compound 2 was found to be enantiomerically pure and identical to the enantiomer 28. The absolute stereochemistry of the enantiomer 28 was determined by Mosher's method, thus establishing the stereochemistry of the active metabolite 2.

Activation of the dormant secondary metabolite production by introducing gentamicin-resistance in a marine-derived Penicillium purpurogenum G59.

PubMed

Chai, Yun-Jing; Cui, Cheng-Bin; Li, Chang-Wei; Wu, Chang-Jing; Tian, Cong-Kui; Hua, Wei

2012-03-01

A new approach to activate silent gene clusters for dormant secondary metabolite production has been developed by introducing gentamicin-resistance to an originally inactive, marine-derived fungal strain Penicillium purpurogenum G59. Upon treatment of the G59 spores with a high concentration of gentamicin in aqueous DMSO, a total of 181 mutants were obtained by single colony isolation. In contrast to the strain G59, the EtOAc extracts of nine mutant cultures showed inhibitory effects on K562 cells, indicating that the nine mutants had acquired capability to produce antitumor metabolites. This was evidenced by TLC and HPLC analysis of EtOAc extracts of G59 and the nine mutants. Further isolation and characterization demonstrated that four antitumor secondary metabolites, janthinone (1), fructigenine A (2), aspterric acid methyl ester (3) and citrinin (4), were newly produced by mutant 5-1-4 compared to the parent strain G59, and which were also not found in the secondary metabolites of other Penicillium purpurogenum strains. However, Compounds 1-4 inhibited the proliferation of K562 cells with inhibition rates of 34.6% (1), 60.8% (2), 31.7% (3) and 67.1% (4) at 100 μg/mL, respectively. The present study demonstrated the effectiveness of a simple, yet practical approach to activate the production of dormant fungal secondary metabolites by introducing acquired resistance to aminoglycoside antibiotics, which could be applied to the studies for eliciting dormant metabolic potential of fungi to obtain cryptic secondary metabolites.

Reorganization of Actin Cytoskeleton by the Phosphoinositide Metabolite Glycerophosphoinositol 4-Phosphate

PubMed Central

Mancini, Raffaella; Piccolo, Enza; Mariggio', Stefania; Filippi, Beatrice Maria; Iurisci, Cristiano; Pertile, Paolo; Berrie, Christopher P.; Corda, Daniela

2003-01-01

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P. PMID:12589050

7,3',4'-Trihydroxyisoflavone, a metabolite of the soy isoflavone daidzein, suppresses ultraviolet B-induced skin cancer by targeting Cot and MKK4.

PubMed

Lee, Dong Eun; Lee, Ki Won; Byun, Sanguine; Jung, Sung Keun; Song, Nury; Lim, Sung Hwan; Heo, Yong-Seok; Kim, Jong Eun; Kang, Nam Joo; Kim, Bo Yeon; Bowden, G Tim; Bode, Ann M; Lee, Hyong Joo; Dong, Zigang

2011-04-22

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3',4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3',4'-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3',4'-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3',4'-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3',4'-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3',4'-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3',4'-THIF, a potential skin cancer chemopreventive agent.

Activation of the Dormant Secondary Metabolite Production by Introducing Gentamicin-Resistance in a Marine-Derived Penicillium purpurogenum G59

PubMed Central

Chai, Yun-Jing; Cui, Cheng-Bin; Li, Chang-Wei; Wu, Chang-Jing; Tian, Cong-Kui; Hua, Wei

2012-01-01

A new approach to activate silent gene clusters for dormant secondary metabolite production has been developed by introducing gentamicin-resistance to an originally inactive, marine-derived fungal strain Penicillium purpurogenum G59. Upon treatment of the G59 spores with a high concentration of gentamicin in aqueous DMSO, a total of 181 mutants were obtained by single colony isolation. In contrast to the strain G59, the EtOAc extracts of nine mutant cultures showed inhibitory effects on K562 cells, indicating that the nine mutants had acquired capability to produce antitumor metabolites. This was evidenced by TLC and HPLC analysis of EtOAc extracts of G59 and the nine mutants. Further isolation and characterization demonstrated that four antitumor secondary metabolites, janthinone (1), fructigenine A (2), aspterric acid methyl ester (3) and citrinin (4), were newly produced by mutant 5-1-4 compared to the parent strain G59, and which were also not found in the secondary metabolites of other Penicillium purpurogenum strains. However, Compounds 1–4 inhibited the proliferation of K562 cells with inhibition rates of 34.6% (1), 60.8% (2), 31.7% (3) and 67.1% (4) at 100 μg/mL, respectively. The present study demonstrated the effectiveness of a simple, yet practical approach to activate the production of dormant fungal secondary metabolites by introducing acquired resistance to aminoglycoside antibiotics, which could be applied to the studies for eliciting dormant metabolic potential of fungi to obtain cryptic secondary metabolites. PMID:22611354

Effect of tibolone and its principal metabolites (3α- and 3β-hydroxy, 3α-sulfate, and 4-ene derivatives) on estrone sulfatase activity in normal and cancerous human breast tissue.

PubMed

Chetrite, Gérard S; Cortes-Prieto, Joaquin; Pasqualini, Jorge R

2011-12-01

Tibolone (Org-OD14) is the active substance of Livial®, a synthetic steroid with the structure 7α,17α-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one, possessing weak tissue-specific estrogenic, progestogenic, and androgenic properties, used to treat menopausal complaints. After oral administration, tibolone is extensively metabolized into the 3α-(Org-4904) and 3β-(Org-30126) hydroxy derivatives with estrogenic properties, its 4-ene (Org-OM38) isomer with progestogenic/androgenic activities, and the 3α-sulfate (Org-34322) derivative, a major biologically inactive circulating form. We compared the dose response of tibolone and its metabolites on estrone sulfatase activity [conversion of estrone sulfate (E1S) to estrone (E1)] in normal and cancerous human breast tissues. Tissue minces were incubated with physiological concentrations of [3H]-E1S (5×10-9M) alone or in the presence of tibolone and its metabolites (concentration range: 5×10-7to 5×10-5M) for 4 h. Tritiated E1, estradiol (E2), and E1S were separated and evaluated quantitatively by thin-layer chromatography. The sulfatase activity was significantly higher in cancerous breast but strongly inhibited by tibolone and the different metabolites, whereas 3α- and 3β-hydroxy derivatives were the most potent inhibitors. This very significant inhibitory effect of tibolone and its principal metabolites on the enzyme involved in E2biosynthesis in the human breast provides interesting perspectives to study the biological responses of these compounds in trials with breast cancer patients.

Activation of the Silent Secondary Metabolite Production by Introducing Neomycin-Resistance in a Marine-Derived Penicillium purpurogenum G59

PubMed Central

Wu, Chang-Jing; Yi, Le; Cui, Cheng-Bin; Li, Chang-Wei; Wang, Nan; Han, Xiao

2015-01-01

Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1–5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1–5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways. PMID:25913704

Activation of the silent secondary metabolite production by introducing neomycin-resistance in a marine-derived Penicillium purpurogenum G59.

PubMed

Wu, Chang-Jing; Yi, Le; Cui, Cheng-Bin; Li, Chang-Wei; Wang, Nan; Han, Xiao

2015-04-22

Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α, 6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1-5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1-5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways.

Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor

PubMed Central

Cherian, Milu T.; Yang, Lei; Chai, Sergio C.; Lin, Wenwei

2016-01-01

The constitutive androstane receptor (CAR) regulates the expression of genes involved in drug metabolism and other processes. A specific inhibitor of CAR is critical for modulating constitutive CAR activity. We recently described a specific small-molecule inhibitor of CAR, CINPA1 (ethyl (5-(diethylglycyl)-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)carbamate), which is capable of reducing CAR-mediated transcription by changing the coregulator recruitment pattern and reducing CAR occupancy at the promoter regions of its target genes. In this study, we showed that CINPA1 is converted to two main metabolites in human liver microsomes. By using cell-based reporter gene and biochemical coregulator recruitment assays, we showed that although metabolite 1 was very weak in inhibiting CAR function and disrupting CAR-coactivator interaction, metabolite 2 was inactive in this regard. Docking studies using the CAR ligand-binding domain structure showed that although CINPA1 and metabolite 1 can bind in the CAR ligand-binding pocket, metabolite 2 may be incapable of the molecular interactions required for binding. These results indicate that the metabolites of CINPA1 may not interfere with the action of CINPA1. We also used in vitro enzyme assays to identify the cytochrome P450 enzymes responsible for metabolizing CINPA1 in human liver microsomes and showed that CINPA1 was first converted to metabolite 1 by CYP3A4 and then further metabolized by CYP2D6 to metabolite 2. Identification and characterization of the metabolites of CINPA1 enabled structure-activity relationship studies of this family of small molecules and provided information to guide in vivo pharmacological studies. PMID:27519550

Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity.

PubMed

Ahluwalia, Vivek; Kumar, Jitendra; Rana, Virendra S; Sati, Om P; Walia, S

2015-01-01

This investigation was undertaken to identify the major secondary metabolite, produced by two Trichoderma harzianum strains (T-4 and T-5) with their antifungal activity against phytopathogenic fungi using poison food technique. The ethyl acetate extract was subjected to column chromatography using n-hexane, ethyl acetate and methanol gradually. Chromatographic separation of ethyl acetate extract of T. harzianum (T-4) resulted in the isolation and identification of palmitic acid (1), 1,8-dihydroxy-3-methylanthraquinone (2), 6-pentyl-2H-pyran-2-one (3), 2(5H)-furanone (4), stigmasterol (5) and β-sitosterol (6), while T. harzianum (T-5) gave palmitic acid (1), 1-hydroxy-3-methylanthraquinone (7), δ-decanolactone (8), 6-pentyl-2H-pyran-2-one (3), ergosterol (9), harzianopyridone (10) and 6-methyl-1,3,8-trihydroxyanthraquinone (11) as major metabolites. Among compounds screened for antifungal activity, compound 10 was found to be most active (EC50 35.9-50.2 μg mL(-1)). In conclusion, the present investigation provided significant information about antifungal activity and compounds isolated from two different strains of T. harzianum obtained from two different Himalayan locations.

Biotransformation of dianabol with the filamentous fungi and β-glucuronidase inhibitory activity of resulting metabolites.

PubMed

Khan, Naik T; Zafar, Salman; Noreen, Shagufta; Al Majid, Abdullah M; Al Othman, Zeid A; Al-Resayes, Saud Ibrahim; Atta-ur-Rahman; Choudhary, M Iqbal

2014-07-01

Biotransformation of the anabolic steroid dianabol (1) by suspended-cell cultures of the filamentous fungi Cunninghamella elegans and Macrophomina phaseolina was studied. Incubation of 1 with C. elegans yielded five hydroxylated metabolites 2-6, while M. phaseolina transformed compound 1 into polar metabolites 7-11. These metabolites were identified as 6β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (2), 15α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (3), 11α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (4), 6β,12β,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (5), 6β,15α,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (6), 17β-hydroxy-17α-methylandrost-1,4-dien-3,6-dione (7), 7β,17β,-dihydroxy-17α-methylandrost-1,4-dien-3-one (8), 15β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (9), 17β-hydroxy-17α-methylandrost-1,4-dien-3,11-dione (10), and 11β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (11). Metabolite 3 was also transformed chemically into diketone 12 and oximes 13, and 14. Compounds 6 and 12-14 were identified as new derivatives of dianabol (1). The structures of all transformed products were deduced on the basis of spectral analyses. Compounds 1-14 were evaluated for β-glucuronidase enzyme inhibitory activity. Compounds 7, 13, and 14 showed a strong inhibition of β-glucuronidase enzyme, with IC50 values between 49.0 and 84.9 μM. Copyright © 2014 Elsevier Inc. All rights reserved.

Metabolic characteristics of 13-cis-retinoic acid (isotretinoin) and anti-tumour activity of the 13-cis-retinoic acid metabolite 4-oxo-13-cis-retinoic acid in neuroblastoma

PubMed Central

Sonawane, Poonam; Cho, Hwang Eui; Tagde, Ashujit; Verlekar, Dattesh; Yu, Alice L; Reynolds, C Patrick; Kang, Min H

2014-01-01

Background and Purpose Isotretinoin (13-cis-retinoic acid; 13-cRA) is a differentiation inducer used to treat minimal residual disease after myeloablative therapy for high-risk neuroblastoma. However, more than 40% of children develop recurrent disease during or after 13-cRA treatment. The plasma concentrations of 13-cRA in earlier studies were considered subtherapeutic while 4-oxo-13-cis-RA (4-oxo-13-cRA), a metabolite of 13-cRA considered by some investigators as inactive, were greater than threefold higher than 13-cRA. We sought to define the metabolic pathways of 13-cRA and investigated the anti-tumour activity of its major metabolite, 4-oxo-13-cRA. Experimental Approach Effects of 13-cRA and 4-oxo-13-cRA on human neuroblastoma cell lines were assessed by DIMSCAN and flow cytometry for cell proliferation, MYCN down-regulation by reverse transcription PCR and immunoblotting, and neurite outgrowth by confocal microscopy. 13-cRA metabolism was determined using tandem MS in human liver microsomes and in patient samples. Key Results Six major metabolites of 13-cRA were identified in patient samples. Of these, 4-oxo-13-cRA was the most abundant, and 4-oxo-13-cRA glucuronide was also detected at a higher level in patients. CYP3A4 was shown to play a major role in catalysing 13-cRA to 4-oxo-13-cRA. In human neuroblastoma cell lines, 4-oxo-13-cRA and 13-cRA were equi-effective at inducing neurite outgrowth, inhibiting proliferation, decreasing MYCN mRNA and protein, and increasing the expression of retinoic acid receptor-β mRNA and protein levels. Conclusions and Implications We showed that 4-oxo-13-cRA is as active as 13-cRA against neuroblastoma cell lines. Plasma levels of both 13-cRA and 4-oxo-13-cRA should be evaluated in pharmacokinetic studies of isotretinoin in neuroblastoma. PMID:25039756

Metabolic characteristics of 13-cis-retinoic acid (isotretinoin) and anti-tumour activity of the 13-cis-retinoic acid metabolite 4-oxo-13-cis-retinoic acid in neuroblastoma.

PubMed

Sonawane, Poonam; Cho, Hwang Eui; Tagde, Ashujit; Verlekar, Dattesh; Yu, Alice L; Reynolds, C Patrick; Kang, Min H

2014-12-01

Isotretinoin (13-cis-retinoic acid; 13-cRA) is a differentiation inducer used to treat minimal residual disease after myeloablative therapy for high-risk neuroblastoma. However, more than 40% of children develop recurrent disease during or after 13-cRA treatment. The plasma concentrations of 13-cRA in earlier studies were considered subtherapeutic while 4-oxo-13-cis-RA (4-oxo-13-cRA), a metabolite of 13-cRA considered by some investigators as inactive, were greater than threefold higher than 13-cRA. We sought to define the metabolic pathways of 13-cRA and investigated the anti-tumour activity of its major metabolite, 4-oxo-13-cRA. Effects of 13-cRA and 4-oxo-13-cRA on human neuroblastoma cell lines were assessed by DIMSCAN and flow cytometry for cell proliferation, MYCN down-regulation by reverse transcription PCR and immunoblotting, and neurite outgrowth by confocal microscopy. 13-cRA metabolism was determined using tandem MS in human liver microsomes and in patient samples. Six major metabolites of 13-cRA were identified in patient samples. Of these, 4-oxo-13-cRA was the most abundant, and 4-oxo-13-cRA glucuronide was also detected at a higher level in patients. CYP3A4 was shown to play a major role in catalysing 13-cRA to 4-oxo-13-cRA. In human neuroblastoma cell lines, 4-oxo-13-cRA and 13-cRA were equi-effective at inducing neurite outgrowth, inhibiting proliferation, decreasing MYCN mRNA and protein, and increasing the expression of retinoic acid receptor-β mRNA and protein levels. We showed that 4-oxo-13-cRA is as active as 13-cRA against neuroblastoma cell lines. Plasma levels of both 13-cRA and 4-oxo-13-cRA should be evaluated in pharmacokinetic studies of isotretinoin in neuroblastoma. © 2014 The British Pharmacological Society.

Secondary Metabolites Produced by an Endophytic Fungus Pestalotiopsis sydowiana and Their 20S Proteasome Inhibitory Activities.

PubMed

Xia, Xuekui; Kim, Soonok; Liu, Changheng; Shim, Sang Hee

2016-07-20

Fungal endophytes have attracted attention due to their functional diversity. Secondary metabolites produced by Pestalotiopsis sydowiana from a halophyte, Phragmites communis Trinus, were investigated. Eleven compounds, including four penicillide derivatives (1-4) and seven α-pyrone analogues (5-10) were isolated from cultures of P. sydowiana. The compounds were identified based on spectroscopic data. The inhibitory activities against the 20S proteasome were evaluated. Compounds 1-3, 5, and 9-10 showed modest proteasome inhibition activities, while compound 8 showed strong activity with an IC50 of 1.2 ± 0.3 μM. This is the first study on the secondary metabolites produced by P. sydowiana and their proteasome inhibitory activities. The endophytic fungus P. sydowiana might be a good resource for proteasome inhibitors.

Anti-inflammatory effects of 4′-demethylnobiletin, a major metabolite of nobiletin

PubMed Central

Rakariyatham, Kanyasiri; Zheng, Jinkai; Guo, Shanshan; Tang, Zhonghai; Zhou, Shuangde; Xiao, Hang

2015-01-01

Nobiletin, a citrus flavonoid has been associated with various beneficial biological activities. 4′-Demethylnobiletin (4DN) is a major metabolite of nobiletin and its tissue level was found to be much higher than that of nobiletin after oral administration of nobiletin in mice. Anti-inflammatory effects of 4DN were studied in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. The results showed 4DN not only dose-dependently inhibited LPS-induced nitric oxide production, but also significantly reduced expression of pro-inflammatory mediators, namely PGE2, IL-1β and IL-6. 4DN potently suppressed the expression of iNOS and COX-2 at both protein and mRNA levels. 4DN also inhibited nuclear translocation of NF-κB and AP-1. Furthermore, we demonstrated that 4DN activated transcription factor Nrf2 and its dependent genes including HO-1 and NQO1 whose expression may contribute to anti-inflammatory effects. The results demonstrated anti-inflammatory effects of 4DN and provided a scientific basis for using nobiletin as a nutraceutical to inhibit inflammation–driven diseases. PMID:26770275

Unique effect of 4-hydroxyestradiol and its methylation metabolites on lipid and cholesterol profiles in ovariectomized female rats.

PubMed

Wang, Pan; Zhu, Bao-Ting

2017-04-05

Animal studies have shown that endogenous estrogens such as 17β-estradiol (E 2 ) can modulate lipid profiles in vivo, and this effect is generally thought to be mediated by the estrogen receptors (ERs). The present study sought to test a hypothesis that some of the endogenous estrogen metabolites that have very weak estrogenic activity may exert some of their modulating effects on lipid metabolism in an ER-independent manner. Using ovariectomized female rats as an in vivo model, we found that 4-hydroxyestradiol (4-OH-E 2 ) has a markedly stronger effect in reducing the adipocyte size and serum cholesterol level in rats compared to E 2 , despite the weaker estrogenic activity of 4-OH-E 2 . Moreover, when E 2 or 4-OH-E 2 is used in combination with ICI-182,780 (an ER antagonist), some of their lipid-modulating effects are not blocked by this antiestrogen. Interestingly, two of the O-methylation metabolites of 4-OH-E 2 , namely, 4-methoxyestradiol and 4-methoxyestrone, which have much weaker estrogenic activity, were also found to have similar lipid-modulating effects compared to 4-OH-E 2 . Mechanistically, up-regulation of the expression of leptin, cytochrome P450 7A1 and LXRα genes is observed in the liver of animals treated with E 2 or 4-OH-E 2 , and the up-regulation is essentially not inhibited by co-treatment with ICI-182,780. These results demonstrate that some of the endogenous E 2 metabolites are functionally important modulators of lipid metabolic profiles in vivo. In addition, our findings indicate that an ER-independent pathway likely mediates some of the lipid-modulating effects of endogenous estrogens and their metabolic derivatives. Copyright © 2017 Elsevier B.V. All rights reserved.

Transporter-Mediated Disposition, Clinical Pharmacokinetics and Cholestatic Potential of Glyburide and Its Primary Active Metabolites.

PubMed

Li, Rui; Bi, Yi-An; Vildhede, Anna; Scialis, Renato J; Mathialagan, Sumathy; Yang, Xin; Marroquin, Lisa D; Lin, Jian; Varma, Manthena V S

2017-07-01

Glyburide is widely used for the treatment of type 2 diabetes. We studied the mechanisms involved in the disposition of glyburide and its pharmacologically active hydroxy metabolites M1 and M2b and evaluated their clinical pharmacokinetics and the potential role in glyburide-induced cholestasis employing physiologically based pharmacokinetic (PBPK) modeling. Transport studies of parent and metabolites in human hepatocytes and transfected cell systems imply hepatic uptake mediated by organic anion-transporting polypeptides. Metabolites are also subjected to basolateral and biliary efflux by P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated proteins, and are substrates to renal organic anion transporter 3. A PBPK model in combination with a Bayesian approach was developed considering the identified disposition mechanisms. The model reasonably described plasma concentration time profiles and urinary recoveries of glyburide and the metabolites, implying the role of multiple transport processes in their pharmacokinetics. Predicted free liver concentrations of the parent (∼30-fold) and metabolites (∼4-fold) were higher than their free plasma concentrations. Finally, all three compounds showed bile salt export pump inhibition in vitro; however, significant in vivo inhibition was not apparent for any compound on the basis of a predicted unbound liver exposure-response effect model using measured in vitro IC 50 values. In conclusion, this study demonstrates the important role of multiple drug transporters in the disposition of glyburide and its active metabolites, suggesting that variability in the function of these processes may lead to pharmacokinetic variability in the parent and the metabolites, potentially translating to pharmacodynamic variability. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Porritoxins, metabolites of Alternaria porri, as anti-tumor-promoting active compounds.

PubMed

Horiuchi, Masayuki; Tokuda, Harukuni; Ohnishi, Keiichiro; Yamashita, Masakazu; Nishino, Hoyoku; Maoka, Takashi

2006-02-01

To search for possible cancer chemopreventive agents from natural sources, we performed primary screening of metabolites of Alternaria porri by examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. The ethyl acetate extract of A. porri showed the inhibitory effect on EBV-EA activation. Three porritoxins (1-3) were obtained as inhibitory active compounds for EBV-EA from ethyl acetate extract. 6-(3',3'-Dimethylallyloxy)-4-methoxy-5-methylphthalide (2) showed the strongest activity among them. Inhibitory effect of porritoxin (1) and (2) was superior to that of beta-carotene, a well-known anti-tumor promoter. Furthermore, the structure-activity correlation of porritoxins and their related compounds were discussed.

Lignans, bacteriocides and organochlorine compounds activate the human pregnane X receptor (PXR)

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

Jacobs, Miriam N.; Nolan, Gail T.; Hood, Steven R.

2005-12-01

The pregnane X receptor (PXR) mediates the induction of enzymes involved in steroid metabolism and xenobiotic detoxification. The receptor is expressed in liver and intestinal tissues and is activated by a wide range of compounds. The ability of a diverse range of dietary compounds to activate PXR-mediated transcription was assayed in HuH7 cells following transient transfection with human PXR (hPXR). The compounds investigated included phytochemicals such as lignans and phytoestrogens, organochlorine dietary contaminants such as polychlorinated biphenyls (PCBs) and triclosan and selected steroid, drug and herbal compounds. The hPXR activation at the top concentrations tested (10 {mu}M) relative to themore » positive control 10 {mu}M rifampicin ranged from 1.3% (trans-resveratrol) to 152% (ICI 182780). Hydroxylated compounds were marginally more potent than the parent compounds (tamoxifen activation was 74.6% whereas 4 hydroxytamoxifen activation was 84.2%) or significantly greater (vitamin D{sub 3} activation was 1.6%, while hydroxylated vitamin D{sub 3} activation was 55.6%). Enterolactone, the metabolite of common dietary lignans, was a medium activator of PXR (35.6%), compared to the lower activation of a parent lignan, secoisolariciresinol (20%). Two non-hydroxylated PCB congeners (PCB 118 and 153), which present a larger fraction of the PCB contamination of fatty foods, activated hPXR by 26.6% and 17%, respectively. The pesticide trans-nonachlor activation was 53.8%, while the widely used bacteriocide triclosan was a medium activator of hPXR at 46.2%. The responsiveness of PXR to activation by lignan metabolites suggests that dietary intake of these compounds may affect the metabolism of drugs that are CYP3A substrates. Additionally, the evidence that organochlorine chemicals, particularly the ubiquitous triclosan, activate hPXR suggests that these environmental chemicals may, in part, exhibit their endocrine disruptor activities by altering PXR

In vitro antimycoplasmal activities of rufloxacin and its metabolite MF 922.

PubMed Central

Furneri, P M; Bisignano, G; Cerniglia, G; Nicoletti, G; Cesana, M; Tempera, G

1994-01-01

The in vitro activities of rufloxacin and its metabolite, MF 922, were compared with those of ofloxacin, ciprofloxacin, erythromycin, and minocycline against Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma fermentans, and Ureaplasma urealyticum. Rufloxacin, MF 922, and ciprofloxacin shared similar activities against all mycoplasmas tested. (MICs for 90% of isolates tested [MIC90s], 0.5 to 4 micrograms/ml. Ofloxacin had the lowest MIC90s for U. urealyticum, M. fermentans, and M. hominis (MIC90s, 0.25 to 1 micrograms/ml) and erythromycin had the lowest MIC90 for M. pneumoniae (MIC90, 0.004 micrograms/ml). PMID:7872762

Extraction of tamoxifen and its metabolites from formalin-fixed, paraffin-embedded tissues: an innovative quantitation method using liquid chromatography and tandem mass spectrometry.

PubMed

Ng, Ella S M; Kangarloo, S Bill; Konno, Mie; Paterson, Alexander; Magliocco, Anthony M

2014-03-01

Tamoxifen is a key therapeutic option for breast cancer treatment. Understanding its complex metabolism and pharmacokinetics is important for dose optimization. We examined the possibility of utilizing archival formalin-fixed paraffin-embedded (FFPE) tissue as an alternative sample source for quantification since well-annotated retrospective samples were always limited. Six 15 μm sections of FFPE tissues were deparaffinized with xylene and purified using solid-phase extraction. Tamoxifen and its metabolites were separated and detected by liquid chromatography-tandem mass spectrometry using multiple-reaction monitoring. This method was linear between 0.4 and 200 ng/g for 4-hydroxy-tamoxifen and endoxifen, and 4-2,000 ng/g for tamoxifen and N-desmethyl-tamoxifen. Inter- and intra-assay precisions were <9 %, and mean accuracies ranged from 81 to 106 %. Extraction recoveries were between 83 and 88 %. The validated method was applied to FFPE tissues from two groups of patients, who received 20 mg/day of tamoxifen for >6 months, and were classified into breast tumor recurrence and non-recurrence. Our preliminary data show that levels of tamoxifen metabolites were significantly lower in patients with recurrent cancer, suggesting that inter-individual variability in tamoxifen metabolism might partly account for the development of cancer recurrence. Nevertheless, other causes such as non-compliance or stopping therapy of tamoxifen could possibly lead to the concentration differences. The ability to successfully study tamoxifen metabolism in such tissue samples will rapidly increase our knowledge of how tamoxifen's action, metabolism and tissue distribution contribute to breast cancer control. However, larger population studies are required to understand the underlying mechanism of tamoxifen metabolism for optimization of its treatment.

Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS

PubMed Central

Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Huestis, Marilyn A

2014-01-01

Background Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. Conclusion For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including Phase I and II metabolites. Metabolite structural information and associated high-resolution mass spectra can be employed for developing clinical and forensic laboratory RCS-4 urine screening methods. PMID:25046048

The daidzein metabolite, 6,7,4'-Trihydroxyisoflavone, is a novel inhibitor of PKCα in suppressing solar UV-induced matrix metalloproteinase 1.

PubMed

Lim, Tae-Gyu; Kim, Jong-Eun; Lee, Sung-Young; Park, Jun Seong; Yeom, Myung Hun; Chen, Hanyong; Bode, Ann M; Dong, Zigang; Lee, Ki Won

2014-11-19

Soy isoflavone is an attractive source of functional cosmetic materials with anti-wrinkle, whitening and skin hydration effects. After consumption, the majority of soy isoflavones are converted to their metabolites in the human gastrointestinal tract. To understand the physiological impact of soy isoflavone on the human body, it is necessary to evaluate and address the biological function of its metabolites. In this study, we investigated the effect of 6,7,4'-trihydroxyisoflavone (6,7,4'-THIF), a major metabolite of daidzein, against solar UV (sUV)-induced matrix metalloproteinases (MMPs) in normal human dermal fibroblasts. MMPs play a critical role in the degradation of collagen in skin, thereby accelerating the aging process of skin. The mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MKK)3/6/p38 and MKK4/c-Jun N-terminal kinases (JNK) signaling pathways are known to modulate MMP-1 function, and their activation by sUV was significantly reduced by 6,7,4'-THIF pretreatment. Our results also indicated that the enzyme activity of protein kinase C (PKC)α, an upstream regulator of MKKs signaling, is suppressed by 6,7,4'-THIF using the in vitro kinase assay. Furthermore, the direct interaction between 6,7,4'-THIF and endogenous PKCα was confirmed using the pull-down assay. Not only sUV-induced MMP-1 expression, but also sUV-induced signaling pathway activation were decreased in PKCα knockdown cells. Overall, we elucidated the inhibitory effect of 6,7,4'-THIF on sUV-induced MMPs and suggest PKCα as its direct molecular target.

Widespread occurrence of neuro-active pharmaceuticals and metabolites in 24 Minnesota rivers and wastewaters

USGS Publications Warehouse

Writer, Jeffrey; Ferrer, Imma; Barber, Larry B.; Thurman, E. Michael

2013-01-01

Concentrations of 17 neuro-active pharmaceuticals and their major metabolites (bupropion, hydroxy-bupropion, erythro-hydrobupropion, threo-hydrobupropion, carbamazepine, 10,11,-dihydro-10,11,-dihydroxycarbamazepine, 10-hydroxy-carbamazepine, citalopram, N-desmethyl-citalopram, fluoxetine, norfluoxetine, gabapentin, lamotrigine, 2-N-glucuronide-lamotrigine, oxcarbazepine, venlafaxine and O-desmethyl-venlafaxine), were measured in treated wastewater and receiving surface waters from 24 locations across Minnesota, USA. The analysis of upstream and downstream sampling sites indicated that the wastewater treatment plants were the major source of the neuro-active pharmaceuticals and associated metabolites in surface waters of Minnesota. Concentrations of parent compound and the associated metabolite varied substantially between treatment plants (concentrations ± standard deviation of the parent compound relative to its major metabolite) as illustrated by the following examples; bupropion and hydrobupropion 700 ± 1000 ng L−1, 2100 ± 1700 ng L−1, carbamazepine and 10-hydroxy-carbamazepine 480 ± 380 ng L−1, 360 ± 400 ng L−1, venlafaxine and O-desmethyl-venlafaxine 1400 ± 1300 ng L−1, 1800 ± 2300 ng L−1. Metabolites of the neuro-active compounds were commonly found at higher or comparable concentrations to the parent compounds in wastewater effluent and the receiving surface water. Neuro-active pharmaceuticals and associated metabolites were detected only sporadically in samples upstream from the effluent outfall. Metabolite to parent ratios were used to evaluate transformation, and we determined that ratios in wastewater were much lower than those reported in urine, indicating that the metabolites are relatively more labile than the parent compounds in the treatment plants and in receiving waters. The widespread occurrence of neuro-active pharmaceuticals and metabolites in Minnesota effluents and surface waters indicate that

7,3′,4′-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses Ultraviolet B-induced Skin Cancer by Targeting Cot and MKK4*

PubMed Central

Lee, Dong Eun; Lee, Ki Won; Byun, Sanguine; Jung, Sung Keun; Song, Nury; Lim, Sung Hwan; Heo, Yong-Seok; Kim, Jong Eun; Kang, Nam Joo; Kim, Bo Yeon; Bowden, G. Tim; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

2011-01-01

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3′,4′-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3′,4′-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3′,4′-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3′,4′-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3′,4′-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3′,4′-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3′,4′-THIF, a potential skin cancer chemopreventive agent. PMID:21378167

Synthesis and Characterization of Bioactive Tamoxifen-conjugated Polymers

PubMed Central

Rickert, Emily L.; Trebley, Joseph P.; Peterson, Anton C.; Morrell, Melinda M.; Weatherman, Ross V.

2008-01-01

Macromolecular conjugates of tamoxifen could perhaps be used to circumvent some of the limitations of the extensively used breast cancer drug. To test the feasibility of these conjugates, a 4-hydroxytamoxifen analog was conjugated to a diaminoalkyl linker and then conjugated to activated esters of a poly(methacrylic acid) polymer synthesized by atom transfer radical polymerization. A polymer conjugated to the 4-hydroxytamoxifen analog with a six carbon linker showed high affinity for both estrogen receptor alpha and estrogen receptor beta and potent antagonism of the estrogen receptor in cell-based transcriptional reporter assays. These results suggest that the conjugation of 4-hydroxytamoxifen to a polymer results in a macromolecular conjugate that can display ligand in a manner that can be recognized by estrogen receptor and still act as a potent antiestrogen in cells. PMID:17929966

Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets β-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1

PubMed Central

Poliakov, Eugenia; Samuel, William; Duncan, Todd; Gutierrez, Danielle B.; Mata, Nathan L.; Redmond, T. Michael

2017-01-01

The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide’s various therapeutic uses will begin to resolve the pleotropic nature of this compound. PMID:28448568

Passage of irinotecan and its active metabolite, SN-38, into human milk.

PubMed

Nakagawa, J; Terui, K; Hosoi, K; Ueno, K; Yokoyama, Y; Hayakari, M

2016-10-01

We measured the levels of irinotecan and its active metabolite, SN-38, in human milk after the administration of irinotecan to assess the potential risks when women treated with irinotecan nurse their infants. Human milk was collected for 6 days starting on the day after irinotecan was administered. The levels of irinotecan and SN-38 in human milk were measured using liquid chromatography-mass spectrometry. Irinotecan was detected on Days 2 and 3 but not after Day 4. A strong signal indicating the presence of SN-38 was detected on Day 2 and the signal was readily detected until Day 7, indicating that SN-38 remained in human milk. Intravenously administered CPT-11 continues to pass into human milk over a prolonged period in the form of its active metabolite, SN-38. The relationship between administration of CPT-11 and SN-38 exposure and toxicity is still not well defined, so patients should avoid nursing their infants while they are being treated with CPT-11. © 2016 John Wiley & Sons Ltd.

Transport of the soy isoflavone daidzein and its conjugative metabolites by the carriers SOAT, NTCP, OAT4, and OATP2B1.

PubMed

Grosser, Gary; Döring, Barbara; Ugele, Bernhard; Geyer, Joachim; Kulling, Sabine E; Soukup, Sebastian T

2015-12-01

Soy isoflavones (IF) are phytoestrogens, which interact with estrogen receptors. They are extensively metabolized by glucuronosyltransferases and sulfotransferases, leading to the modulation of their estrogenic activity. It can be assumed that this biotransformation also has a crucial impact on the uptake of IF by active or passive cellular transport mechanisms, but little is known about the transport of IF phase II metabolites into the cell. Therefore, transport assays for phase II metabolites of daidzein (DAI) were carried out using HEK293 cell lines transfected with five human candidate carriers, i.e., organic anion transporter OAT4, sodium-dependent organic anion transporter (SOAT), Na(+)-taurocholate cotransporting polypeptide (NTCP), apical sodium-dependent bile acid transporter ASBT, and organic anion transporting polypeptide OATP2B1. Cellular uptake was monitored by UHPLC-DAD. DAI monosulfates were transported by the carriers NTCP and SOAT in a sodium-dependent manner, while OAT4-HEK293 cells revealed a partly sodium-dependent transport for these compounds. In contrast, DAI-7,4'-disulfate was only taken up by NTCP-HEK293 cells. DAI-7-glucuronide, but not DAI-4'-glucuronide, was transported exclusively by OATP2B1 in a sodium-independent manner. DAI-7-glucuronide-4'-sulfate, DAI-7-glucoside, and DAI were no substrate of any of the tested carriers. In addition, the inhibitory potency of the DAI metabolites toward estrone-sulfate (E1S) uptake of the above-mentioned carriers was determined. In conclusion, human SOAT, NTCP, OATP2B1, and OAT4 were identified as carriers for the DAI metabolites. Several metabolites were able to inhibit carrier-dependent E1S uptake. These findings might contribute to a better understanding of the bioactivity of IF especially in case of hormone-related cancers.

Mutagenic activity and metabolites in the urine of workers exposed to trinitrotoluene (TNT).

PubMed Central

Ahlborg, G; Einistö, P; Sorsa, M

1988-01-01

Urine samples taken after work and after a free weekend from 50 workers employed in various activities in a chemical plant manufacturing explosives were analysed. On the basis of hygienic surveys, the subjects were divided into three categories of exposure to trinitrotoluene (TNT). The urine analyses consisted of gas chromatographic identification of TNT and its two metabolites, 4-ADNT and 2-ADNT, and a determination of the mutagenic activity. Two frame shift detector strains of Salmonella typhimurium were used, TA 98 and TA 98 NR, the latter being deficient in endogenous nitroreductase activity. On the basis of previous results on TNT mutagenicity, no exogeneous metabolic system was used to test the urine concentrates. Both tester strains showed that the mean urinary mutagenic activity was higher in the after work samples than in post weekend samples from the same subjects, showing that bacterial nitroreductase activity was not significantly responsible for the mutagenicity, although the response was higher with strain TA 98 than with TA 98 NR. The interindividual variation in urine mutagenicity was high, however, and the difference between the two sampling times was statistically significant (p less than 0.05) only for the high exposed group (workers in trotyl foundry and sieve house). Correlation between urinary mutagenicity and concentration of TNT in urine was poor; correlation was significant only with the urinary concentration of 4-ADNT. The correlation between urinary TNT and both metabolites was good (p less than 0.001). These results suggest that analysis of 4-ADNT in urine would be a sufficient biological measure for controlling exposure to TNT. PMID:3378017

Secondary metabolites profiles and antioxidant activities of germinated brown and red rice

NASA Astrophysics Data System (ADS)

Nurnaistia, Y.; Aisyah, S.; Munawaroh, H. S. H.; Zackiyah

2018-05-01

The research aims to investigate the effect of germination on the secondary metabolite profiles and antioxidant activity of brown and red rice. The germination was performed by using a simple laboratory-scale machine that was designed and optimized to provide conditions that support the germination process. The germination was carried out for 2 days in dark conditions at 26°C and 99% humidity. Analysis of the secondary metabolite profile of ungerminated and germinated rice was performed using LC-MS. The antioxidant activities of ungerminated and germinated rice were done by using DPPH method. The results showed that the profiles of secondary metabolites of brown and red rice changed after germination. Some peaks were found to be induced in the germinated rice. However, some peaks were also loss during germination. The antioxidant activity of brown rice was slightly increased due to the germination, from 11.2% to 22.5%. Meanwhile the antioxidant activity of red rice was decreased after germination, from 73.8% to 60.0%.

Allocation of secondary metabolites, photosynthetic capacity, and antioxidant activity of Kacip Fatimah (Labisia pumila Benth) in response to CO2 and light intensity.

PubMed

Ibrahim, Mohd Hafiz; Jaafar, Hawa Z E; Karimi, Ehsan; Ghasemzadeh, Ali

2014-01-01

A split plot 3 by 4 experiment was designed to investigate and distinguish the relationships among production of secondary metabolites, soluble sugar, phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity, leaf gas exchange, chlorophyll content, antioxidant activity (DPPH), and lipid peroxidation under three levels of CO2 (400, 800, and 1200 μ mol/mol) and four levels of light intensity (225, 500, 625, and 900 μ mol/m(2)/s) over 15 weeks in Labisia pumila. The production of plant secondary metabolites, sugar, chlorophyll content, antioxidant activity, and malondialdehyde content was influenced by the interactions between CO2 and irradiance. The highest accumulation of secondary metabolites, sugar, maliondialdehyde, and DPPH activity was observed under CO2 at 1200 μ mol/mol + light intensity at 225 μ mol/m(2)/s. Meanwhile, at 400 μ mol/mol CO2 + 900 μ mol/m(2)/s light intensity the production of chlorophyll and maliondialdehyde content was the highest. As CO2 levels increased from 400 to 1200 μ mol/mol the photosynthesis, stomatal conductance, f v /f m (maximum efficiency of photosystem II), and PAL activity were enhanced. The production of secondary metabolites displayed a significant negative relationship with maliondialdehyde indicating lowered oxidative stress under high CO2 and low irradiance improved the production of plant secondary metabolites that simultaneously enhanced the antioxidant activity (DPPH), thus improving the medicinal value of Labisia pumila under this condition.

Decoy peptide targeted to Toll-IL-1R domain inhibits LPS and TLR4-active metabolite morphine-3 glucuronide sensitization of sensory neurons.

PubMed

Allette, Yohance M; Kim, Youngsook; Randolph, Aaron L; Smith, Jared A; Ripsch, Matthew S; White, Fletcher A

2017-06-16

Accumulating evidence indicates that Toll-like receptor (TLR) signaling adapter protein interactions with Toll/Interleukin-1 Receptor (TIR) domains present in sensory neurons may modulate neuropathic pain states. Following ligand interaction with TLRs, TIR serves to both initiate intracellular signaling and facilitate recruitment of signaling adapter proteins to the intracytoplasmic domain. Although TLR TIR is central to a number of TLR signaling cascades, its role in sensory neurons is poorly understood. In this study we investigated the degree to which TLR TIR decoy peptide modified to include a TAT sequence (Trans-Activator of Transcription gene in HIV; TAT-4BB) affected LPS-induced intracellular calcium flux and excitation in sensory neurons, and behavioral changes due to TLR4 active metabolite, morphine-3-glucuronide (M3G) exposure in vivo. TAT-4BB inhibited LPS-induced calcium changes in a majority of sensory neurons and decreased LPS-dependent neuronal excitability in small diameter neurons. Acute systemic administration of the TAT-4BB reversed M3G-induced tactile allodynia in a dose-dependent manner but did not affect motor activity, anxiety or responses to noxious thermal stimulus. These data suggest that targeting TLR TIR domains may provide novel pharmacological targets to reduce or reverse TLR4-dependent pain behavior in the rodent.

Antifungal activity of microbial secondary metabolites.

PubMed

Coleman, Jeffrey J; Ghosh, Suman; Okoli, Ikechukwu; Mylonakis, Eleftherios

2011-01-01

Secondary metabolites are well known for their ability to impede other microorganisms. Reanalysis of a screen of natural products using the Caenorhabditis elegans-Candida albicans infection model identified twelve microbial secondary metabolites capable of conferring an increase in survival to infected nematodes. In this screen, the two compound treatments conferring the highest survival rates were members of the epipolythiodioxopiperazine (ETP) family of fungal secondary metabolites, acetylgliotoxin and a derivative of hyalodendrin. The abundance of fungal secondary metabolites indentified in this screen prompted further studies investigating the interaction between opportunistic pathogenic fungi and Aspergillus fumigatus, because of the ability of the fungus to produce a plethora of secondary metabolites, including the well studied ETP gliotoxin. We found that cell-free supernatant of A. fumigatus was able to inhibit the growth of Candida albicans through the production of a secreted product. Comparative studies between a wild-type and an A. fumigatus ΔgliP strain unable to synthesize gliotoxin demonstrate that this secondary metabolite is the major factor responsible for the inhibition. Although toxic to organisms, gliotoxin conferred an increase in survival to C. albicans-infected C. elegans in a dose dependent manner. As A. fumigatus produces gliotoxin in vivo, we propose that in addition to being a virulence factor, gliotoxin may also provide an advantage to A. fumigatus when infecting a host that harbors other opportunistic fungi.

Antifungal Activity of Microbial Secondary Metabolites

PubMed Central

Okoli, Ikechukwu; Mylonakis, Eleftherios

2011-01-01

Secondary metabolites are well known for their ability to impede other microorganisms. Reanalysis of a screen of natural products using the Caenorhabditis elegans-Candida albicans infection model identified twelve microbial secondary metabolites capable of conferring an increase in survival to infected nematodes. In this screen, the two compound treatments conferring the highest survival rates were members of the epipolythiodioxopiperazine (ETP) family of fungal secondary metabolites, acetylgliotoxin and a derivative of hyalodendrin. The abundance of fungal secondary metabolites indentified in this screen prompted further studies investigating the interaction between opportunistic pathogenic fungi and Aspergillus fumigatus, because of the ability of the fungus to produce a plethora of secondary metabolites, including the well studied ETP gliotoxin. We found that cell-free supernatant of A. fumigatus was able to inhibit the growth of Candida albicans through the production of a secreted product. Comparative studies between a wild-type and an A. fumigatus ΔgliP strain unable to synthesize gliotoxin demonstrate that this secondary metabolite is the major factor responsible for the inhibition. Although toxic to organisms, gliotoxin conferred an increase in survival to C. albicans-infected C. elegans in a dose dependent manner. As A. fumigatus produces gliotoxin in vivo, we propose that in addition to being a virulence factor, gliotoxin may also provide an advantage to A. fumigatus when infecting a host that harbors other opportunistic fungi. PMID:21966496

The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites.

PubMed

Guyonnet, Julien P; Vautrin, Florian; Meiffren, Guillaume; Labois, Clément; Cantarel, Amélie A M; Michalet, Serge; Comte, Gilles; Haichar, Feth El Zahar

2017-04-01

The aim of this study was to determine (i) whether plant nutritional strategy affects the composition of primary metabolites exuded into the rhizosphere and (ii) the impact of exuded metabolites on denitrification activity in soil. We answered this question by analysing primary metabolite content extracted from the root-adhering soil (RAS) and the roots of three grasses representing different nutrient management strategies: conservative (Festuca paniculata), intermediate (Bromus erectus) and exploitative (Dactylis glomerata). We also investigated the impact of primary metabolites on soil microbial denitrification enzyme activity without carbon addition, comparing for each plant RAS and bulk soils. Our data show that plant nutritional strategy impacts on primary metabolite composition of root extracts or RAS. Further we show, for the first time, that RAS-extracted primary metabolites are probably better indicators to explain plant nutrient strategy than root-extracted ones. In addition, our results show that some primary metabolites present in the RAS were well correlated with soil microbial denitrification activity with positive relationships found between denitrification and the presence of some organic acids and negative ones with the presence of xylose. We demonstrated that the analysis of primary metabolites extracted from the RAS is probably more pertinent to evaluate the impact of plant on soil microbial community functioning. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Reappraising the structures and distribution of metabolites from black aspergilli containing uncommon 2-benzyl-4H-pyran-4-one and 2-benzylpyridin-4(1H)-one systems

PubMed Central

Henrikson, Jon C.; Ellis, Trevor K.; King, Jarrod B.; Cichewicz, Robert H.

2011-01-01

To date, natural products containing 2-benzyl-4H-pyran-4-one and 2-benzylpyridin-4(1H)-one substructures have been encountered in relatively few fungi outside of the black aspergilli clade. While exploring the occurrence of these compounds among Aspergillus spp., it was determined that the structures of the unusual furopyrrols tensidols A and B (5 and 6) and JBIR-86 and JBIR-87 (9 and 10) were incorrect and should be reassigned as 2-benzyl-4H-pyran-4-ones (7, 8, 11e, and 12, respectively). The origin of the unique N-phenyl groups in the 2-benzylpyridin-4(1H)-ones nygerones A and B (1 and 2) was also examined and it was established that N-phenylamides added to the culture medium were suitable substrates for generating these metabolites; however, this phenomenon remained limited to a single fungus in our collection (Aspergillus niger ATCC 1015). A variety of 2-benzyl-4H-pyran-4-ones and 2-benzylpyridin-4(1H)-ones were detected among the black aspergilli, but only pestalamide B (13) was found in all eleven of the tested strains. These metabolites, as well as a group of synthetic analogues demonstrated weak antifungal activity against several Candida strains, Aspergillus flavus, and Aspergillus fumigatus. PMID:21854017

Smart coumarin-tagged imprinted polymers for the rapid detection of tamoxifen.

PubMed

Ray, Judith V; Mirata, Fosca; Pérollier, Celine; Arotcarena, Michel; Bayoudh, Sami; Resmini, Marina

2016-03-01

A signalling molecularly imprinted polymer was synthesised for easy detection of tamoxifen and its metabolites. 6-Vinylcoumarin-4-carboxylic acid (VCC) was synthesised from 4-bromophenol to give a fluorescent monomer, designed to switch off upon binding of tamoxifen. Clomiphene, a chlorinated analogue, was used as the template for the imprinting, and its ability to quench the coumarin fluorescence when used in a 1:1 ratio was demonstrated. Tamoxifen and 4-hydroxytamoxifen were also shown to quench coumarin fluorescence. Imprinted and non-imprinted polymers were synthesised using VCC, methacrylic acid as a backbone monomer and ethylene glycol dimethacrylate as cross-linker, and were ground and sieved to particle sizes ranging between 45 and 25 μm. Rebinding experiments demonstrate that the imprinted polymer shows very strong affinity for both clomiphene and tamoxifen, while the non-imprinted polymer shows negligible rebinding. The fluorescence of the imprinted polymer is quenched by clomiphene, tamoxifen and 4-hydroxytamoxifen. The switch off in fluorescence of the imprinted polymer under these conditions could also be detected under a UV lamp with the naked eye, making this matrix suitable for applications when coupled with a sample preparation system.

Sulindac metabolites inhibit epidermal growth factor receptor activation and expression.

PubMed

Pangburn, Heather A; Kraus, Hanna; Ahnen, Dennis J; Rice, Pamela L

2005-09-02

Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a decreased mortality from colorectal cancer (CRC). NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2) signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF) receptor (EGFR). HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068), total EGFR, phosphorylated ERK1/2 (pERK1/2), total ERK1/2, activated caspase-3, and alpha-tubulin. EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. These results suggest that downregulation of EGFR signaling by sulindac metabolites may

[Isolation, identification and structural characterization of secondary metabolites from amarine sponge-derived rare actinobacterium Dermacoccus sp. X4].

PubMed

Zhang, Yanfeng; Xu, Yong; Chen, Lei; Hu, Jun; Zhang, Xuecheng; Fang, Wei; Fang, Zemin; Xiao, Yazhong

2016-05-25

We isolated and identified the symbiotic and adnascent microorganisms from an unidentified sponge collected from 10-meter-deep seawater of the Paracel Islands in China. A total of 16 strains were obtained and identified. Through bacteriostatic activity assay, one of the strains, Dermacoccus sp. X4, was found to effectively inhibit the growth of Staphylococcus aureus. Subsequently, its secondary metabolites were purified by silica gel partition, octadecylsilane (ODS) reverse phase, Sephadex™LH-20 size exclusion, and C18 reverse phase chromatography. Using liquid chromatography, mass spectrometry, and nuclear magnetic resonance, three of the purified compounds were structurally characterized to be one 3-(4-hydroxybenzyl) hexahydropyrrolo [1,2-a]pyrazine-1,4-dione and two indole acid glycerides. This is the first report about indole acid glyceride isolated from microbial secondary metabolites, enriching marine drug candidate resources.

Diversity of Secondary Metabolites from Marine Bacillus Species: Chemistry and Biological Activity

PubMed Central

Mondol, Muhammad Abdul Mojid; Shin, Hee Jae; Islam, Mohammad Tofazzal

2013-01-01

Marine Bacillus species produce versatile secondary metabolites including lipopeptides, polypeptides, macrolactones, fatty acids, polyketides, and isocoumarins. These structurally diverse compounds exhibit a wide range of biological activities, such as antimicrobial, anticancer, and antialgal activities. Some marine Bacillus strains can detoxify heavy metals through reduction processes and have the ability to produce carotenoids. The present article reviews the chemistry and biological activities of secondary metabolites from marine isolates. Side by side, the potential for application of these novel natural products from marine Bacillus strains as drugs, pesticides, carotenoids, and tools for the bioremediation of heavy metal toxicity are also discussed. PMID:23941823

Allocation of Secondary Metabolites, Photosynthetic Capacity, and Antioxidant Activity of Kacip Fatimah (Labisia pumila Benth) in Response to CO2 and Light Intensity

PubMed Central

Jaafar, Hawa Z. E.; Karimi, Ehsan; Ghasemzadeh, Ali

2014-01-01

A split plot 3 by 4 experiment was designed to investigate and distinguish the relationships among production of secondary metabolites, soluble sugar, phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity, leaf gas exchange, chlorophyll content, antioxidant activity (DPPH), and lipid peroxidation under three levels of CO2 (400, 800, and 1200 μmol/mol) and four levels of light intensity (225, 500, 625, and 900 μmol/m2/s) over 15 weeks in Labisia pumila. The production of plant secondary metabolites, sugar, chlorophyll content, antioxidant activity, and malondialdehyde content was influenced by the interactions between CO2 and irradiance. The highest accumulation of secondary metabolites, sugar, maliondialdehyde, and DPPH activity was observed under CO2 at 1200 μmol/mol + light intensity at 225 μmol/m2/s. Meanwhile, at 400 μmol/mol CO2 + 900 μmol/m2/s light intensity the production of chlorophyll and maliondialdehyde content was the highest. As CO2 levels increased from 400 to 1200 μmol/mol the photosynthesis, stomatal conductance, f v/f m (maximum efficiency of photosystem II), and PAL activity were enhanced. The production of secondary metabolites displayed a significant negative relationship with maliondialdehyde indicating lowered oxidative stress under high CO2 and low irradiance improved the production of plant secondary metabolites that simultaneously enhanced the antioxidant activity (DPPH), thus improving the medicinal value of Labisia pumila under this condition. PMID:24683336

Induction of cytochrome P450 1A1 in MCF-7 human breast cancer cells by 4-chlorobiphenyl (PCB3) and the effects of its hydroxylated metabolites on cellular apoptosis

PubMed Central

Ptak, Anna; Ludewig, Gabriele; Rak, Agnieszka; Nadolna, Weronika; Bochenek, Michał; Gregoraszczuk, Ewa L

2010-01-01

Several studies suggest an involvement of PCBs in breast cancer formation, but the results are ambiguous and the mechanisms not clear. We propose that local activation of cytochrome P450 enzymes, CYP1A1 and CYP1B1 by PCB3, may generate active metabolites which affect apoptosis and thereby promote mammary carcinogenesis. To test this hypothesis MCF-7 human breast cancer cells were exposed to 300 nM PCB3 and its hydroxylated metabolites, 4OH-PCB and 3,4diOH-PCB3. The enzyme activity for CYP1A1 was assayed using the EROD assay, and CYP1A1 and CYP1B1 protein expression by western blotting. PCB3 increased CYP1A1 activity (~1.5fold) and protein levels within 6 hrs after exposure. No effect on CYP1B1 protein expression was observed. The effects of PCB3 and both its metabolites on staurosporine-induced apoptosis were determined by measuring DNA fragmentation using ELISA and TUNEL assays, and by measuring caspase-8 and caspase-9 activity. We found that PCB3 and both of its hydroxylated metabolites had no effect on caspase-8 and caspase-9 activity when cells were grown in medium deprived of estrogen, but reduced caspase-9 activity when cells were grown in medium supplemented with serum containing estradiol. Interestingly, a decrease of DNA fragmentation was observed upon treatment with 3,4diOH-PCB3 in both culture conditions, suggesting that 3,4diOH-PCB3 affects a caspase-independent pathway of cell death. In summary, interactions of PCB3 and its metabolites with estradiol by yet unknown mechanisms inhibit caspase 9-related apoptosis and additional, other death pathways are affected by the catechol metabolite 3,4diOH-PCB3. These anti-apoptotic effects and the change in metabolic activity may contribute to the carcinogenic effect of PCBs. PMID:19604582

Antimicrobial and Cytotoxic Activity of Extracts of Ferula heuffelii Griseb. ex Heuff. and Its Metabolites.

PubMed

Pavlović, Ivan; Petrović, Silvana; Milenković, Marina; Stanojković, Tatjana; Nikolić, Dejan; Krunić, Aleksej; Niketić, Marjan

2015-10-01

The antimicrobial and cytotoxic activities of isolates (CHCl3 and MeOH extracts and selected metabolites) obtained from the underground parts of the Balkan endemic plant Ferula heuffelii Griseb. ex Heuff. were assessed. The CHCl3 and MeOH extracts exhibited moderate antimicrobial activity, being more pronounced against Gram-positive than Gram-negative bacteria, especially against Staphylococcus aureus (MIC=12.5 μg/ml for both extracts) and Micrococcus luteus (MIC=50 and 12.5 μg/ml, resp.). Among the tested metabolites, (6E)-1-(2,4-dihydroxyphenyl)-3,7,11-trimethyl-3-vinyldodeca-6,10-dien-1-one (2) and (2S*,3R*)-2-[(3E)-4,8-dimethylnona-3,7-dien-1-yl]-2,3-dihydro-7-hydroxy-2,3-dimethylfuro[3,2-c]coumarin (4) demonstrated the best antimicrobial activity. Compounds 2 and 4 both strongly inhibited the growth of M. luteus (MIC=11.2 and 5.2 μM, resp.) and Staphylococcus epidermidis (MIC=22.5 and 10.5 μM, resp.) and compound 2 additionally also the growth of Bacillus subtilis (MIC=11.2 μM). The cytotoxic activity of the isolates was tested against three human cancer cell lines, viz., cervical adenocarcinoma (HeLa), chronic myelogenous leukemia (K562), and breast cancer (MCF-7) cells. The CHCl3 extract exhibited strong cytotoxic activity against all cell lines (IC50 <11.0 μg/ml). All compounds strongly inhibited the growth of the K562 and HeLa cell lines. Compound 4 exhibited also a strong activity against the MCF-7 cell line, comparable to that of cisplatin (IC50 =22.32±1.32 vs. 18.67±0.75μM). Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

Strategies to enhance biologically active-secondary metabolites in cell cultures of Artemisia - current trends.

PubMed

Ali, Mohammad; Abbasi, Bilal Haider; Ahmad, Nisar; Khan, Haji; Ali, Gul Shad

2017-11-01

The genus Artemisia has been utilized worldwide due to its immense potential for protection against various diseases, especially malaria. Artemisia absinthium, previously renowned for its utilization in the popular beverage absinthe, is gaining resurgence due to its extensive pharmacological activities. Like A. annua, this species exhibits strong biological activities like antimalarial, anticancer and antioxidant. Although artemisinin was found to be the major metabolite for its antimalarial effects, several flavonoids and terpenoids are considered to possess biological activities when used alone and also to synergistically boost the bioavailability of artemisinin. However, due to the limited quantities of these metabolites in wild plants, in vitro cultures were established and strategies have been adopted to enhance medicinally important secondary metabolites in these cultures. This review elaborates on the traditional medicinal uses of Artemisia species and explains current trends to establish cell cultures of A. annua and A. absinthium for enhanced production of medicinally important secondary metabolites.

Long-Chain Metabolites of Vitamin E: Metabolic Activation as a General Concept for Lipid-Soluble Vitamins?

PubMed

Schubert, Martin; Kluge, Stefan; Schmölz, Lisa; Wallert, Maria; Galli, Francesco; Birringer, Marc; Lorkowski, Stefan

2018-01-12

Vitamins E, A, D and K comprise the class of lipid-soluble vitamins. For vitamins A and D, a metabolic conversion of precursors to active metabolites has already been described. During the metabolism of vitamin E, the long-chain metabolites (LCMs) 13'-hydroxychromanol (13'-OH) and 13'-carboxychromanol (13'-COOH) are formed by oxidative modification of the side-chain. The occurrence of these metabolites in human serum indicates a physiological relevance. Indeed, effects of the LCMs on lipid metabolism, apoptosis, proliferation and inflammatory actions as well as tocopherol and xenobiotic metabolism have been shown. Interestingly, there are several parallels between the actions of the LCMs of vitamin E and the active metabolites of vitamin A and D. The recent findings that the LCMs exert effects different from that of their precursors support their putative role as regulatory metabolites. Hence, it could be proposed that the mode of action of the LCMs might be mediated by a mechanism similar to vitamin A and D metabolites. If the physiological relevance and this concept of action of the LCMs can be confirmed, a general concept of activation of lipid-soluble vitamins via their metabolites might be deduced.

Swelling and Eicosanoid Metabolites Differentially Gate TRPV4 Channels in Retinal Neurons and Glia

PubMed Central

Ryskamp, Daniel A.; Jo, Andrew O.; Frye, Amber M.; Vazquez-Chona, Felix; MacAulay, Nanna; Thoreson, Wallace B.

2014-01-01

Activity-dependent shifts in ionic concentrations and water that accompany neuronal and glial activity can generate osmotic forces with biological consequences for brain physiology. Active regulation of osmotic gradients and cellular volume requires volume-sensitive ion channels. In the vertebrate retina, critical support to volume regulation is provided by Müller astroglia, but the identity of their osmosensor is unknown. Here, we identify TRPV4 channels as transducers of mouse Müller cell volume increases into physiological responses. Hypotonic stimuli induced sustained [Ca2+]i elevations that were inhibited by TRPV4 antagonists and absent in TRPV4−/− Müller cells. Glial TRPV4 signals were phospholipase A2- and cytochrome P450-dependent, characterized by slow-onset and Ca2+ waves, and, in excess, were sufficient to induce reactive gliosis. In contrast, neurons responded to TRPV4 agonists and swelling with fast, inactivating Ca2+ signals that were independent of phospholipase A2. Our results support a model whereby swelling and proinflammatory signals associated with arachidonic acid metabolites differentially gate TRPV4 in retinal neurons and glia, with potentially significant consequences for normal and pathological retinal function. PMID:25411497

Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications

PubMed Central

Ren, Lili; Chen, Fang; Feng, Yuqian

2016-01-01

Ginkgo biloba is a typical relic plant that rarely suffers from pest hazards. This study analyzed the pattern of G. biloba pest hazards in Beijing; tested the antifeedant activity of G. biloba extracts, including ginkgo flavonoids, ginkgolide, and bilobalide, against Hyphantria cunea larvae; determined the activities of glutathione transferase (GSTs), acetylcholinesterase (AChE), carboxylesterase (CarE) and mixed-functional oxidase (MFO), in larvae after feeding on these G. biloba secondary metabolites; and screened for effective botanical antifeedants in the field. In this study, no indicators of insect infestation were found for any of the examined leaves of G. biloba; all tested secondary metabolites showed significant antifeedant activity and affected the activity of the four larval detoxifying enzymes. Ginkgolide had the highest antifeedant activity and the most significant effect on the detoxifying enzymes (P<0.05). Spraying leaves with G. biloba extracts or ginkgolide both significantly repelled H. cunea larvae in the field (P<0.05), although the former is more economical and practical. This study investigated the antifeedant activity of G. biloba secondary metabolites against H. cunea larvae, and the results provide new insights into the mechanism of G. biloba pest resistance. This study also developed new applications of G. biloba secondary metabolites for effective pest control. PMID:27214257

Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications.

PubMed

Pan, Long; Ren, Lili; Chen, Fang; Feng, Yuqian; Luo, Youqing

2016-01-01

Ginkgo biloba is a typical relic plant that rarely suffers from pest hazards. This study analyzed the pattern of G. biloba pest hazards in Beijing; tested the antifeedant activity of G. biloba extracts, including ginkgo flavonoids, ginkgolide, and bilobalide, against Hyphantria cunea larvae; determined the activities of glutathione transferase (GSTs), acetylcholinesterase (AChE), carboxylesterase (CarE) and mixed-functional oxidase (MFO), in larvae after feeding on these G. biloba secondary metabolites; and screened for effective botanical antifeedants in the field. In this study, no indicators of insect infestation were found for any of the examined leaves of G. biloba; all tested secondary metabolites showed significant antifeedant activity and affected the activity of the four larval detoxifying enzymes. Ginkgolide had the highest antifeedant activity and the most significant effect on the detoxifying enzymes (P<0.05). Spraying leaves with G. biloba extracts or ginkgolide both significantly repelled H. cunea larvae in the field (P<0.05), although the former is more economical and practical. This study investigated the antifeedant activity of G. biloba secondary metabolites against H. cunea larvae, and the results provide new insights into the mechanism of G. biloba pest resistance. This study also developed new applications of G. biloba secondary metabolites for effective pest control.

Differential effects of clinically used derivatives and metabolites of artemisinin in the activation of constitutive androstane receptor isoforms

PubMed Central

Burk, O; Piedade, R; Ghebreghiorghis, L; Fait, JT; Nussler, AK; Gil, JP; Windshügel, B; Schwab, M

2012-01-01

BACKGROUND AND PURPOSE Widespread resistance to antimalarial drugs requires combination therapies with increasing risk of pharmacokinetic drug–drug interactions. Here, we explore the capacity of antimalarial drugs to induce drug metabolism via activation of constitutive androstane receptors (CAR) by ligand binding. EXPERIMENTAL APPROACH A total of 21 selected antimalarials and 11 major metabolites were screened for binding to CAR isoforms using cellular and in vitro CAR-coactivator interaction assays, combined with in silico molecular docking. Identified ligands were further characterized by cell-based assays and primary human hepatocytes were used to elucidate induction of gene expression. KEY RESULTS Only two artemisinin derivatives arteether and artemether, the metabolite deoxyartemisinin and artemisinin itself demonstrated agonist binding to the major isoforms CAR1 and CAR3, while arteether and artemether were also inverse agonists of CAR2. Dihydroartemisinin and artesunate acted as weak inverse agonists of CAR1. While arteether showed the highest activities in vitro, it was less active than artemisinin in inducing hepatic CYP3A4 gene expression in hepatocytes. CONCLUSIONS AND IMPLICATIONS Artemisinin derivatives and metabolites differentially affect the activities of CAR isoforms and of the pregnane X receptor (PXR). This negates a common effect of these drugs on CAR/PXR-dependent induction of drug metabolism and further provides an explanation for artemisinin consistently inducing cytochrome P450 genes in vivo, whereas arteether and artemether do not. All these drugs are metabolized very rapidly, but only artemisinin is converted to an enzyme-inducing metabolite. For better understanding of pharmacokinetic drug–drug interaction possibilities, the inducing properties of artemisinin metabolites should be considered. PMID:22577882

Screening of marine fungus from Nanji Island and activity of their metabolites against pathogenic Vibrio from Pseudosciaena crocea

NASA Astrophysics Data System (ADS)

Zhao, Shujiang; Li, Shuping; Liu, Huihui; Zhao, Qian; Wang, Jieyou; Yan, Maocang

2012-09-01

Seventy-eight marine fungal strains were isolated from sediment samples collected off the coast of Nanji Island, Wenzhou, Zhejiang Province, China. Antibacterial screening using the agar disc method showed that 19 of the isolated strains could inhibit at least one pathogenic V ibrio from P seudosciaena crocea. Subsequent screening confirmed that nine strains produced antibacterial metabolites that had activity against one or several types of pathogenic V ibrio. Strain NJ0104 had the widest antimicrobial spectrum and strong activity, particularly against Vibrio parahaemolyticus-MM0810072. A preliminary study of NJ0104 antibacterial metabolites demonstrated that they had thermal stability up to 80°C, ultraviolet stability up to 40 min and pH stability between 4.0-7.0. In addition, the antibacterial metabolites were readily soluble in butanol. To identify the specific strain, the ITS-5.8S rDNA regions of NJ0104 were PCR amplified and sequenced. Based on the combination of phenotypic and genotypic data, the strain was identified as Arthrinium sp.

Hypouricaemic action of mangiferin results from metabolite norathyriol via inhibiting xanthine oxidase activity.

PubMed

Niu, Yanfen; Liu, Jia; Liu, Hai-Yang; Gao, Li-Hui; Feng, Guo-Hua; Liu, Xu; Li, Ling

2016-09-01

Context Mangiferin has been reported to possess a potential hypouricaemic effect. However, the pharmacokinetic studies in rats showed that its oral bioavailability was only 1.2%, suggesting that mangiferin metabolites might exert the action. Objective The hypouricaemic effect and the xanthine oxidase inhibition of mangiferin and norathyriol, a mangiferin metabolite, were investigated. Inhibition of norathyriol analogues (compounds 3-9) toward xanthine oxidase was also evaluated. Materials and methods For a dose-dependent study, mangiferin (1.5-6.0 mg/kg) and norathyriol (0.92-3.7 mg/kg) were administered intragastrically to mice twice daily for five times. For a time-course study, mice received mangiferin and norathyriol both at a single dose of 7.1 μmol/kg. In vitro, inhibition of test compounds (2.4-2.4 mM) against xanthine oxidase activity was evaluated by the spectrophotometrical method. The inhibition type was identified from Lineweaver-Burk plots. Results Norathyriol (0.92, 1.85 and 3.7 mg/kg) dose dependently decreased the serum urate levels by 27.0, 33.6 and 37.4%, respectively. The action was more potent than that of mangiferin at the low dose, but was equivalent at the higher doses. Additionally, the hypouricaemic action of them exhibited a time dependence. In vitro, norathyriol markedly inhibited the xanthine oxidase activities, with the IC50 value of 44.6 μM, but mangiferin did not. The kinetic studies showed that norathyriol was an uncompetitive inhibitor by Lineweaver-Burk plots. The structure-activity relationships exhibited that three hydroxyl groups in norathyriol at the C-1, C-3 and C-6 positions were essential for maintaining xanthine oxidase inhibition. Discussion and conclusion Norathyriol was responsible for the hypouricaemic effect of mangiferin via inhibiting xanthine oxidase activity.

Secondary metabolites from Ganoderma.

PubMed

Baby, Sabulal; Johnson, Anil John; Govindan, Balaji

2015-06-01

Ganoderma is a genus of medicinal mushrooms. This review deals with secondary metabolites isolated from Ganoderma and their biological significance. Phytochemical studies over the last 40years led to the isolation of 431 secondary metabolites from various Ganoderma species. The major secondary compounds isolated are (a) C30 lanostanes (ganoderic acids), (b) C30 lanostanes (aldehydes, alcohols, esters, glycosides, lactones, ketones), (c) C27 lanostanes (lucidenic acids), (d) C27 lanostanes (alcohols, lactones, esters), (e) C24, C25 lanostanes (f) C30 pentacyclic triterpenes, (g) meroterpenoids, (h) farnesyl hydroquinones (meroterpenoids), (i) C15 sesquiterpenoids, (j) steroids, (k) alkaloids, (l) prenyl hydroquinone (m) benzofurans, (n) benzopyran-4-one derivatives and (o) benzenoid derivatives. Ganoderma lucidum is the species extensively studied for its secondary metabolites and biological activities. Ganoderma applanatum, Ganoderma colossum, Ganoderma sinense, Ganoderma cochlear, Ganoderma tsugae, Ganoderma amboinense, Ganoderma orbiforme, Ganoderma resinaceum, Ganoderma hainanense, Ganoderma concinna, Ganoderma pfeifferi, Ganoderma neo-japonicum, Ganoderma tropicum, Ganoderma australe, Ganoderma carnosum, Ganoderma fornicatum, Ganoderma lipsiense (synonym G. applanatum), Ganoderma mastoporum, Ganoderma theaecolum, Ganoderma boninense, Ganoderma capense and Ganoderma annulare are the other Ganoderma species subjected to phytochemical studies. Further phytochemical studies on Ganoderma could lead to the discovery of hitherto unknown biologically active secondary metabolites. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Secondary Metabolites from Marine Microorganisms. I. Secondary Metabolites from Marine Actinomycetes].

PubMed

Orlova, T I; Bulgakova, V G; Polin, A N

2015-01-01

Review represents data on new active metabolites isolated from marine actinomycetes published in 2007 to 2014. Marine actinomycetes are an unlimited source of novel secondary metabolites with various biological activities. Among them there are antibiotics, anticancer compounds, inhibitors of biochemical processes.

Long-Chain Metabolites of Vitamin E: Metabolic Activation as a General Concept for Lipid-Soluble Vitamins?

PubMed Central

Schubert, Martin; Kluge, Stefan; Schmölz, Lisa; Wallert, Maria

2018-01-01

Vitamins E, A, D and K comprise the class of lipid-soluble vitamins. For vitamins A and D, a metabolic conversion of precursors to active metabolites has already been described. During the metabolism of vitamin E, the long-chain metabolites (LCMs) 13′-hydroxychromanol (13′-OH) and 13′-carboxychromanol (13′-COOH) are formed by oxidative modification of the side-chain. The occurrence of these metabolites in human serum indicates a physiological relevance. Indeed, effects of the LCMs on lipid metabolism, apoptosis, proliferation and inflammatory actions as well as tocopherol and xenobiotic metabolism have been shown. Interestingly, there are several parallels between the actions of the LCMs of vitamin E and the active metabolites of vitamin A and D. The recent findings that the LCMs exert effects different from that of their precursors support their putative role as regulatory metabolites. Hence, it could be proposed that the mode of action of the LCMs might be mediated by a mechanism similar to vitamin A and D metabolites. If the physiological relevance and this concept of action of the LCMs can be confirmed, a general concept of activation of lipid-soluble vitamins via their metabolites might be deduced. PMID:29329238

Characterization of the methemoglobin forming metabolites of benzocaine and lidocaine.

PubMed

Hartman, Neil; Zhou, Hongfei; Mao, Jinzhe; Mans, Daniel; Boyne, Michael; Patel, Vikram; Colatsky, Thomas

2017-05-01

1. Topical anesthesia with benzocaine or lidocaine occasionally causes methemoglobinemia, an uncommon but potentially fatal disorder where the blood has a reduced ability to transport oxygen. Previous in vitro studies using human whole blood have shown that benzocaine causes more methemoglobin (MetHb) formation than lidocaine, and that both compounds require metabolic transformation to form the MetHb producing species. In the current investigation, the active species of benzocaine forming the MetHb was investigated. 2. HPLC analysis of benzocaine samples incubated with human hepatic S9 showed the formation of a peak with the same UV spectrum and retention time as benzocaine hydroxylamine (BenzNOH). To confirm the activity of BenzNOH, MetHb production following exposure to the compound was determined in whole human blood using an Avoximeter 4000 CO-oximeter. 3. BenzNOH produced MetHb in a concentration dependent manner without the need for metabolic activation. Benzocaine in the presence of metabolic activation required a concentration of 500 μM to produce a similar degree of MetHb formation as 20 μM BenzNOH without activation. Previous work suggested that two metabolites of lidocaine may also form MetHb; N-hydroxyxylidine and 4-hydroxyxylidine. Of these two metabolites 4-hydroxyxylidine produced the most MetHb in whole blood in vitro in the absence of metabolic activation, however BenzNOH produced up to 14.2 times more MetHb than 4-hydroxyxylidine at a similar concentration. 4. These results suggest that the ability of benzocaine to form MetHb is likely to be mediated through its hydroxylamine metabolite and that this metabolite is inherently more active than the potentially MetHb-forming metabolites of lidocaine.

Dipyrone metabolite 4-MAA induces hypothermia and inhibits PGE2-dependent and -independent fever while 4-AA only blocks PGE2-dependent fever

PubMed Central

Malvar, David do C; Aguiar, Fernando A; Vaz, Artur de L L; Assis, Débora C R; de Melo, Miriam C C; Jabor, Valquíria A P; Kalapothakis, Evanguedes; Ferreira, Sérgio H; Clososki, Giuliano C; de Souza, Glória E P

2014-01-01

BACKGROUND AND PURPOSE The antipyretic and hypothermic prodrug dipyrone prevents PGE2-dependent and -independent fever induced by LPS from Escherichia coli and Tityus serrulatus venom (Tsv) respectively. We aimed to identify the dipyrone metabolites responsible for the antipyretic and hypothermic effects. EXPERIMENTAL APPROACH Male Wistar rats were treated i.p. with indomethacin (2 mg·kg−1), dipyrone, 4-methylaminoantipyrine (4-MAA), 4-aminoantipyrine (4-AA) (60–360 mg·kg−1), 4-formylaminoantipyrine, 4-acethylaminoantipyrine (120–360 mg·kg−1) or vehicle 30 min before i.p. injection of LPS (50 μg·kg−1), Tsv (150 μg·kg−1) or saline. Rectal temperatures were measured by tele-thermometry and dipyrone metabolite concentrations determined in the plasma, CSF and hypothalamus by LC-MS/MS. PGE2 concentrations were determined in the CSF and hypothalamus by elisa. KEY RESULTS In contrast to LPS, Tsv-induced fever was not followed by increased PGE2 in the CSF or hypothalamus. The antipyretic time-course of 4-MAA and 4-AA on LPS-induced fever overlapped with the period of the highest concentrations of 4-MAA and 4-AA in the hypothalamus, CSF and plasma. These metabolites reduced LPS-induced fever and the PGE2 increase in the plasma, CSF and hypothalamus. Only 4-MAA inhibited Tsv-induced fever. The higher doses of dipyrone and 4-MAA also induced hypothermia. CONCLUSIONS AND IMPLICATIONS The presence of 4-MAA and 4-AA in the CSF and hypothalamus was associated with PGE2 synthesis inhibition and a decrease in LPS-induced fever. 4-MAA was also shown to be an antipyretic metabolite for PGE2-independent fever induced by Tsv suggesting that it is responsible for the additional antipyretic mechanism of dipyrone. Moreover, 4-MAA is the hypothermic metabolite of dipyrone. PMID:24712707

Dipyrone metabolite 4-MAA induces hypothermia and inhibits PGE2 -dependent and -independent fever while 4-AA only blocks PGE2 -dependent fever.

PubMed

Malvar, David do C; Aguiar, Fernando A; Vaz, Artur de L L; Assis, Débora C R; de Melo, Miriam C C; Jabor, Valquíria A P; Kalapothakis, Evanguedes; Ferreira, Sérgio H; Clososki, Giuliano C; de Souza, Glória E P

2014-08-01

The antipyretic and hypothermic prodrug dipyrone prevents PGE2 -dependent and -independent fever induced by LPS from Escherichia coli and Tityus serrulatus venom (Tsv) respectively. We aimed to identify the dipyrone metabolites responsible for the antipyretic and hypothermic effects. Male Wistar rats were treated i.p. with indomethacin (2 mg·kg(-1) ), dipyrone, 4-methylaminoantipyrine (4-MAA), 4-aminoantipyrine (4-AA) (60-360 mg·kg(-1) ), 4-formylaminoantipyrine, 4-acethylaminoantipyrine (120-360 mg·kg(-1) ) or vehicle 30 min before i.p. injection of LPS (50 μg·kg(-1) ), Tsv (150 μg·kg(-1) ) or saline. Rectal temperatures were measured by tele-thermometry and dipyrone metabolite concentrations determined in the plasma, CSF and hypothalamus by LC-MS/MS. PGE2 concentrations were determined in the CSF and hypothalamus by elisa. In contrast to LPS, Tsv-induced fever was not followed by increased PGE2 in the CSF or hypothalamus. The antipyretic time-course of 4-MAA and 4-AA on LPS-induced fever overlapped with the period of the highest concentrations of 4-MAA and 4-AA in the hypothalamus, CSF and plasma. These metabolites reduced LPS-induced fever and the PGE2 increase in the plasma, CSF and hypothalamus. Only 4-MAA inhibited Tsv-induced fever. The higher doses of dipyrone and 4-MAA also induced hypothermia. The presence of 4-MAA and 4-AA in the CSF and hypothalamus was associated with PGE2 synthesis inhibition and a decrease in LPS-induced fever. 4-MAA was also shown to be an antipyretic metabolite for PGE2 -independent fever induced by Tsv suggesting that it is responsible for the additional antipyretic mechanism of dipyrone. Moreover, 4-MAA is the hypothermic metabolite of dipyrone. © 2014 The British Pharmacological Society.

Bisphenol A and its analogs: Do their metabolites have endocrine activity?

PubMed

Gramec Skledar, Darja; Peterlin Mašič, Lucija

2016-10-01

Structural analogs of bisphenol A are commonly used as its alternatives in industrial and commercial applications. Nevertheless, the question arises whether the use of other bisphenols is justified as replacements for bisphenol A in mass production of plastic materials. To evaluate the influence of metabolic reactions on endocrine activities of bisphenols, we conducted a systematic review of the literature. Knowledge about the metabolic pathways and enzymes involved in metabolic biotransformations is essential for understanding and predicting mechanisms of toxicity. Bisphenols are metabolized predominantly by the glucuronidation reaction, which is considered their most important detoxification pathway, as based on current knowledge, glucuronides do not have activity on endocrine receptors. In contrast, several oxidative metabolites of bisphenols with enhanced endocrine activities are presented, and these findings indicate that oxidative metabolites of bisphenols can still have endocrine activities in humans. Copyright © 2016 Elsevier B.V. All rights reserved.

Urinary metabolites of isorhynchophylline in rats and their neuroprotective activities in the HT22 cell assay

PubMed Central

Chen, Fangfang; Qi, Wen; Sun, Jiahong; Simpkins, James W.; Yuan, Dan

2015-01-01

Isorhynchophylline is one of the major alkaloids from the Uncaria hook possessing the effects of lowered blood pressure, vasodilatation and protection against ischemia-induced neuronal damage. However, the metabolic pathway of isorhynchophylline has not been fully reported yet. In this paper, the metabolism of isorhynchophylline was investigated in rats. Five metabolites were isolated by using solvent extraction and repeated chromatographic methods, and identified by spectroscopic methods including UV, MS, NMR and CD experiments. Three new compounds were identified as 5-oxoisorhynchophyllic acid-22-O-β-D-glucuronide (M1), 17-O-demethyl-16,17-dihydro isorhynchophylline (M2) and 5-oxoisorhynchophyllic acid (M4) together with two known compounds isorhynchophylline (M0) and rhynchophylline (M3). Possible metabolic pathways of isorhynchophylline are proposed. Furthermore, the activity assay for all the metabolites showed that isorhynchophylline (M0) exhibited potent neuroprotective effects against glutamate-induced HT22 cell death. However, little or weak neuroprotective activities were observed for M1–M4. Our present study is important to further understand its metabolic fate and disposition in humans. PMID:24910000

Identification of Furan Metabolites Derived from Cysteine-cis-2-Butene-1,4-Dial-Lysine Crosslinks

PubMed Central

Lu, Ding; Peterson, Lisa A.

2010-01-01

Furan is a rodent hepatotoxicant and carcinogen. Since this compound is an important industrial intermediate and has been detected in heat-processed foods and smoke, humans are likely exposed to this toxic compound. Characterization of urinary metabolites of furan will lead to the development of biomarkers to assess human health risks associated with furan exposure. Previous studies indicate that furan is oxidized to a reactive α, β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA), in a reaction catalyzed by cytochrome P450. Five previously characterized metabolites are derived from the reaction of BDA with cellular nucleophiles such as glutathione and protein. They include the mono-glutathione reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine cyclic sulfide and its downstream metabolite, S-[1-(1,3-dicarboxypropyl)-1H-pyrrol-3-yl]methylthiol as well as R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid and N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-L-cysteine and its sulfoxide. The last two compounds are downstream metabolites of a BDA-derived cysteine-lysine crosslink, S-[1-(5-amino-5-carboxypentyl)-1H-pyrrol-3-yl]-L-cysteine. In this report, we present the characterization of seven additional urinary furan metabolites, all of which are derived from this crosslink. The cysteinyl residue is subject to several biotransformation reactions, including N-acetylation and S-oxidation. Alternatively, it can undergo β-elimination followed by S-methylation to a methylthiol intermediate that is further oxidized to a sulfoxide. The lysine portion of the crosslink is either N-acetylated or undergoes an oxidative transamination reaction to generate an α-ketoacid metabolite that undergoes oxidative decarboxylation. Some of these metabolites are among the most abundant furan metabolites present in urine as judged by LC-MS/MS analysis, indicating that the oxidation of furan to BDA and BDA�

Antimalarial Activity of Plant Metabolites.

PubMed

Pan, Wen-Hui; Xu, Xin-Ya; Shi, Ni; Tsang, Siu Wai; Zhang, Hong-Jie

2018-05-06

Malaria, as a major global health problem, continues to affect a large number of people each year, especially those in developing countries. Effective drug discovery is still one of the main efforts to control malaria. As natural products are still considered as a key source for discovery and development of therapeutic agents, we have evaluated more than 2000 plant extracts against Plasmodium falciparum . As a result, we discovered dozens of plant leads that displayed antimalarial activity. Our phytochemical study of some of these plant extracts led to the identification of several potent antimalarial compounds. The prior comprehensive review article entitled “Antimalarial activity of plant metabolites” by Schwikkard and Van Heerden (2002) reported structures of plant-derived compounds with antiplasmodial activity and covered literature up to the year 2000. As a continuation of this effort, the present review covers the antimalarial compounds isolated from plants, including marine plants, reported in the literature from 2001 to the end of 2017. During the span of the last 17 years, 175 antiplasmodial compounds were discovered from plants. These active compounds are organized in our review article according to their plant families. In addition, we also include ethnobotanical information of the antimalarial plants discussed.

A derivatization-enhanced detection strategy in mass spectrometry: analysis of 4-hydroxybenzoates and their metabolites after keratinocytes are exposed to UV radiation

PubMed Central

Lee, Yi-Hsuan; Lin, Ying-Chi; Feng, Chia-Hsien; Tseng, Wei-Lung; Lu, Chi-Yu

2017-01-01

4-Hydroxybenzoate is a phenolic derivative of alkyl benzoates and is a widely used preservative in cosmetic and pharmaceutical products. The presence of 4-hydroxybenzoates in the human body may result from the use of pharmaceutical and personal care products. These compounds are also known to exhibit estrogenic and genotoxic activities. The potential adverse effects of these compounds include endocrine disruption, oxidative and DNA damage, contact dermatitis, and allergic reactions. This study used two mass spectrometry methods that are applicable when using a derivatization-enhanced detection strategy (DEDS) to screen 4-hydroxybenzoates and their metabolites. Chemical derivatization was used to enhance the detection of these compounds. To evaluate the metabolic process triggered by UV radiation, human keratinocyte HaCaT cells treated with these 4-hydroxybenzoates were further exposed to UVA, UVB and UVC radiation. Metabolites transformed by human keratinocytes in the chemical derivatization procedure were identified by a nano ultra-performance liquid chromatographic system (nanoUPLC) coupled with LTQ Orbitrap. The experiments confirmed the feasibility of this method for identifying 4-hydroxybenzoate metabolites and for high-throughput screening of 4-hydroxybenzoate in commercial products (50 samples) by the DEDS. PMID:28057923

Intracellular studies of the nucleoside reverse transcriptase inhibitor active metabolites: a review.

PubMed

Rodriguez Orengo, J F; Santana, J; Febo, I; Diaz, C; Rodriguez, J L; Garcia, R; Font, E; Rosario, O

2000-03-01

Nucleoside reverse transcriptase inhibitors (NRTIs) plasma concentrations do not correlate with clinical efficacy or toxicity. These agents need to be phosphorylated to become active against HIV-infection. Thus, the characterization of the NRTIs intracellular metabolite pharmacological parameters will provide a better understanding that could lead to the development of more rational dose regimens in the HIV-infected population. Furthermore, intracellular measurements of NRTIs may provide a better marker with respect to clinical efficacy and toxicity than plasma concentrations. Thus, in this article we review the latest information regarding the intracellular pharmacological parameters of zidovudine (ZDV) and lamivudine (3TC) active metabolites in HIV-infected patients including the results from our recent clinical studies. We will start the discussion with ZDV and 3TC clinical efficacy, followed by systemic pharmacokinetics studies. We will then discuss the in vitro and in vivo intracellular studies with particular emphasis in the method development to measure these metabolites and we will conclude with the most current data from our clinical trials.

Marine Invertebrate Metabolites with Anticancer Activities: Solutions to the “Supply Problem”

PubMed Central

Gomes, Nelson G. M.; Dasari, Ramesh; Chandra, Sunena; Kiss, Robert; Kornienko, Alexander

2016-01-01

Marine invertebrates provide a rich source of metabolites with anticancer activities and several marine-derived agents have been approved for the treatment of cancer. However, the limited supply of promising anticancer metabolites from their natural sources is a major hurdle to their preclinical and clinical development. Thus, the lack of a sustainable large-scale supply has been an important challenge facing chemists and biologists involved in marine-based drug discovery. In the current review we describe the main strategies aimed to overcome the supply problem. These include: marine invertebrate aquaculture, invertebrate and symbiont cell culture, culture-independent strategies, total chemical synthesis, semi-synthesis, and a number of hybrid strategies. We provide examples illustrating the application of these strategies for the supply of marine invertebrate-derived anticancer agents. Finally, we encourage the scientific community to develop scalable methods to obtain selected metabolites, which in the authors’ opinion should be pursued due to their most promising anticancer activities. PMID:27213412

Influence of Sulforaphane Metabolites on Activities of Human Drug-Metabolizing Cytochrome P450 and Determination of Sulforaphane in Human Liver Cells.

PubMed

Vanduchova, Alena; Tomankova, Veronika; Anzenbacher, Pavel; Anzenbacherova, Eva

2016-12-01

The influence of metabolites of sulforaphane, natural compounds present in broccoli (Brassica oleracea var. botrytis italica) and in other cruciferous vegetables, on drug-metabolizing cytochrome P450 (CYP) enzymes in human liver microsomes and possible entry of sulforaphane into human hepatic cells were investigated. Metabolites studied are compounds derived from sulforaphane by the mercapturic acid pathway (conjugation with glutathione and by following reactions), namely sulforaphane glutathione and sulforaphane cysteine conjugates and sulforaphane-N-acetylcysteine. Their possible effect on four drug-metabolizing CYP enzymes, CYP3A4 (midazolam 1'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), CYP1A2 (7-ethoxyresorufin O-deethylation), and CYP2B6 (7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation), was tested. Inhibition of four prototypical CYP activities by sulforaphane metabolites was studied in pooled human liver microsomes. Sulforaphane metabolites did not considerably affect biological function of drug-metabolizing CYPs in human liver microsomes except for CYP2D6, which was found to be inhibited down to 73-78% of the original activity. Analysis of the entry of sulforaphane into human hepatocytes was done by cell disruption by sonication, methylene chloride extraction, and modified high-performance liquid chromatography method. The results have shown penetration of sulforaphane into the human hepatic cells.

Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression in Jurkat Cells.

PubMed

Pan, Yao; Wei, Xuetao; Hao, Weidong

2015-08-28

Trichloroethylene (TCE) is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells.

Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression inJurkat Cells

PubMed Central

Pan, Yao; Wei, Xuetao; Hao, Weidong

2015-01-01

Trichloroethylene (TCE) is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells. PMID:26343699

The antibacterial activity of syringopicroside, its metabolites and natural analogues from Syringae Folium.

PubMed

Zhou, Zhengyuan; Han, Na; Liu, Zhihui; Song, Zehai; Wu, Peng; Shao, Jingxuan; Zhang, Jia Ming; Yin, Jun

2016-04-01

In the present study, the in vitro antibacterial activity of an effective fraction (ESF) from Syringae Folium (SF) on Methicillin-resistant Staphylococcus aureus (MRSA) was evaluated and then its in vivo activity was evaluated by using the MRSA-infected mouse peritonitis model. The ESF showed a significant in vitro and in vivo activity on decreasing the Minimum Inhibitory Concentrations (MICs) and increasing the survival rate of mouse from 42.8% to 100%. Six iridoid glucosides (IGs) of ESF were characterized by UPLC-TOF-MS method and also isolated by column chromatography. Most of them showed in vitro anti MRSA activity. Syringopicroside (Sy), the major compound of IGs, was found to increase the survival rate from 42.8% to 92.8% of the MRSA-infected mouse, which revealed Sy is also the main active components of ESF. In order to know why the effect of oral administration of SF is better than its injections in clinic and the metabolites of Sy, seven metabolites of Sy were isolated from rat urine and identified on the basis of NMR and MS spectra. Most of metabolites possessed stronger in vitro anti-MRSA activity than that of Sy, which furtherly proved the clinical result. Copyright © 2016 Elsevier B.V. All rights reserved.

Mutagenicity of 1-nitropyrene metabolites from lung S9.

PubMed

King, L C; Kohan, M J; Ball, L M; Lewtas, J

1984-04-01

The mutagenicity of 1-nitropyrene metabolites from rabbit lung S9 incubates was evaluated using the Salmonella typhimurium plate incorporation assay with strain TA98, with and without Aroclor-induced rat liver S9. The following metabolites were isolated, identified and quantitated by HPLC: 1-nitropyrene -4,5- or -9,10-dihydrodiol (K-DHD), N-acetyl-1-aminopyrene ( NAAP ), 1-aminopyrene (1-AMP), 10-hydroxy-1-nitropyrene, 4-, 5-, 6-, 8- or 9-monohydroxy-1-nitropyrene (phenols) and 3-hydroxy-1-nitropyrene. The predominant metabolites formed by lung S9 incubates were K-DHD, 3-OH-1-nitropyrene and phenols. All of the metabolites were mutagenic in the absence of the exogenous rat liver S9 metabolic activation system, and several, including two unidentified metabolites were more potent than the parent 1-nitropyrene. The mutagenicity of 3 of the metabolites ( NAAP , 10-OH-1-nitropyrene and phenols) were enhanced by S9 while most of the other metabolites were less mutagenic in the presence of S9. These results indicate that lung tissue is capable of both oxidative and reductive metabolism which produced mutagenic metabolites, several of which were more potent than the parent compound, 1-NP.

Effect of storage time on metabolite profile and alpha-glucosidase inhibitory activity of Cosmos caudatus leaves - GCMS based metabolomics approach.

PubMed

Javadi, Neda; Abas, Faridah; Mediani, Ahmed; Abd Hamid, Azizah; Khatib, Alfi; Simoh, Sanimah; Shaari, Khozirah

2015-09-01

Cosmos caudatus, which is a commonly consumed vegetable in Malaysia, is locally known as "Ulam Raja". It is a local Malaysian herb traditionally used as a food and medicinal herb to treat several maladies. Its bioactive or nutritional constituents consist of a wide range of metabolites, including glucosinolates, phenolics, amino acids, organic acids, and sugars. However, many of these metabolites are not stable and easily degraded or modified during storage. In order to investigate the metabolomics changes occurring during post-harvest storage, C. caudatus samples were subjected to seven different storage times (0 hours, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, and 12 hours) at room temperature. As the model experiment, the metabolites identified by gas chromatography-mass spectrometry (GC-MS) were correlated with α-glucosidase inhibitory activity analyzed with multivariate data analysis (MVDA) to find out the variation among samples and metabolites contributing to the activity. Orthogonal partial least squares (OPLS) analysis was applied to investigate the metabolomics changes. A profound chemical alteration, both in primary and secondary metabolites, was observed. The α-tocopherol, catechin, cyclohexen-1-carboxylic acid, benzoic acid, myo-inositol, stigmasterol, and lycopene compounds were found to be the discriminating metabolites at early storage; however, sugars such as sucrose, α-d-galactopyranose, and turanose were detected, which was attributed to the discriminating metabolites for late storage. The result shows that the MVDA method is a promising technique to identify biomarker compounds relative to storage at different times. Copyright © 2015. Published by Elsevier B.V.

Pharmacologically active plant metabolites as survival strategy products.

PubMed

Attardo, C; Sartori, F

2003-01-01

The fact that plant organisms produce chemical substances that are able to positively or negatively interfere with the processes which regulate human life has been common knowledge since ancient times. One of the numerous possible examples in the infusion of Conium maculatum, better known as Hemlock, a plant belonging to the family umbelliferae, used by the ancient Egyptians to cure skin diseases. The current official pharmacopoeia includes various chemical substances produced by secondary plant metabolisms. For example, the immunosuppressive drugs used to prevent organ transplant rejection and the majority of antibiotics are metabolites produced by fungal organisms, pilocarpin, digitalis, strophantus, salicylic acid and curare are examples of plant organism metabolites. For this reason, there has been an increase in research into plants, based on information on their medicinal use in the areas where they grow. The study of plants in relation to local culture and traditions is known as "ethnobotany". Careful study of the behaviour of sick animals has also led to the discovery of medicinal plants. The study of this subject is known as "zoopharmacognosy". The aim of this article is to discuss the fact that "ad hoc" production of such chemical substances, defined as "secondary metabolites", is one of the modes in which plant organisms respond to unfavourable environmental stimuli, such as an attack by predatory phytophagous animals or an excessive number of plant individuals, even of the same species, in a terrain. In the latter case, the plant organisms produce toxic substances, called "allelopathic" which limit the growth of other individuals. "Secondary metabolites" are produced by metabolic systems that are shunts of the primary systems which, when required, may be activated from the beginning, or increased to the detriment of others. The study of the manner in which such substances are produced is the subject of a new branch of learning called "ecological

Early phenylpropanoid biosynthetic steps in Cannabis sativa: link between genes and metabolites.

PubMed

Docimo, Teresa; Consonni, Roberto; Coraggio, Immacolata; Mattana, Monica

2013-06-28

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data.

Early Phenylpropanoid Biosynthetic Steps in Cannabis sativa: Link between Genes and Metabolites

PubMed Central

Docimo, Teresa; Consonni, Roberto; Coraggio, Immacolata; Mattana, Monica

2013-01-01

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data. PMID:23812081

Identification of human cell responses to benzene and benzene metabolites.

PubMed

Gillis, Bruce; Gavin, Igor M; Arbieva, Zarema; King, Stephen T; Jayaraman, Sundararajan; Prabhakar, Bellur S

2007-09-01

Benzene is a common air pollutant and confirmed carcinogen, especially in reference to the hematopoietic system. In the present study we analyzed cytokine/chemokine production by, and gene expression induction in, human peripheral blood mononuclear cells upon their exposure to the benzene metabolites catechol, hydroquinone, 1,2,4-benzenetriol, and p-benzoquinone. Protein profiling showed that benzene metabolites can stimulate the production of chemokines, the proinflammatory cytokines TNF-alpha and IL-6, and the Th2 cytokines IL-4 and IL-5. Activated cells showed concurrent suppression of anti-inflammatory cytokine IL-10 expression. We also identified changes in global gene expression patterns in response to benzene metabolite challenges by using high-density oligonucleotide microarrays. Treatment with 1,2,4-benzenetriol resulted in the suppression of genes related to the regulation of protein expression and a concomitant activation of genes that encode heat shock proteins and cytochrome P450 family members. Protein and gene expression profiling identified unique human cellular responses upon exposure to benzene and benzene metabolites.

Temporally-Controlled Site-Specific Recombination in Zebrafish

PubMed Central

Hans, Stefan; Kaslin, Jan; Freudenreich, Dorian; Brand, Michael

2009-01-01

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms. PMID:19247481

Arsenite and its metabolites, MMA(III) and DMA(III), modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice.

PubMed

Medina-Díaz, I M; Estrada-Muñiz, E; Reyes-Hernández, O D; Ramírez, P; Vega, L; Elizondo, G

2009-09-01

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA(III) induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA(III) increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA(III) induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

Urinary Estrogen Metabolites, Active and Sedentary Behaviors, and Breast Cancer Risk

Cancer.gov

A cross-sectional study of approximately 600 postmenopausal controls in the Breast Cancer Case-Control Study in Poland to assess urinary estrogen metabolites in relation to accelerometer-based measures of active and sedentary behaviors

Chemopreventive Activities of Sulforaphane and Its Metabolites in Human Hepatoma HepG2 Cells.

PubMed

Liu, Peng; Wang, Wei; Zhou, Zhigang; Smith, Andrew J O; Bowater, Richard P; Wormstone, Ian Michael; Chen, Yuqiong; Bao, Yongping

2018-05-09

Sulforaphane (SFN) exhibits chemopreventive effects through various mechanisms. However, few studies have focused on the bioactivities of its metabolites. Here, three metabolites derived from SFN were studied, known as sulforaphane glutathione, sulforaphane cysteine and sulforaphane- N -acetylcysteine. Their effects on cell viability, DNA damage, tumorigenicity, cell migration and adhesion were measured in human hepatoma HepG2 cells, and their anti-angiogenetic effects were determined in a 3D co-culture model of human umbilical vein endothelial cells (HUVECs) and pericytes. Results indicated that these metabolites at high doses decreased cancer cell viability, induced DNA damage and inhibited motility, and impaired endothelial cell migration and tube formation. Additionally, pre-treatment with low doses of SFN metabolites protected against H₂O₂ challenge. The activation of the nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway and the induction of intracellular glutathione (GSH) played an important role in the cytoprotective effects of SFN metabolites. In conclusion, SFN metabolites exhibited similar cytotoxic and cytoprotective effects to SFN, which proves the necessity to study the mechanisms of action of not only SFN but also of its metabolites. Based on the different tissue distribution profiles of these metabolites, the most relevant chemical forms can be selected for targeted chemoprevention.

Baicalin, a metabolite of baicalein with antiviral activity against dengue virus

PubMed Central

Moghaddam, Ehsan; Teoh, Boon-Teong; Sam, Sing-Sin; Lani, Rafidah; Hassandarvish, Pouya; Chik, Zamri; Yueh, Andrew; Abubakar, Sazaly; Zandi, Keivan

2014-01-01

Baicalin, a flavonoid derived from Scutellaria baicalensis, is the main metabolite of baicalein released following administration in different animal models and human. We previously reported the antiviral activity of baicalein against dengue virus (DENV). Here, we examined the anti-DENV properties of baicalin in vitro, and described the inhibitory potentials of baicalin at different steps of DENV-2 (NGC strain) replication. Our in vitro antiviral experiments showed that baicalin inhibited virus replication at IC50 = 13.5 ± 0.08 μg/ml with SI = 21.5 following virus internalization by Vero cells. Baicalin exhibited virucidal activity against DENV-2 extracellular particles at IC50 = 8.74 ± 0.08 μg/ml and showed anti-adsorption effect with IC50 = 18.07 ± 0.2 μg/ml. Our findings showed that baicalin as the main metabolite of baicalein exerting in vitro anti-DENV activity. Further investigations on baicalein and baicalin to deduce its antiviral therapeutic effects are warranted. PMID:24965553

Identification of 4-oxo-13-cis-retinoic acid as the major metabolite of 13-cis-retinoic acid in human blood.

PubMed

Vane, F M; Buggé, C J

1981-01-01

The metabolites of 13-cis-retinoic acid (Accutane) were investigated in blood samples from human volunteers on chronic treatment for dermatological disorders. The major metabolite was isolated by reverse-phase high-pressure liquid chromatography and identified as 4-oxo-13-cis-retinoic acid by comparison of its mass and NMR spectra to the spectra of the reference compound. 4-Oxo-all-trans-retinoic acid was also identified, but the extent to which this compound was a metabolite of 13-cis-retinoic acid or an artifactual isomerization product of the major metabolite is unknown. Chromatographic data suggested that small amounts of 13-cis-retinoic acid, 4-hydroxy-13-cis-retinoic acid, and dioxygenated metabolites of 13-cis-retinoic acid may also be present in the blood. This study indicates that a major metabolic pathway of 13-cis-retinoic acid in humans is oxidation at C4 of the cyclohexenyl group.

Serotonergic Neurotoxic Thioether Metabolites of 3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”): Synthesis, Isolation and Characterization of Diastereoisomers

PubMed Central

Pizarro, Nieves; de la Torre, Rafael; Joglar, Jesús; Okumura, Noriko; Perfetti, Ximena; Lau, Serrine S.; Monks, Terrence J.

2014-01-01

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a synthetic recreational drug of abuse that produces long-term toxicity associated with the degeneration of serotonergic nerve terminals. In various animal models direct administration of MDMA into the brain fails to reproduce the serotonergic neurotoxicity, implying a requirement for the systemic metabolism and bioactivation of MDMA. Catechol-thioether metabolites of MDMA, formed via oxidation of 3,4-dihydroxymetamphetamine and 3,4-dihydroxyamphetamine (HHMA and HHA) and subsequent conjugation with glutathione (GSH), are selective serotonergic neurotoxicants when administered directly into brain. Moreover, following systemic administration of MDMA, the thioether adducts are present in rat brain dialysate. MDMA contains a stereogenic center, and is consumed as a racemate. Interestingly, different pharmacological properties have been attributed to the two enantiomers, (S)-MDMA being the most active in the central nervous system and responsible for the entactogenic effects, and most likely also for the neurodegeneration. The present study focused on the synthesis and stereochemical analysis of the neurotoxic MDMA thioether metabolites, 5-(glutathion-S-yl)-HHMA, 5-(N-acetylcysteine-S-yl)-HHMA, 2,5-bis-(glutathion-S-yl)-HHMA and 2,5-bis-(N-acetylcysteine-S-yl)-HHMA. Both enzymatic and electrochemical syntheses were explored, and methodologies for analytical and semi-preparative diastereoisomeric separation of MDMA thioether conjugates by HPLC-CEAS and HPLC-UV respectively were developed. Synthesis, diastereoisomeric separation, and unequivocal identification of the thioether conjugates of MDMA provide the chemical tools necessary for appropriate toxicological and metabolic studies on MDMA metabolites contributing to its neurotoxicity. PMID:19548351

Pharmacokinetics of endoxifen and tamoxifen in female mice: implications for comparative in vivo activity studies.

PubMed

Reid, Joel M; Goetz, Matthew P; Buhrow, Sarah A; Walden, Chad; Safgren, Stephanie L; Kuffel, Mary J; Reinicke, Kathryn E; Suman, Vera; Haluska, Paul; Hou, Xiaonan; Ames, Matthew M

2014-12-01

Reduced CYP2D6 metabolism and low Z-endoxifen (ENDX) concentrations may increase the risk of breast cancer recurrence in tamoxifen (TAM)-treated women. Little is known regarding the differences between TAM and ENDX murine pharmacokinetics or the effect of administration route on plasma concentrations of each drug. The pharmacokinetics of TAM and ENDX were characterized in female mice. For subcutaneous [s.c.] and oral TAM (4, 10 and 20 mg/kg), TAM AUC increased in a linear manner, but concentrations of the active metabolites [ENDX and 4-hydroxytamoxifen (4HT)] remained low. For oral TAM (20 mg), 4HT concentrations were tenfold greater (>25 ng/ml) than achievable in TAM-treated humans. Both oral (10-200 mg/kg) and s.c. (2.5-25 mg/kg) ENDX·HCl resulted in a greater than dose-proportional increase in AUC, with eightfold greater ENDX concentrations than an equivalent TAM dose. ENDX accumulated in plasma after 5-day dosing of 25 or 100 mg/kg ENDX·HCl and exceeded target concentrations of 0.1 and 1.0 μM, respectively, by twofold to fourfold. In murine models, oral ENDX yields substantially higher ENDX concentrations, compared to TAM. The low 4HT and ENDX concentrations observed in mice receiving s.c. TAM mirror the TAM pharmacokinetics in humans with impaired CYP2D6 metabolism. These data support the ongoing development of ENDX as a novel agent for the endocrine treatment of ER-positive breast cancer.

CYP2C19*17 increases clopidogrel-mediated platelet inhibition but does not alter the pharmacokinetics of the active metabolite of clopidogrel.

PubMed

Pedersen, Rasmus Steen; Nielsen, Flemming; Stage, Tore Bjerregaard; Vinholt, Pernille Just; el Achwah, Alaa Bilal; Damkier, Per; Brosen, Kim

2014-11-01

The aim of the present study was to determine the impact of CYP2C19*17 on the pharmacokinetics and pharmacodynamics of the active metabolite of clopidogrel and the pharmacokinetics of proguanil. Thus, we conducted an open-label two-phase cross-over study in 31 healthy male volunteers (11 CYP2C19*1/*1, 11 CYP2C19*1/*17 and nine CYP2C19*17/*17). In Phase A, the pharmacokinetics of the derivatized active metabolite of clopidogrel (CAMD) and platelet function were determined after administration of a single oral dose of 600 mg clopidogrel (Plavix; Sanofi-Avensis, Horsholm, Denmark). In Phase B, the pharmacokinetics of proguanil and its metabolites cycloguanil and 4-chlorphenylbiguanide (4-CPB) were determined in 29 of 31 subjects after a single oral dose of 200 mg proguanil given as the combination drug Malarone (GlaxoSmithKline Pharma, Brondby, Denmark). Significant correlations were found between the area under the time-concentration curve (AUC0-∞ ) of CAMD and both the absolute ADP-induced P2Y12 receptor-activated platelet aggregation (r = -0.60, P = 0.0007) and the percentage inhibition of aggregation (r = 0.59, P = 0.0009). In addition, the CYP2C19*17/*17 and CYP2C19*1/*17 genotype groups had significantly higher percentage inhibition of platelet aggregation compared with the CYP2C19*1/*1 subjects (geometric mean percentage inhibition of 84%, 73% and 63%, respectively; P = 0.014). Neither the absolute ADP-induced P2Y12 receptor-activated platelet aggregation, exposure to CAMD nor the pharmacokinetic parameters of proguanil, cycloguanil and 4-CPB exhibited any significant differences among the genotype groups. In conclusion, carriers of CYP2C19*17 exhibit higher percentage inhibition of platelet aggregation, but do not have significantly lower absolute P2Y12 receptor-activated platelet aggregation or higher exposure to the active metabolite after a single oral administration of 600 mg clopidogrel. © 2014 Wiley Publishing Asia Pty Ltd.

Secondary metabolites from Sida rhombifolia L. (Malvaceae) and the vasorelaxant activity of cryptolepinone.

PubMed

Chaves, Otemberg Souza; Gomes, Roosevelt Albuquerque; Tomaz, Anna Cláudia de Andrade; Fernandes, Marianne Guedes; das Graças Mendes, Leônidas; de Fátima Agra, Maria; Braga, Valdir Andrade; de Fátima Vanderlei de Souza, Maria

2013-03-01

The phytochemical study of Sida rhombifolia L. (Malvaceae) led to the isolation through chromatographic techniques of eleven secondary metabolites: sitosterol (1a) and stigmasterol (1b), sitosterol-3-O-b-D-glucopyranoside (2a) and stigmasterol-3-O-b-D-glucopyranoside (2b), phaeophytin A (3), 17³-ethoxypheophorbide A (4), 13²-hydroxy phaeophytin B (5), 17³-ethoxypheophorbide B (6), 5,7-dihydroxy-4'-methoxyflavone (7), cryptolepinone (8) and a salt of cryptolepine (9). Their structures were identified by ¹H- and ¹³C-NMR using one- and two-dimensional techniques. In addition, the vasorelaxant activity of cryptolepinone in rat mesenteric artery rings is reported herein for the first time.

Effects of APOE ε4, age, and HIV on glial metabolites and cognitive deficits.

PubMed

Chang, Linda; Jiang, Caroline; Cunningham, Eric; Buchthal, Steven; Douet, Vanessa; Andres, Marilou; Ernst, Thomas

2014-06-17

We aimed to evaluate the combined effects of HIV and APOE ε4 allele(s) on glial metabolite levels, and on known cognitive deficits associated with either condition, across the ages. One hundred seventy-seven participants, primarily of white and mixed race (97 seronegative subjects: aged 44.7 ± 1.3 years, 85 [87.6%] men, 28 [28.9%] APOE ε4+; 80 HIV+ subjects: aged 47.3 ± 1.1 years, 73 [91.3%] men, 23 [28.8%] APOE ε4+), were assessed cross-sectionally for metabolite concentrations using proton magnetic resonance spectroscopy in 4 brain regions and for neuropsychological performance. Frontal white matter myo-inositol was elevated in subjects with HIV across the age span but showed age-dependent increase in seronegative subjects, especially in APOE ε4+ carriers. In contrast, only seronegative APOE ε4+ subjects showed elevated myo-inositol in parietal cortex. All APOE ε4+ subjects had lower total creatine in basal ganglia. While all HIV subjects showed greater cognitive deficits, HIV+ APOE ε4+ subjects had the poorest executive function, fluency memory, and attention/working memory. Higher myo-inositol levels were associated with poorer fine motor function across all subjects, slower speed of information processing in APOE ε4+ subjects, and worse fluency in HIV+ APOE ε4+ subjects. In frontal white matter of subjects with HIV, the persistent elevation and lack of normal age-dependent increase in myo-inositol suggest that persistent glial activation attenuated the typical antagonistic pleiotropic effects of APOE ε4 on neuroinflammation. APOE ε4 negatively affects energy metabolism in brain regions rich in dopaminergic synapses. The combined effects of HIV infection and APOE ε4 may lead to greater cognitive deficits, especially in those with greater neuroinflammation. APOE ε4 allele(s) may be a useful genetic marker to identify white and mixed-race HIV subjects at risk for cognitive decline. © 2014 American Academy of Neurology.

[Optimising azathioprine treatment: determination of thiopurine methyltransferase activity and thiopurine metabolites].

PubMed

Alvarez Beltran, M; Infante Pina, D; Tormo Carnicé, R; Segarra Cantón, O; Redecillas Ferreiro, S

2009-02-01

Individualised doses of azathioprine (AZA) may be prescribed by monitoring the levels of the enzyme thiopurine methyltransferase (TPMT). The measurements of thiopurine metabolites of AZA, 6-thioguanine (6-TGN) and 6-methylmercaptopurine (6-MMP), have also been reported as new markers of AZA activity. To describe TPMT phenotype in our population and to establish a relationship between thiopurine metabolites,and therapeutic activity and adverse effects. Data on TPMT were retrospectively collected from 107 patients, and 6-TGN and 6-MMP levels in 18 patients currently on treatment with AZA (Crohn's disease 5, ulcerative colitis 5, autoimmune hepatitis 5). Mean value of TPMT was 20.19U/ml. None of the patients had a TPMT activity<5U/ml. Of the 18 patients on treatment, 13 showed sub-therapeutic levels of 6-TGN (<235pmol/8x10(8) red blood cells). Clinical remission was maintained in 45% of patients. Mean levels of 6-TGN in patients with clinical remission were 259pmol/8x10(8) red blood cells versus 209pmol/8x10(8) red blood cells in non-responders (p=0.37). There was an inverse relationship (r=-0.28) between TPMT and 6-TGN levels. Toxic effects occurred in 6 of 18 patients, with leukopenia in 5 and hyperamylasemia in 1. Determination of TPMT and monitoring of thiopurine metabolites allows AZA treatment to be optimised, although further studies are necessary to establish therapeutic effectiveness and toxicity ranges.

Urinary pesticide metabolites in school students from northern Thailand.

PubMed

Panuwet, Parinya; Prapamontol, Tippawan; Chantara, Somporn; Barr, Dana B

2009-05-01

We evaluated exposure to pesticides among secondary school students aged 12-13 years old in Chiang Mai Province, Thailand. Pesticide-specific urinary metabolites were used as biomarkers of exposure for a variety of pesticides, including organophosphorus insecticides, synthetic pyrethroid insecticides and selected herbicides. We employed a simple solid-phase extraction with analysis using isotope dilution high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). A total of 207 urine samples from Thai students were analyzed for 18 specific pesticide metabolites. We found 14 metabolites in the urine samples tested; seven of them were detected with a frequency > or=17%. The most frequently detected metabolites were 2-[(dimethoxyphosphorothioyl) sulfanyl] succinic acid (malathion dicarboxylic acid), para-nitrophenol (PNP), 3,5,6-trichloro-2-pyridinol (TPCY; metabolite of chlorpyrifos), 2,4-dichlorophenoxyacetic acid (2,4-D), cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acids (c-DCCA and t-DCCA; metabolite of permethrin) and 3-phenoxybenzoic acid (3-PBA; metabolite of pyrethroids). The students were classified into 4 groups according to their parental occupations: farmers (N=60), merchants and traders (N=39), government and company employees (N=52), and laborers (N=56). Children of farmers had significantly higher urinary concentrations of pyrethroid insecticide metabolites than did other children (p<0.05). Similarly, children of agricultural families had significantly higher pyrethroid metabolite concentrations. Males had significantly higher values of PNP (Mann-Whitney test, p=0.009); however, no other sex-related differences were observed. Because parental occupation and agricultural activities seemed to have little influence on pesticide levels, dietary sources were the likely contributors to the metabolite levels observed.

Thiopurine methyltransferase genotype–phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia

PubMed Central

Lennard, Lynne; Cartwright, Cher Suzanne; Wade, Rachel; Richards, Susan M; Vora, Ajay

2013-01-01

Aims In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype–genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation. Methods We measured TPMT (activity, as units ml−1 packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 108 RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97. Results Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P < 0.0001). At diagnosis genotype–phenotype was discordant. During chemotherapy the overall concordance was 92%, but this fell to 55% in the intermediate activity cohort (45% had wild-type genotypes). For both thiopurines TGN concentrations differed by TPMT status. For mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P < 0.0001), whilst median MeMPNs, products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients. Conclusions In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy. PMID:23252716

Thiopurine methyltransferase genotype-phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia.

PubMed

Lennard, Lynne; Cartwright, Cher Suzanne; Wade, Rachel; Richards, Susan M; Vora, Ajay

2013-07-01

In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype-genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation. We measured TPMT (activity, as units ml(-1) packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 10(8) RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97. Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P < 0.0001). At diagnosis genotype-phenotype was discordant. During chemotherapy the overall concordance was 92%, but this fell to 55% in the intermediate activity cohort (45% had wild-type genotypes). For both thiopurines TGN concentrations differed by TPMT status. For mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P < 0.0001), whilst median MeMPNs, products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients. In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

The pH-dependent local anesthetic activity of diethylaminoethanol, a procaine metabolite.

PubMed

Butterworth, J F; Lief, P A; Strichartz, G R

1988-04-01

To test whether the products of procaine hydrolysis have local anesthetic actions resembling those of procaine, the authors compared the ability of procaine and its metabolites diethylaminoethanol (DEAE) and para-aminobenzoic acid (PABA) to block compound action potentials in excised, desheathed frog and rat sciatic nerves. Studies were performed in solutions of impermeant buffers at pH 7.4 (corresponding to mammalian physiologic pH) and at pH 9.2 (close to the pKa of procaine and DEAE) to test for extracellular pH-dependent increases in drug permeation and potency. Both procaine and DEAE inhibited compound action potentials at pH 7.4 and 9.2 in a reversible and dose-dependent manner, and both were approximately ten-fold more potent at pH 9.2 than at pH 7.4, procaine inhibiting the action potential height by 50% at 0.15 mM (pH 9.2) and 1.1 mM (pH 7.4), DEAE at 4 mM (pH 9.2) and 70 mM (pH 7.4). In contrast, PABA at concentrations up to 25 mM and at either pH failed to inhibit compound action potentials, and did not modify the effects of DEAE when both drugs were given together. Procaine produced greater use-dependent block at the higher pH and at higher stimulation rates (100 Hz vs. 40 Hz); DEAE produced almost no use-dependent block. These observations suggest: 1) that DEAE might account for some of the neuropharmacologic activity of procaine in techniques that favor the accumulation of metabolites (such as those requiring large doses or prolonged infusions); and 2) that alkalinization of procaine and DEAE solutions appears to increase their potency for both resting and use-dependent block of action potentials.

Biotransformation of Bisphenol AF to Its Major Glucuronide Metabolite Reduces Estrogenic Activity

PubMed Central

Yin, Jie; Zhang, Jing; Feng, Yixing; Shao, Bing

2013-01-01

Bisphenol AF (BPAF), an endocrine disrupting chemical, can induce estrogenic activity through binding to estrogen receptor (ER). However, the metabolism of BPAF in vivo and the estrogenic activity of its metabolites remain unknown. In the present study, we identified four metabolites including BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrated and BPAF sulfate in the urine of Sprague-Dawley (SD) rats. BPAF-G was further characterized by nuclear magnetic resonance (NMR). After treatment with a single dose of BPAF, BPAF was metabolized rapidly to BPAF-G, as detected in the plasma of SD rats. Biotransformation of BPAF to BPAF-G was confirmed with human liver microsomes (HLM), and Vmax of glucuronidation for HLM was 11.6 nmol/min/mg. We also found that BPAF glucuronidation could be mediated through several human recombinant UDP-glucuronosyltransferases (UGTs) including UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, among which UGT2B7 showed the highest efficiency of glucuronidation. To explain the biological function of BPAF biotransformation, the estrogenic activities of BPAF and BPAF-G were evaluated in ER-positive breast cancer T47D and MCF7 cells. BPAF significantly stimulates ER-regulated gene expression and cell proliferation at the dose of 100 nM and 1 μM in breast cancer cells. However, BPAF-G did not show any induction of estrogenic activity at the same dosages, implying that formation of BPAF-G is a potential host defense mechanism against BPAF. Based on our study, biotransformation of BPAF to BPAF-G can eliminate BPAF-induced estrogenic activity, which is therefore considered as reducing the potential threat to human beings. PMID:24349450

Isolation and identification of metabolites of osthole in rats.

PubMed

Lv, X; Wang, C-Y; Hou, J; Zhang, B-J; Deng, S; Tian, Y; Huang, S-S; Zhang, H-L; Shu, X-H; Zhen, Y-H; Liu, K-X; Yao, J-H; Ma, X-C

2012-11-01

Osthole (Ost), one of the major components of Cnidium monnieri (L.) Cusson, is had the structure of an isopentenoxy-coumarin with a range of pharmacological activities. In the present study, the metabolism of Ost in male Sprague-Dawley rats was investigated by identifying Ost metabolites excreted in rat urine. Following an oral dose of 40 mg/kg Ost, 10 phase I and 3 phase II metabolites were isolated from the urine of rats, and their structures identified on the basis of a range of spectroscopic data, including 2D-NMR techniques. These metabolites were fully characterized as 5'-hydroxyl-osthole (M-1), osthenol (M-2), 4'-hydroxyl-osthole (M-3), 3, 5'-dihydroxyl-osthole (M-4), 5'-hydroxyl-osthenol (M-5), 4'-hydroxyl-2', 3'-dihydro-osthenol (M-6), 4'-hydroxyl-osthenol (M-7), 3, 4'-dihydroxyl-osthole (M-8), 2', 3'-dihydroxyl-osthole (M-9), 5'-hydroxyl-2', 3'-dihydroosthole (M-10), osthenol-7-O-β-D-glucuronide (M-11), osthole-4'-O-β-D-glucuronide (M-12) and osthole-5'-O-β-D-glycuronate (M-13). This is the first identification of M-1, M-3 to M-13 in vivo. On the basis of the metabolites profile, a possible metabolic pathway for Ost metabolism in rats has been proposed. This is the first systematic study on the phases I and II metabolites of 8-isopentenoxy-coumarin derivative.

Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

PubMed

Siddiqui, Mahwish; Ahmad, Malik Shoaib; Wahab, Atia-Tul-; Yousuf, Sammer; Fatima, Narjis; Naveed Shaikh, Nimra; Rahman, Atta-Ur-; Choudhary, M Iqbal

2017-01-01

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6β,10β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11β,17β-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of β-glucuronidase enzyme (IC50 = 42.98 ± 1.24 μM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 μM). Its transformed products 3 (IC50 = 79.09 ± 0.06 μM), and 8 (IC50 = 70.09 ± 0.05 μM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 μM), and its metabolite 8 (IC50 = 34.16 ± 5.3 μM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 μM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 μM) and 4 (IC50 = 152.5 ± 2.15 μM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 μM), and its transformed products 2 (IC50 = 43.3 ± 7.7 μM), 3 (IC50 = 65.6 ± 2.5 μM), and 4 (IC50 = 89.4 ± 2.7 �

Three New and Eleven Known Unusual C25 Steroids: Activated Production of Silent Metabolites in a Marine-Derived Fungus by Chemical Mutagenesis Strategy using Diethyl Sulphate

PubMed Central

Xia, Ming-Wen; Cui, Cheng-Bin; Li, Chang-Wei; Wu, Chang-Jing

2014-01-01

Three new (1–3) and 11 known (4–14) C25 steroids with an unusual bicyclo[4.4.1]A/B ring system were isolated by tracing newly produced metabolites in the EtOAc extract of an antitumor mutant AD-1-2 obtained by the diethyl sulphate (DES) mutagenesis of a marine-derived Penicillium purpurogenum G59. HPLC-PDAD-UV and HPLC-ESI-MS analyses indicated that the G59 strain did not produce these metabolites and the production of 1–14 in the mutant AD-1-2 extract was caused by the activation of silent metabolites in the original G59 strain by DES mutagenesis. The structures of the new compounds, named antineocyclocitrinols A (1) and B (2) and 23-O-methylantineocyclocitrinol (3), including their absolute configurations were determined by various spectroscopic methods, especially the NMR and Mo2-induced CD analyses. Compounds 1–3 provide the first examples of the C25 bicyclo[4.4.1]A/B ring steroids with the Z-configuration of 20,22-double bond. All of 1–14 weakly inhibited several human cancer cell lines to varying extents. These results provided additional examples for the successful application of the chemical mutagenesis strategy using DES to discover new compounds by activating silent metabolites in fungal isolates and supported also the effectiveness and usefulness of this new strategy. PMID:24633254

Effects of selexipag and its active metabolite in contrasting the profibrotic myofibroblast activity in cultured scleroderma skin fibroblasts.

PubMed

Cutolo, Maurizio; Ruaro, Barbara; Montagna, Paola; Brizzolara, Renata; Stratta, Emanuela; Trombetta, Amelia Chiara; Scabini, Stefano; Tavilla, Pier Paolo; Parodi, Aurora; Corallo, Claudio; Giordano, Nicola; Paolino, Sabrina; Pizzorni, Carmen; Sulli, Alberto; Smith, Vanessa; Soldano, Stefano

2018-05-02

Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). Selexipag is a prostacyclin receptor agonist synthesized for the treatment of pulmonary arterial hypertension. The study investigated the possibility for selexipag and its active metabolite (ACT-333679) to downregulate the profibrotic activity in primary cultures of SSc fibroblasts/myofibroblasts and the fibrogenic signaling molecules involved. Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3 rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 μM to 0.3 μM) or ACT-333679 (from 10 μM to 0.1 μM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679-treated cells by western botting. Selexipag and ACT-333679 significantly reduced protein synthesis and gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, whereas FN was significantly downregulated at the protein level. Interestingly, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts. Selexipag and mainly its active metabolite ACT-333679 were found for the first time to potentially interfere with the profibrotic activity of cultured SSc fibroblasts

Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice.

PubMed

Lewandowski, M; Chui, Y C; Levi, P; Hodgson, E

1991-02-01

A simple and sensitive method for the separation of 14C-labelled acetanilide, 4-hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBA2/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [14C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.

Arsenite and its metabolites, MMA{sup III} and DMA{sup III}, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

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

Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half themore » drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA{sup III} induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA{sup III} increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA{sup III} induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.« less

Activation of the reticulothalamic cholinergic pathway by the major metabolites of aniracetam.

PubMed

Nakamura, K; Shirane, M

1999-09-10

The aim of the study was to further investigate the effects of aniracetam, a cognition enhancer, and its metabolites on the brain cholinergic system. We measured choline acetyltransferase activity and acetylcholine release using in vivo brain microdialysis in stroke-prone spontaneously hypertensive rats (SHRSP). The enzyme activity in the pons-midbrain and hippocampus, and basal acetylcholine release in the nucleus reticularis thalami were lower in SHRSP than in age-matched Wistar Kyoto rats, indicating central cholinergic deficits in SHRSP. Repeated treatment of aniracetam (50 mg/kg p.o. x 11 for 6 days) preferentially increased the enzyme activity in the thalamus, whereas decreased it in the striatum. Among the metabolites of aniracetam, local perfusion of N-anisoyl-gamma-aminobutyric acid (GABA, 0.1 and/or 1 microM) and p-anisic acid (1 microM) into the nucleus reticularis thalami, dorsal hippocampus and prefrontal cortex of SHRSP produced a significant but delayed increase of acetylcholine release. We failed, however, to find any effect of aniracetam itself. A direct injection of N-anisoyl-GABA (1 nmol) into the pedunculopontine tegmental nucleus of SHRSP enhanced the release in the nucleus reticularis thalami. Thus, these data prove that aniracetam can facilitate central cholinergic neurotransmission via both metabolites. Based on its pharmacokinetic profile, N-anisoyl-GABA may contribute to the clinical effects of aniracetam, mainly by acting on the reticulothalamic cholinergic pathway.

Amplified and in situ detection of redox-active metabolite using a biobased redox capacitor.

PubMed

Kim, Eunkyoung; Gordonov, Tanya; Bentley, William E; Payne, Gregory F

2013-02-19

Redox cycling provides a mechanism to amplify electrochemical signals for analyte detection. Previous studies have shown that diverse mediators/shuttles can engage in redox-cycling reactions with a biobased redox capacitor that is fabricated by grafting redox-active catechols onto a chitosan film. Here, we report that redox cycling with this catechol-chitosan redox capacitor can amplify electrochemical signals for detecting a redox-active bacterial metabolite. Specifically, we studied the redox-active bacterial metabolite pyocyanin that is reported to be a virulence factor and signaling molecule for the opportunistic pathogen P. aeruginosa. We demonstrate that redox cycling can amplify outputs from various electrochemical methods (cyclic voltammetry, chronocoulometry, and differential pulse voltammetry) and can lower the detection limit of pyocyanin to 50 nM. Further, the compatibility of this biobased redox capacitor allows the in situ monitoring of the production of redox-active metabolites (e.g., pyocyanin) during the course of P. aeruginosa cultivation. We anticipate that the amplified output of redox-active virulence factors should permit an earlier detection of life-threatening infections by the opportunistic pathogen P. aeruginosa while the "bio-compatibility" of this measurement approach should facilitate in situ study of the spatiotemporal dynamics of bacterial redox signaling.

The role of 17β-estradiol metabolites in chromium-induced oxidative stress.

PubMed

Sawicka, Ewa; Długosz, Anna

2017-01-01

The increasing incidence of estrogen-dependent breast cancer and the presence in the environment of a large number of factors that interact with estrogen receptors have sparked interest in chemical influences on estrogen-dependent processes. In a previous work, the authors examined the interaction of estradiol with chromium. In the present article the importance of estradiol biotransformation in these interactions is investigated. There is no information in the available literature about the role of metabolites in exposure to chromium. It seems important because estradiol metabolites have various carcinogenic abilities and their formation during biotransformation could be increased or decreased by environmental enzyme inducers or inhibitors. The metabolites could play a detoxifying role or create a toxic synergism in free radical processes induced by chromium VI (CrVI). The aim of this study was to evaluate the influence of 2 17β-estradiol metabolites - 4-hydroxyestradiol (4-OHE2) and 16α-hydroxyestrone (16α-OHE1) - in conditions of oxidative stress caused by CrVI. Human blood, erythrocytes or mitochondria isolated from human placentas after natural deliveries were used in the experiments. The influence of CrVI, 4-OHE2 and 16-OHE1 on thiobarbituric acid reactive substances (TBARS), the hydroxyl radical (•OH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and the interactions of the metabolites exposed to chromium expressed by these factors were examined. 4-OHE2 reduced the level of TBARS induced by CrVI in mitochondria (p < 0.05) and in erythrocytes (p < 0.05), and increased SOD activity (p < 0.05). 16α-OHE1 increased the activity of GST in erythrocytes exposed to CrVI (p < 0.05). The metabolites do not have toxic interactions with CrVI. On the contrary, they exhibited a protective effect. The mechanism of protection varied: 4-OHE2 decreased TBARS and increased SOD activity, while 16α-OHE1 increased GST

Activation of dormant secondary metabolite production by introducing neomycin resistance into the deep-sea fungus, Aspergillus versicolor ZBY-3.

PubMed

Dong, Yuan; Cui, Cheng-Bin; Li, Chang-Wei; Hua, Wei; Wu, Chang-Jing; Zhu, Tian-Jiao; Gu, Qian-Qun

2014-07-29

A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(D-Pro-D-Phe) (1), cyclo(D-Tyr-D-Pro) (2), phenethyl 5-oxo-L-prolinate (3), cyclo(L-Ile-L-Pro) (4), cyclo(L-Leu-L-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1-6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal

Human Metabolites of Cannabidiol: A Review on Their Formation, Biological Activity, and Relevance in Therapy

PubMed Central

Ujváry, István; Hanuš, Lumír

2016-01-01

Abstract Cannabidiol (CBD), the main nonpsychoactive constituent of Cannabis sativa, has shown a wide range of therapeutically promising pharmacological effects either as a sole drug or in combination with other drugs in adjunctive therapy. However, the targets involved in the therapeutic effects of CBD appear to be elusive. Furthermore, scarce information is available on the biological activity of its human metabolites which, when formed in pharmacologically relevant concentration, might contribute to or even account for the observed therapeutic effects. The present overview summarizes our current knowledge on the pharmacokinetics and metabolic fate of CBD in humans, reviews studies on the biological activity of CBD metabolites either in vitro or in vivo, and discusses relevant drug–drug interactions. To facilitate further research in the area, the reported syntheses of CBD metabolites are also catalogued. PMID:28861484

Human Metabolites of Cannabidiol: A Review on Their Formation, Biological Activity, and Relevance in Therapy.

PubMed

Ujváry, István; Hanuš, Lumír

2016-01-01

Cannabidiol (CBD), the main nonpsychoactive constituent of Cannabis sativa , has shown a wide range of therapeutically promising pharmacological effects either as a sole drug or in combination with other drugs in adjunctive therapy. However, the targets involved in the therapeutic effects of CBD appear to be elusive. Furthermore, scarce information is available on the biological activity of its human metabolites which, when formed in pharmacologically relevant concentration, might contribute to or even account for the observed therapeutic effects. The present overview summarizes our current knowledge on the pharmacokinetics and metabolic fate of CBD in humans, reviews studies on the biological activity of CBD metabolites either in vitro or in vivo , and discusses relevant drug-drug interactions. To facilitate further research in the area, the reported syntheses of CBD metabolites are also catalogued.

Ultra-high performance liquid chromatography tandem mass spectrometric method for the determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in dried blood spots--development, validation and clinical application during breast cancer adjuvant therapy.

PubMed

Antunes, Marina Venzon; Raymundo, Suziane; de Oliveira, Vanessa; Staudt, Dilana Elisabeth; Gössling, Gustavo; Peteffi, Giovana Piva; Biazús, Jorge Villanova; Cavalheiro, José Antônio; Tre-Hardy, Marie; Capron, Arnaud; Haufroid, Vincent; Wallemacq, Pierre; Schwartsmann, Gilberto; Linden, Rafael

2015-01-01

A LC-MSMS method for the simultaneous determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in dried blood spots samples was developed and validated. The method employs an ultrasound-assisted liquid extraction and a reversed phase separation in an Acquity(®) C18 column (150×2.1 mm, 1.7 µm). Mobile phase was a mixture of formic acid 0.1% (v/v) pH 2.7 and acetonitrile (gradient from 60:40 to 50:50, v/v). Total analytical run time was 8 min. Precision assays showed CV % lower than 10.75% and accuracy in the range 94.5 to 110.3%. Mean analytes recoveries from DBS ranged from 40% to 92%. The method was successfully applied to 91 paired clinical DBS and plasma samples. Dried blood spots concentrations were highly correlated to plasma, with rs>0.83 (P<0.01). Median estimated plasma concentrations after hematocrit and partition factor adjustment were: TAM 123.3 ng mL(-1); NDT 267.9 ng mL(-1), EDF 10.0 ng mL(-1) and HTF 1.3 ng mL(-1,) representing in average 98 to 104% of the actually measured concentrations. The DBS method was able to identify 96% of patients with plasma EDF concentrations below the clinical threshold related to better prognosis (5.9 ng mL(-1)). The procedure has adequate analytical performance and can be an efficient tool to optimize adjuvant breast cancer treatment, especially in resource limited settings. Copyright © 2014 Elsevier B.V. All rights reserved.

Cytotoxicity of lapachol metabolites produced by probiotics.

PubMed

Oliveira Silva, E; Cruz de Carvalho, T; Parshikov, I A; Alves dos Santos, R; Silva Emery, F; Jacometti Cardoso Furtado, N A

2014-07-01

Probiotics are currently added to a variety of functional foods to provide health benefits to the host and are commonly used by patients with gastrointestinal complaints or diseases. The therapeutic effects of lapachol continue to inspire studies to obtain derivatives with improved bioactivity and lower unwanted effects. Therefore, the general goal of this study was to show that probiotics are able to convert lapachol and are important to assess the effects of bacterial metabolism on drug performance and toxicity. The microbial transformations of lapachol were carried out by Bifidobacterium sp. and Lactobacillus acidophilus and different metabolites were produced in mixed and isolated cultures. The cytotoxic activities against breast cancer and normal fibroblast cell lines of the isolated metabolites (4α-hydroxy-2,2-dimethyl-5-oxo-2,3,4,4α,5,9β-hexahydroindeno[1,2-β]pyran-9β-carboxilic acid, a new metabolite produced by mixed culture and dehydro-α-lapachone produced by isolated cultures) were assessed and compared with those of lapachol. The new metabolite displayed a lower activity against a breast cancer cell line (IC50 = 532.7 μmol l(-1) ) than lapachol (IC50 = 72.3 μmol l(-1) ), while dehydro-α-lapachone (IC50 = 10.4 μmol l(-1) ) displayed a higher activity than lapachol. The present study is the first to demonstrate that probiotics are capable of converting lapachol into the most effective cytotoxic compound against a breast cancer cell line. Probiotics have been used in dairy products to promote human health and have the ability to metabolize drugs and other xenobiotics. Naphthoquinones, such as lapachol, are considered privileged scaffolds due to their high propensity to interact with biological targets. The present study is the first to demonstrate that probiotics are capable of converting lapachol into the most effective cytotoxic compound against a breast cancer cell line. The developed approach highlights the importance of probiotics to assess

The Relative Amounts and Identification of Some 2,4-Dichlorophenoxyacetic Acid Metabolites Isolated from Soybean Cotyledon Callus Cultures 1

PubMed Central

Feung, Chao-Shieung; Hamilton, Robert H.; Witham, Francis H.; Mumma, Ralph O.

1972-01-01

Soybean (Glycine max L.) cotyledon callus grown on radioactive 2,4-dichlorophenoxyacetic acid (2,4-D-1-14C) as an auxin produced 2,4-D metabolites, which qualitatively and quantitatively changed with time. Water soluble fractions from the tissue exhibited a steady increase in radioactivity during the course of 24 days. Following β-glucosidase treatment, at least eight aglycones were obtained from the water soluble fraction of the tissue after 8 days. The metabolite, 4-hydroxy-2,5-dichlorophenoxyacetic acid was the most abundant aglycone during the entire 32 day growth period while 4-hydroxy-2,3-dichlorophenoxyacetic acid was detected as a minor metabolite. Radioactivity in the ether soluble acidic fractions reached a maximum of 82% of the total in the tissue after 2 days. The level then decreased to 44% by the end of 24 days. A total of seven ether soluble components were detected. In addition to 2,4-D glutamic acid, which was detected in high amounts after 24 hours, 2,4-D aspartic acid was found to be the most abundant ether soluble metabolite after longer time periods. Mass spectral data and a fragmentation pattern are presented for 2,4-D aspartic acid. PMID:16658138

Review on the targeted conjugation of anticancer drugs doxorubicin and tamoxifen with synthetic polymers for drug delivery.

PubMed

Sanyakamdhorn, S; Agudelo, D; Tajmir-Riahi, H A

2017-08-01

In this review, the binding and loading efficacy (LE) of anticancer drugs doxorubicin (DOX), tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox) with several synthetic polymers poly(ethylene glycol) (PEG), methoxypoly (ethylene glycol) polyamidoamine (mPEG-PAMAM-G3), and polyamidoamine (PAMAM-G4) dendrimers were compared in aqueous solution at pH 7.4. The results of multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling of conjugated drug-polymer were examined. Structural analysis showed that drug-polymer conjugation occurs mainly via H-bonding and hydrophobic contacts. The order of binding is PAMAM-G4 > mPEG-PAMAM-G3 > PEG-6000 with 4-hydroxttamoxifen forming more stable conjugate than tamoxifen and endoxifen. Doxorubicin shows stronger affinity for PAMAM-G4 than tamoxifen and its metabolites. The drug LE was 30-55%. TEM showed significant changes in the carrier morphology upon drug encapsulation. Modeling also showed that drug is located in the surface and in the internal cavities of PAMAM with DOX forming more stable polymer conjugates.

Anthocyanins and their gut metabolites reduce the adhesion of monocyte to TNFα-activated endothelial cells at physiologically relevant concentrations.

PubMed

Krga, Irena; Monfoulet, Laurent-Emmanuel; Konic-Ristic, Aleksandra; Mercier, Sylvie; Glibetic, Maria; Morand, Christine; Milenkovic, Dragan

2016-06-01

An increasing number of evidence suggests a protective role of dietary anthocyanins against cardiovascular diseases. Anthocyanins' extensive metabolism indicates that their metabolites could be responsible for the protective effects associated with consumption of anthocyanin-rich foods. The aim of this work was to investigate the effect of plasma anthocyanins and their metabolites on the adhesion of monocytes to TNFα-activated endothelial cells and on the expression of genes encoding cell adhesion molecules. Human umbilical vein endothelial cells (HUVECs) were exposed to circulating anthocyanins: cyanidin-3-arabinoside, cyanidin-3-galactoside, cyanidin-3-glucoside, delphinidin-3-glucoside, peonidin-3-glucoside, anthocyanin degradation product: 4-hydroxybenzaldehyde, or to their gut metabolites: protocatechuic, vanillic, ferulic and hippuric acid, at physiologically-relevant concentrations (0.1-2 μM) and time of exposure. Both anthocyanins and gut metabolites decreased the adhesion of monocytes to HUVECs, with a magnitude ranging from 18.1% to 47%. The mixture of anthocyanins and that of gut metabolites also reduced monocyte adhesion. However, no significant effect on the expression of genes encoding E-selectin, ICAM1 and VCAM1 was observed, suggesting that other molecular targets are involved in the observed effect. In conclusion, this study showed the potency of anthocyanins and their gut metabolites to modulate the adhesion of monocytes to endothelial cells, the initial step in atherosclerosis development, under physiologically-relevant conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel Carbonyl Analogues of Tamoxifen: Design, Synthesis, and Biological Evaluation

NASA Astrophysics Data System (ADS)

Kasiotis, Konstantinos M.; Lambrinidis, George; Fokialakis, Nikolas; Tzanetou, Evangelia N.; Mikros, Emmanuel; Haroutounian, Serkos A.

2017-09-01

Aim of this work was to provide tamoxifen analogues with enhanced estrogen receptor binding affinity. Hence, several derivatives were prepared using an efficient triarylethylenes synthetic protocol. The novel compounds bioactivity was evaluated through the determination of their receptor binding affinity and their agonist/antagonist activity against breast cancer tissue using a MCF-7 cell-based assay. Phenyl esters 6a,b and 8a,b exhibited binding affinity to both ERα and ERβ higher than 4-hydroxytamoxifen while compounds 13 and 14 have shown cellular antiestrogenic activity similar to 4-hydroxytamoxifen and the known estrogen receptor inhibitor ICI182,780. Theoretical calculations and molecular modelling were applied to investigate, support and explain the biological profile of the new compounds. The relevant data indicated an agreement between calculations and demonstrated biological activity allowing to extract useful structure-activity relationships. Results herein underline that modifications of tamoxifen structure still provide molecules with substantial activity, as portrayed in the inhibition of MCF-7 cells proliferation.

Linking diet, physical activity, cardiorespiratory fitness and obesity to serum metabolite networks: findings from a population-based study

PubMed Central

Floegel, A; Wientzek, A; Bachlechner, U; Jacobs, S; Drogan, D; Prehn, C; Adamski, J; Krumsiek, J; Schulze, M B; Pischon, T; Boeing, H

2014-01-01

Objective: It is not yet resolved how lifestyle factors and intermediate phenotypes interrelate with metabolic pathways. We aimed to investigate the associations between diet, physical activity, cardiorespiratory fitness and obesity with serum metabolite networks in a population-based study. Methods: The present study included 2380 participants of a randomly drawn subcohort of the European Prospective Investigation into Cancer and Nutrition-Potsdam. Targeted metabolomics was used to measure 127 serum metabolites. Additional data were available including anthropometric measurements, dietary assessment including intake of whole-grain bread, coffee and cake and cookies by food frequency questionnaire, and objectively measured physical activity energy expenditure and cardiorespiratory fitness in a subsample of 100 participants. In a data-driven approach, Gaussian graphical modeling was used to draw metabolite networks and depict relevant associations between exposures and serum metabolites. In addition, the relationship of different exposure metabolite networks was estimated. Results: In the serum metabolite network, the different metabolite classes could be separated. There was a big group of phospholipids and acylcarnitines, a group of amino acids and C6-sugar. Amino acids were particularly positively associated with cardiorespiratory fitness and physical activity. C6-sugar and acylcarnitines were positively associated with obesity and inversely with intake of whole-grain bread. Phospholipids showed opposite associations with obesity and coffee intake. Metabolite networks of coffee intake and obesity were strongly inversely correlated (body mass index (BMI): r=−0.57 and waist circumference: r=−0.59). A strong positive correlation was observed between metabolite networks of BMI and waist circumference (r=0.99), as well as the metabolite networks of cake and cookie intake with cardiorespiratory fitness and intake of whole-grain bread (r=0.52 and r=0

Linking diet, physical activity, cardiorespiratory fitness and obesity to serum metabolite networks: findings from a population-based study.

PubMed

Floegel, A; Wientzek, A; Bachlechner, U; Jacobs, S; Drogan, D; Prehn, C; Adamski, J; Krumsiek, J; Schulze, M B; Pischon, T; Boeing, H

2014-11-01

It is not yet resolved how lifestyle factors and intermediate phenotypes interrelate with metabolic pathways. We aimed to investigate the associations between diet, physical activity, cardiorespiratory fitness and obesity with serum metabolite networks in a population-based study. The present study included 2380 participants of a randomly drawn subcohort of the European Prospective Investigation into Cancer and Nutrition-Potsdam. Targeted metabolomics was used to measure 127 serum metabolites. Additional data were available including anthropometric measurements, dietary assessment including intake of whole-grain bread, coffee and cake and cookies by food frequency questionnaire, and objectively measured physical activity energy expenditure and cardiorespiratory fitness in a subsample of 100 participants. In a data-driven approach, Gaussian graphical modeling was used to draw metabolite networks and depict relevant associations between exposures and serum metabolites. In addition, the relationship of different exposure metabolite networks was estimated. In the serum metabolite network, the different metabolite classes could be separated. There was a big group of phospholipids and acylcarnitines, a group of amino acids and C6-sugar. Amino acids were particularly positively associated with cardiorespiratory fitness and physical activity. C6-sugar and acylcarnitines were positively associated with obesity and inversely with intake of whole-grain bread. Phospholipids showed opposite associations with obesity and coffee intake. Metabolite networks of coffee intake and obesity were strongly inversely correlated (body mass index (BMI): r = -0.57 and waist circumference: r = -0.59). A strong positive correlation was observed between metabolite networks of BMI and waist circumference (r = 0.99), as well as the metabolite networks of cake and cookie intake with cardiorespiratory fitness and intake of whole-grain bread (r = 0.52 and r = 0.50; respectively). Lifestyle factors

Activation of the Sigma-1 receptor by haloperidol metabolites facilitates brain-derived neurotrophic factor secretion from human astroglia

PubMed Central

Dalwadi, Dhwanil A.; Kim, Seongcheol; Schetz, John A.

2017-01-01

Glial cells play a critical role in neuronal support which includes the production and release of the neurotrophin brain-derived neurotrophic factor (BDNF). Activation of the sigma-1 receptor (S1R) has been shown to attenuate inflammatory stress-mediated brain injuries, and there is emerging evidence that this may involve a BDNF-dependent mechanism. In this report we studied S1R-mediated BDNF release from human astrocytic glial cells. Astrocytes express the S1R, which mediates BDNF release when stimulated with the prototypical S1R agonists 4-PPBP and (+)-SKF10047. This effect could be antagonized by a selective concentration of the S1R antagonist BD1063. Haloperidol is known to have high affinity interactions with the S1R, yet it was unable to facilitate BDNF release. Remarkably, however, two metabolites of haloperidol, haloperidol I and haloperidol II (reduced haloperidol), were discovered to facilitate BDNF secretion and this effect was antagonized by BD1063. Neither 4-PPBP, nor either of the haloperidol metabolites affected the level of BDNF mRNA as assessed by qPCR. These results demonstrate for the first time that haloperidol metabolites I and II facilitate the secretion of BDNF from astrocytes by acting as functionally selective S1R agonists. PMID:28188803

Coupling of 2,4,6-trinitrotoluene (TNT) metabolites onto humic monomers by a new laccase from Trametes modesta.

PubMed

Nyanhongo, Gibson S; Couto, Susana Rodríguez; Guebitz, Georg M

2006-06-01

During degradation of trinitrotoluene (TNT) by Trametes modesta, addition of humic monomers prevented the accumulation of all major stable TNT metabolites (aminodinitrotoluenes [AMDNT]) by at least 92% in the presence of 200 mM ferulic acid and guaiacol. Acute toxicity tests with individual TNT metabolites and in T. modesta cultures supplemented with 200 microM TNT demonstrated that the TNT biodegradation process lead to less toxic metabolites. Toxicity decreased in the order TNT>4-HADNT (4-hydroxylaminodinitrotoluene)>2-HADNT>2,6-DNT (2,6-dinitrotoluene)>2',2',6,6-azoxytetranitrotoluene>4-AMDNT>2-AMDNT>2,4-diamninonitrotoluene (2,4-DAMNT) while 2,4-DNT and 2,6-DAMNT were the least toxic. Ferulic acid is the best candidate for immobilization TNT biodegradation metabolites since it prevented the accumulation of AMDNTs in cultures during TNT biodegradation and its products were less toxic. All humic monomers were very effective in immobilizing 2-HADNT [100%], 4-HADNT [100%] and 2,2,6,6-azoxytetranitrotoluene [100%]. Two distinct laccase isoenzymes (LTM1 and LTM2) potentially involved in immobilization of TNT degradation products were purified to electrophoretic homogeneity. LTM1 and LTM2 have molecular weights of 77.6 and 52.5 kDa, are 18% and 24% glycosylated, have pI values of 3.6 and 4.2, respectively. Both enzymes oxidized all the typical laccase substrates tested. LTM1 showed highest kinetic constants (K(m)=0.03 microM; K(cat)=8.8 4x 10(7)s(-1)) with syringaldazine as substrate.

The metabolism of primaquine to its active metabolite is dependent on CYP 2D6.

PubMed

Pybus, Brandon S; Marcsisin, Sean R; Jin, Xiannu; Deye, Gregory; Sousa, Jason C; Li, Qigui; Caridha, Diana; Zeng, Qiang; Reichard, Gregory A; Ockenhouse, Christian; Bennett, Jason; Walker, Larry A; Ohrt, Colin; Melendez, Victor

2013-06-20

The efficacy of the 8-aminoquinoline (8AQ) drug primaquine (PQ) has been historically linked to CYP-mediated metabolism. Although to date no clear evidence exists in the literature that unambiguously assigns the metabolic pathway or specific metabolites necessary for activity, recent literature suggests a role for CYP 2D6 in the generation of redox active metabolites. In the present study, the specific CYP 2D6 inhibitor paroxetine was used to assess its effects on the production of specific phenolic metabolites thought to be involved in PQ efficacy. Further, PQ causal prophylactic (developing liver stage) efficacy against Plasmodium berghei in CYP 2D knockout mice was assessed in comparison with a normal C57 background and with humanized CYP 2D6 mice to determine the direct effects of CYP 2D6 metabolism on PQ activity. PQ exhibited no activity at 20 or 40 mg/kg in CYP 2D knockout mice, compared to 5/5 cures in normal mice at 20 mg/kg. The activity against developing liver stages was partially restored in humanized CYP 2D6 mice. These results unambiguously demonstrate that metabolism of PQ by CYP 2D6 is essential for anti-malarial causal prophylaxis efficacy.

Activation and products of the cryptic secondary metabolite biosynthetic gene clusters by rifampin resistance (rpoB) mutations in actinomycetes.

PubMed

Tanaka, Yukinori; Kasahara, Ken; Hirose, Yutaka; Murakami, Kiriko; Kugimiya, Rie; Ochi, Kozo

2013-07-01

A subset of rifampin resistance (rpoB) mutations result in the overproduction of antibiotics in various actinomycetes, including Streptomyces, Saccharopolyspora, and Amycolatopsis, with H437Y and H437R rpoB mutations effective most frequently. Moreover, the rpoB mutations markedly activate (up to 70-fold at the transcriptional level) the cryptic/silent secondary metabolite biosynthetic gene clusters of these actinomycetes, which are not activated under general stressful conditions, with the exception of treatment with rare earth elements. Analysis of the metabolite profile demonstrated that the rpoB mutants produced many metabolites, which were not detected in the wild-type strains. This approach utilizing rifampin resistance mutations is characterized by its feasibility and potential scalability to high-throughput studies and would be useful to activate and to enhance the yields of metabolites for discovery and biochemical characterization.

The oxygen-centered radicals scavenging activity of sulfasalazine and its metabolites. A direct protection of the bowel.

PubMed

Prónai, L; Yukinobu, I; Láng, I; Fehér, J

1992-01-01

Oxygen-centered radicals, such as superoxide (O2-) and hydroxyl radicals (.OH) generated by phagocytes have been suggested to be involved in the pathogenesis of chronic inflammations of the bowel, such as Crohn's disease and colitis ulcerosa. Recently, sulfasalazine (SASP) and its metabolites have been reported to exert their effects as a direct scavenger of oxygen-centered radicals in the bowel. To scavenge oxygen-centered radicals in vivo, however, SASP and its metabolites have to react with O2- and/or .OH in vitro very rapidly, furthermore they have to reach an appropriate (possible millimolar) concentration range at the site of inflammation. To test this possibility, we investigated the direct O2- and .OH scavenging activity of SASP and its metabolites using the specific electron paramagnetic resonance/spin trapping method, and we compared the 50% inhibition rates of SASP and its metabolites with their known concentrations in the bowel and in the human plasma. It was found that SASP and its metabolites, such as 5-amino-salicylic acid (5-ASA), and acetyl-5-amino-salicylic acid (AC-5-ASA), but not sulfapyridine (SP) and acetyl-sulfapyridine (Ac-SP) have a direct O2- and .OH scavenging activity in vitro systems. Among the compounds, SASP and 5-ASA can reach a concentration which is appropriate to scavenge oxygen-centered radicals in the bowel but not in the human plasma. It was concluded that the in vivo antiinflammatory effects of SASP and its metabolites are, at least partly, due to the direct oxygen-centered scavenging activity of these drugs.

Effects of nelfinavir and its M8 metabolite on lymphocyte P-glycoprotein activity during antiretroviral therapy.

PubMed

Donahue, John P; Dowdy, David; Ratnam, Krishna K; Hulgan, Todd; Price, James; Unutmaz, Derya; Nicotera, Janet; Raffanti, Steven; Becker, Mark; Haas, David W

2003-01-01

The efflux pump P-glycoprotein decreases drug penetration into cells and tissues. To determine whether nelfinavir or its metabolites inhibit P-glycoprotein in lymphocytes from a healthy volunteer, whole blood cells from human immunodeficiency virus-negative donors were incubated either in human plasma to which nelfinavir or its M8 metabolite were added ex vivo or in plasma from human immunodeficiency virus-positive patients receiving nelfinavir. The 50% P-glycoprotein inhibitory concentrations of purified nelfinavir and M8 were 10.9 micromol/L and 29.5 micromol/L, respectively, for CD4(+) T cells and 19.3 micromol/L and >48 micromol/L, respectively, for CD8(+) T cells. Significant inhibitory activity was present in plasma from 27 of 46 patients (59%) receiving nelfinavir. Plasma nelfinavir concentrations correlated with percent inhibition on CD4(+) (rho = 0.85, P <.0001) and CD8(+) (rho = 0.83, P <.0001) T cells. The M8 concentrations correlated weakly with both inhibition and nelfinavir concentrations. On the basis of our findings in lymphocytes from a healthy volunteer exposed to plasma from human immunodeficiency virus-positive patients, we believe it is likely that CD4(+) and CD8(+) lymphocytes in patients receiving nelfinavir as therapy for human immunodeficiency virus may have P-glycoprotein inhibited by plasma concentrations of nelfinavir.

Selective Synthesis and Biological Evaluation of Sulfate-Conjugated Resveratrol Metabolites

PubMed Central

Hoshino, Juma; Park, Eun-Jung; Kondratyuk, Tamara P.; Marler, Laura; Pezzuto, John M.; van Breemen, Richard B.; Mo, Shunyan; Li, Yongchao; Cushman, Mark

2010-01-01

Five resveratrol sulfate metabolites were synthesized and assessed for activities known to be mediated by resveratrol: inhibition of tumor necrosis factor (TNF)-α-induced NFκB activity, cylcooxygenases (COX-1 and COX-2), aromatase, nitric oxide production in endotoxin-stimulated macrophages, and proliferation of KB or MCF7 cells, induction of quinone reductase 1 (QR1), accumulation in the sub-G1 phase of the cell cycle, and quenching of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. Two metabolites showed activity in these assays; the 3-sulfate exhibited QR1 induction, DPPH free radical scavenging, and COX-1 and COX-2 inhibitory activities, and the 4′-sulfate inhibited NFκB induction, as well as COX-1 and COX-2 activities. Resveratrol, as well as its 3′-sulfate and 4-sulfate, inhibit NO production by NO scavenging and down-regulation of iNOS expression in RAW 264.7 cells. Resveratrol sulfates displayed low antiproliferative activity and negligible uptake in MCF7 cells. PMID:20527891

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

PubMed

Mills, Evanna L; Ryan, Dylan G; Prag, Hiran A; Dikovskaya, Dina; Menon, Deepthi; Zaslona, Zbigniew; Jedrychowski, Mark P; Costa, Ana S H; Higgins, Maureen; Hams, Emily; Szpyt, John; Runtsch, Marah C; King, Martin S; McGouran, Joanna F; Fischer, Roman; Kessler, Benedikt M; McGettrick, Anne F; Hughes, Mark M; Carroll, Richard G; Booty, Lee M; Knatko, Elena V; Meakin, Paul J; Ashford, Michael L J; Modis, Louise K; Brunori, Gino; Sévin, Daniel C; Fallon, Padraic G; Caldwell, Stuart T; Kunji, Edmund R S; Chouchani, Edward T; Frezza, Christian; Dinkova-Kostova, Albena T; Hartley, Richard C; Murphy, Michael P; O'Neill, Luke A

2018-04-05

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

Pleiotropic mechanisms facilitated by resveratrol and its metabolites

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

Calamini, Barbara; Ratia, Kiira; Malkowski, Michael G.

2010-07-01

Resveratrol has demonstrated cancer chemopreventive activity in animal models and some clinical trials are underway. In addition, resveratrol was shown to promote cell survival, increase lifespan and mimic caloric restriction, thereby improving health and survival of mice on high-calorie diet. All of these effects are potentially mediated by the pleiotropic interactions of resveratrol with different enzyme targets including COX-1 (cyclo-oxygenase-1) and COX-2, NAD{sup +}-dependent histone deacetylase SIRT1 (sirtuin 1) and QR2 (quinone reductase 2). Nonetheless, the health benefits elicited by resveratrol as a direct result of these interactions with molecular targets have been questioned, since it is rapidly and extensivelymore » metabolized to sulfate and glucuronide conjugates, resulting in low plasma concentrations. To help resolve these issues, we tested the ability of resveratrol and its metabolites to modulate the function of some known targets in vitro. In the present study, we have shown that COX-1, COX-2 and QR2 are potently inhibited by resveratrol, and that COX-1 and COX-2 are also inhibited by the resveratrol 4{prime}-O-sulfate metabolite. We determined the X-ray structure of resveratrol bound to COX-1 and demonstrate that it occupies the COX active site similar to other NSAIDs (non-steroidal anti-inflammatory drugs). Finally, we have observed that resveratrol 3- and 4?-O-sulfate metabolites activate SIRT1 equipotently to resveratrol, but that activation is probably a substrate-dependent phenomenon with little in vivo relevance. Overall, the results of this study suggest that in vivo an interplay between resveratrol and its metabolites with different molecular targets may be responsible for the overall beneficial health effects previously attributed only to resveratrol itself.« less

Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms.

PubMed

Bellin, Daniel L; Sakhtah, Hassan; Rosenstein, Jacob K; Levine, Peter M; Thimot, Jordan; Emmett, Kevin; Dietrich, Lars E P; Shepard, Kenneth L

2014-01-01

Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites that are produced by microbial biofilms and can affect their development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. 'Images' over a 3.25 × 0.9 mm(2) area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify and quantify (for concentrations as low as 2.6 μM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression.

Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms

PubMed Central

Bellin, Daniel L.; Sakhtah, Hassan; Rosenstein, Jacob K.; Levine, Peter M.; Thimot, Jordan; Emmett, Kevin; Dietrich, Lars E. P.; Shepard, Kenneth L.

2014-01-01

Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites produced by microbial biofilms, which can drastically affect colony development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. “Images” over a 3.25 × 0.9 mm area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify, and quantify (for concentrations as low as 2.6 μM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression. PMID:24510163

Characterization of the active site properties of CYP4F12.

PubMed

Eksterowicz, John; Rock, Dan A; Rock, Brooke M; Wienkers, Larry C; Foti, Robert S

2014-10-01

Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Activation of the sigma-1 receptor by haloperidol metabolites facilitates brain-derived neurotrophic factor secretion from human astroglia.

PubMed

Dalwadi, Dhwanil A; Kim, Seongcheol; Schetz, John A

2017-05-01

Glial cells play a critical role in neuronal support which includes the production and release of the neurotrophin brain-derived neurotrophic factor (BDNF). Activation of the sigma-1 receptor (S1R) has been shown to attenuate inflammatory stress-mediated brain injuries, and there is emerging evidence that this may involve a BDNF-dependent mechanism. In this report we studied S1R-mediated BDNF release from human astrocytic glial cells. Astrocytes express the S1R, which mediates BDNF release when stimulated with the prototypical S1R agonists 4-PPBP and (+)-SKF10047. This effect could be antagonized by a selective concentration of the S1R antagonist BD1063. Haloperidol is known to have high affinity interactions with the S1R, yet it was unable to facilitate BDNF release. Remarkably, however, two metabolites of haloperidol, haloperidol I and haloperidol II (reduced haloperidol), were discovered to facilitate BDNF secretion and this effect was antagonized by BD1063. Neither 4-PPBP, nor either of the haloperidol metabolites affected the level of BDNF mRNA as assessed by qPCR. These results demonstrate for the first time that haloperidol metabolites I and II facilitate the secretion of BDNF from astrocytes by acting as functionally selective S1R agonists. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antioxidant Enzyme Activities and Secondary Metabolite Profiling of Oil Palm Seedlings Treated with Combination of NPK Fertilizers Infected with Ganoderma boninense.

PubMed

Sahebi, Mahbod; Hanafi, Mohamed M; Mohidin, Hasmah; Rafii, M Y; Azizi, Parisa; Idris, Abu Seman; Fariz, A; Abiri, Rambod; Taheri, Sima; Moradpoor, Mehdi

2018-01-01

Oil palm ( Elaeis guineensis Jacq) is one of the major sources of edible oil. Reducing the effect of Ganoderma, main cause of basal stem rot (BSR) on oil palm, is the main propose of this study. Understanding the oil palm defense mechanism against Ganoderma infection through monitoring changes in the secondary metabolite compounds levels before/after infection by Ganoderma under different fertilizing treatment is required. Oil palm requires macro- and microelements for growth and yield. Manipulating the nutrient for oil palm is a method to control the disease. The 3-4-month-old oil palm seedlings were given different macronutrient treatments to evaluate induction of defense related enzymes and production of secondary metabolite compounds in response to G. boninense inoculation. The observed trend of changes in the infected and uninfected seedlings was a slightly higher activity for β -1,3-glucanases, chitinase, peroxidase, and phenylalanine ammonia-lyase during the process of pathogenesis. It was found that PR proteins gave positive response to the interaction between oil palm seedlings and Ganoderma infection. Although the responses were activated systematically, they were short-lasting as the changes in enzymes activities appeared before the occurrence of visible symptoms. Effect of different nutrients doses was obviously observed among the results of the secondary metabolite compounds. Many identified/unidentified metabolite compounds were presented, of which some were involved in plant cell defense mechanism against pathogens, mostly belonging to alkaloids with bitter-tasting nitrogenous-compounds, and some had the potential to be used as new markers to detect basal stem rot at the initial step of disease.

Antioxidant Enzyme Activities and Secondary Metabolite Profiling of Oil Palm Seedlings Treated with Combination of NPK Fertilizers Infected with Ganoderma boninense

PubMed Central

Mohidin, Hasmah; Idris, Abu Seman; Fariz, A.; Abiri, Rambod; Taheri, Sima; Moradpoor, Mehdi

2018-01-01

Oil palm (Elaeis guineensis Jacq) is one of the major sources of edible oil. Reducing the effect of Ganoderma, main cause of basal stem rot (BSR) on oil palm, is the main propose of this study. Understanding the oil palm defense mechanism against Ganoderma infection through monitoring changes in the secondary metabolite compounds levels before/after infection by Ganoderma under different fertilizing treatment is required. Oil palm requires macro- and microelements for growth and yield. Manipulating the nutrient for oil palm is a method to control the disease. The 3-4-month-old oil palm seedlings were given different macronutrient treatments to evaluate induction of defense related enzymes and production of secondary metabolite compounds in response to G. boninense inoculation. The observed trend of changes in the infected and uninfected seedlings was a slightly higher activity for β-1,3-glucanases, chitinase, peroxidase, and phenylalanine ammonia-lyase during the process of pathogenesis. It was found that PR proteins gave positive response to the interaction between oil palm seedlings and Ganoderma infection. Although the responses were activated systematically, they were short-lasting as the changes in enzymes activities appeared before the occurrence of visible symptoms. Effect of different nutrients doses was obviously observed among the results of the secondary metabolite compounds. Many identified/unidentified metabolite compounds were presented, of which some were involved in plant cell defense mechanism against pathogens, mostly belonging to alkaloids with bitter-tasting nitrogenous-compounds, and some had the potential to be used as new markers to detect basal stem rot at the initial step of disease. PMID:29721500

In Vitro Antibacterial Activities of AF 3013, the Active Metabolite of Prulifloxacin, against Nosocomial and Community Italian Isolates

PubMed Central

Montanari, Maria Pia; Mingoia, Marina; Varaldo, Pietro Emanuele

2001-01-01

AF 3013, the active metabolite of prulifloxacin, was tested to determine its inhibitory and bactericidal activities against 396 nosocomial and 258 community Italian isolates. Compared with that of ciprofloxacin, its activity (assessed in MIC and minimal bactericidal concentration tests) was generally similar or greater against gram-positive bacteria and greater against gram-negative bacteria. In time-kill assays using selected isolates, its bactericidal activity was comparable to that of ciprofloxacin. PMID:11709353

Ex vivo permeation of tamoxifen and its 4-OH metabolite through rat intestine from lecithin/chitosan nanoparticles.

PubMed

Barbieri, S; Buttini, F; Rossi, A; Bettini, R; Colombo, P; Ponchel, G; Sonvico, F; Colombo, G

2015-08-01

Tamoxifen citrate is an anticancer drug slightly soluble in water. Administered orally, it shows great intra- and inter-patient variations in bioavailability. We developed a nanoformulation based on phospholipid and chitosan able to efficiently load tamoxifen and showing an enzyme triggered release. In this work the permeation of tamoxifen released from lecithin/chitosan nanoparticles across excised rat intestinal wall mounted in an Ussing chamber was investigated. Compared to tamoxifen citrate suspension, the amount of the drug permeated using the nanoformulation was increased from 1.5 to 90 times, in absence or in presence of pancreatin or lipase, respectively. It was also evidenced the formation of an active metabolite of tamoxifen, 4-hydroxy tamoxifen, however, the amount of metabolite permeated remained roughly constant in all experiments. The effect of enzymes on intestinal permeation of tamoxifen was shown only when tamoxifen-loaded nanoparticles were in intimate contact with the mucosal surface. The encapsulation of tamoxifen in lecithin/chitosan nanoparticles improved the non-metabolized drug passing through the rat intestinal tissue via paracellular transport. Copyright © 2015 Elsevier B.V. All rights reserved.

Benzene's metabolites alter c-MYB activity via reactive oxygen species in HD3 cells.

PubMed

Wan, Joanne; Winn, Louise M

2007-07-15

Benzene is a known leukemogen that is metabolized to form reactive intermediates and reactive oxygen species (ROS). The c-Myb oncoprotein is a transcription factor that has a critical role in hematopoiesis. c-Myb transcript and protein have been overexpressed in a number of leukemias and cancers. Given c-Myb's role in hematopoiesis and leukemias, it is hypothesized that benzene interferes with the c-Myb signaling pathway and that this involves ROS. To investigate our hypothesis, we evaluated whether benzene, 1,4-benzoquinone, hydroquinone, phenol, and catechol generated ROS in chicken erythroblast HD3 cells, as measured by 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (DCFDA) and dihydrorhodamine-123 (DHR-123), and whether the addition of 100 U/ml of the antioxidating enzyme superoxide dismutase (SOD) could prevent ROS generation. Reduced to oxidized glutathione ratios (GSH:GSSG) were also assessed as well as hydroquinone and benzoquinone's effects on c-Myb protein levels and activation of a transiently transfected reporter construct. Finally we attempted to abrogate benzene metabolite mediated increases in c-Myb activity with the use of SOD. We found that benzoquinone, hydroquinone, and catechol increased DCFDA fluorescence, increased DHR-123 fluorescence, decreased GSH:GSSG ratios, and increased reporter construct expression after 24 h of exposure. SOD was able to prevent DCFDA fluorescence and c-Myb activity caused by benzoquinone and hydroquinone only. These results are consistent with other studies, which suggest metabolite differences in benzene-mediated toxicity. More importantly, this study supports the hypothesis that benzene may mediate its toxicity through ROS-mediated alterations in the c-Myb signaling pathway.

Effects of clopidogrel on the pharmacokinetics of sibutramine and its active metabolites.

PubMed

Bae, Jung-Woo; Jang, Choon-Gon; Lee, Seok-Young

2011-12-01

Sibutramine is metabolized by the enzymes CYP2B6 and CYP2C19 into 2 active metabolites, M1 (mono-desmethyl sibutramine) and M2 (di-desmethyl sibutramine). Clopidogrel is a mechanism-based inhibitor of CYP2B6 and CYP2C19. In this study, 13 extensive metabolizers of CYP2B6 and CYP2C19 were evaluated to clarify whether clopidogrel inhibits the formation of the active metabolites of sibutramine. In the control phase, each subject received a 15-mg oral dose of sibutramine. After a washout period of 2 weeks, in the clopidogrel phase, the subjects received 300 mg of clopidogrel on the first day and then 75-mg once daily for 6 days. One hour after the last dosing of clopidogrel, all subjects received 15-mg of sibutramine. Compared with the control phase, the mean sibutramine and M1 plasma concentrations were higher after clopidogrel treatment. Clopidogrel significantly increased the half-life (242% of control phase) and area under the plasma concentration-time curve from 0 to infinity (AUC(inf)) (227% of control phase) of sibutramine and decreased the apparent oral clearance (31% of control phase) of sibutramine. Pharmacokinetic analysis showed significant increases in the AUC(inf) (162% of control phase) of M1. The CYP2B6 and CYP2C19 inhibitor clopidogrel significantly inhibited the formations of M1 from sibutramine and M2 from sibutramine by 37% and 64%, respectively. Therefore, CYP2B6 and CYP2C19 are in vivo catalysts for the formation of the 2 active metabolites of sibutramine.

Bisphenol-A alters microbiota metabolites derived from aromatic amino acids and worsens disease activity during colitis.

PubMed

DeLuca, Jennifer Aa; Allred, Kimberly F; Menon, Rani; Riordan, Rebekah; Weeks, Brad R; Jayaraman, Arul; Allred, Clinton D

2018-06-01

Inflammatory bowel disease is a complex collection of disorders. Microbial dysbiosis as well as exposure to toxins including xenoestrogens are thought to be risk factors for inflammatory bowel disease development and relapse. Bisphenol-A has been shown to exert estrogenic activity in the colon and alter intestinal function, but the role that xenoestrogens, such as bisphenol-A , play in colonic inflammation has been previously described but with conflicting results. We investigated the ability of bisphenol-A to exacerbate colonic inflammation and alter microbiota metabolites derived from aromatic amino acids in an acute dextran sulfate sodium-induced colitis model. Female C57BL/6 mice were ovariectomized and exposed to bisphenol-A daily for 15 days. Disease activity measures include body weight, fecal consistency, and rectal bleeding. Colons were scored for inflammation, injury, and nodularity. Alterations in the levels of microbiota metabolites derived from aromatic amino acids known to reflect phenotypic changes in the gut microbiome were analyzed. Bisphenol-A exposure increased mortality and worsened disease activity as well as inflammation and nodularity scores in the middle colon region following dextran sulfate sodium exposure. Unique patterns of metabolites were associated with bisphenol-A consumption. Regardless of dextran sulfate sodium treatment, bisphenol-A reduced levels of tryptophan and several metabolites associated with decreased inflammation in the colon. This is the first study to show that bisphenol-A treatment alone can reduce microbiota metabolites derived from aromatic amino acids in the colon which may be associated with increased colonic inflammation and inflammatory bowel disease. Impact statement As rates of inflammatory bowel disease rise, discovery of the mechanisms related to the development of these conditions is important. Environmental exposure is hypothesized to play a role in etiology of the disease, as are alterations in the gut

Metabolites related to renal function, immune activation, and carbamylation are associated with muscle composition in older adults.

PubMed

Lustgarten, Michael S; Fielding, Roger A

2017-12-15

Reduced skeletal muscle density in older adults is associated with insulin resistance, decreased physical function, and an increased all-cause mortality risk. To elucidate mechanisms that may underlie the maintenance of skeletal muscle density, we conducted a secondary analysis of previously published muscle composition and serum metabolomic data in 73 older adults (average age, 78y). Multivariable-adjusted linear regression was used to examine associations between 321 metabolites with muscle composition, defined as the ratio between normal density (NDM) with low density (LDM) thigh muscle cross sectional area (NDM/LDM). Sixty metabolites were significantly (p≤0.05 and q<0.30) associated with NDM/LDM. Decreased renal function and the immune response have been previously linked with reduced muscle density, but the mechanisms underlying these connections are less clear. Metabolites that were significantly associated with muscle composition were then tested for their association with circulating markers of renal function (blood urea nitrogen, creatinine, uric acid), and with the immune response (neutrophils/lymphocytes) and activation (kynurenine/tryptophan). 43 significant NDM/LDM metabolites (including urea) were co-associated with at least 1 marker of renal function; 23 of these metabolites have been previously identified as uremic solutes. The neutrophil/lymphocyte ratio was significantly associated with NDM/LDM (β±SE: -0.3±0.1, p=0.01, q=0.04). 35 significant NDM/LDM metabolites were co-associated with immune activation. Carbamylation (defined as homocitrulline/lysine) was identified as a pathway that may link renal function and immune activation with muscle composition, as 29 significant NDM/LDM metabolites were co-associated with homocitrulline/lysine, with at least 2 markers of renal function, and with kynurenine/tryptophan. When considering that elevated urea and uremic metabolites have been linked with an increased systemic microbial burden, that

Phytoremediation of carbamazepine and its metabolite 10,11-epoxycarbamazepine by C3 and C4 plants.

PubMed

Ryšlavá, Helena; Pomeislová, Alice; Pšondrová, Šárka; Hýsková, Veronika; Smrček, Stanislav

2015-12-01

The anticonvulsant drug carbamazepine is considered as an indicator of sewage water pollution: however, its uptake by plants and effect on metabolism have not been sufficiently documented, let alone its metabolite (10,11-epoxycarbamazepine). In a model system of sterile, hydroponically cultivated Zea mays (as C4 plant) and Helianthus annuus (as C3 plant), the uptake and effect of carbamazepine and 10,11-epoxycarbamazepine were studied in comparison with those of acetaminophen and ibuprofen. Ibuprofen and acetaminophen were effectively extracted from drug-supplemented media by both plants, while the uptake of more hydrophobic carbamazepine was much lower. On the other hand, the carbamazepine metabolite, 10,11-epoxycarbamazepine, was, unlike sunflower, willingly taken up by maize plants (after 96 h 88 % of the initial concentration) and effectively stored in maize tissues. In addition, the effect of the studied pharmaceuticals on the plant metabolism (enzymes of Hatch-Slack cycle, peroxidases) was followed. The activity of bound peroxidases, which could cause xylem vessel lignification and reduction of xenobiotic uptake, was at the level of control plants in maize leaves contrary to sunflower. Therefore, our results indicate that maize has the potential to remove 10,11-epoxycarbamazepine from contaminated soils.

Diclofenac and metabolite pharmacokinetics in children.

PubMed

van der Marel, Caroline D; Anderson, Brian J; Rømsing, Janne; Jacqz-Aigrain, Evelyne; Tibboel, Dick

2004-06-01

Data concerning metabolism of diclofenac in children are limited to intravenous and enteric coated oral formulations. There are no data examining diclofenac or its hydroxyl metabolite pharmacokinetics after rectal administration in children. Infants (n = 26) undergoing tonsillectomy were given diclofenac 2 mg.kg(-1) followed by 1 mg.kg(-1) 8 h as suppository formulation for postoperative analgesia. Serum was assayed for diclofenac, 4'-hydroxydiclofenac and 5'-hydroxydiclofenac concentrations during the procedure and 1, 2 and 4 h postoperatively. The formation clearances of diclofenac to hydroxyl metabolites were estimated using nonlinear mixed effects models. A single compartment, first order absorption and first order elimination model was used to describe diclofenac pharmacokinetics. Published data from 11 children given enteric-coated diclofenac tablets were used to assess relative bioavailability. Mean (sd) age and weight of the patients were 4.5 (1.5) years and 20.5 (4.1) kg. The formation clearance to 4'-hydroxydiclofenac (% CV) and to 5'-hydroxydiclofenac were 8.41 (8.1) and 3.41 (113) l.h(-1) respectively, standardized to a 70 kg person using allometric '1/4 power' models. Clearance by other routes contributed 33.0 (64) l.h(-1) 70 kg(-1). Elimination clearance of hydroxyl metabolites was fixed at 27.5 l.h(-1) 70 kg(-1). The volumes of distribution of parent diclofenac and its hydroxyl metabolite were 22.8 (19.0) and 45.3 (l.70) kg(-1). The suppository formulation had an absorption half-life of 0.613 (33.2) h with a lag time of 0.188 (24.9) h. Interoccasion variability of formation clearance to 4'-hydroxydiclofenac, diclofenac volume of distribution, absorption half-time and lag time for the suppository was 36%, 55%, 14% and 119%, respectively. The relative bioavailability of the suppository compared with an enteric-coated tablet was 1.26. The formation clearance of the active metabolite 4'-hydroxydiclofenac contributed 19% of total clearance (44.82 l.h(-1

Cytotoxic Cytochalasins and Other Metabolites from Xylariaceae sp. FL0390, a Fungal Endophyte of Spanish Moss.

PubMed

Xu, Ya-Ming; Bashyal, Bharat P; Liu, Mangping X; Espinosa-Artiles, Patricia; U'Ren, Jana M; Arnold, A Elizabeth; Gunatilaka, A A Leslie

2015-10-01

Two new metabolites, 6-oxo-12-norcytochalasin D (1) and 4,5-di-isobutyl-2(1H)-pyrimidinone (2), together with seven known metabolites, cytochalasins D (3), Q (4), and N (5), 12-hydroxyzygosporin G (6), heptelidic acid chlorohydrin (7), (+)-heptelidic acid (8), and trichoderonic acid A (9), were isolated from Xylariaceae sp. FL0390, a fungal endophyte inhabiting Spanish moss, Tillandsia usneoides. Metabolite 1 is the first example of a 12-norcytochalasin. All metabolites, except 2 and 9, showed cytotoxic activity in a panel of five human tumor cell lines with IC50S of 0.2-5.0 μM.

Identification of Sildenafil (Viagra) and Its Metabolite (UK 103,320) in Six Aviation Fatalities

DTIC Science & Technology

2006-02-01

Identification of Sildenafil ( Viagra ®) and Its Metabolite (UK-103,320) in Six Aviation Fatalities Robert D. Johnson Russell J. Lewis Civil...DOT/FAA/AM-06/3 4. Title and Subtitle 5. Report Date February 2006 Identification of Sildenafil ( Viagra ®) and Its Metabolite (UK-103,320...report presents a rapid and reliable method for the identification and quantitation of sildenafil ( Viagra ®) and its active metabolite, UK-103,320. This

Degraded protein adducts of cis-2-butene-1,4-dial are urinary and hepatocyte metabolites of furan.

PubMed

Lu, Ding; Sullivan, Mathilde M; Phillips, Martin B; Peterson, Lisa A

2009-06-01

Furan is a liver toxicant and carcinogen in rodents. On the basis of these observations and the large potential for human exposure, furan has been classified as a possible human carcinogen. The mechanism of tumor induction by furan is unknown. However, the toxicity requires cytochrome P450-catalyzed oxidation of furan. The product of this oxidation, cis-2-butene-1,4-dial (BDA), reacts readily with glutathione, amino acids, and DNA and is a bacterial mutagen in Ames assay strain TA104. Characterization of the urinary metabolites of furan is expected to provide information regarding the structure(s) of the reactive metabolite(s). Recently, several urinary metabolites have been identified. We reported the presence of a monoglutathione-BDA reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinylglycine cyclic sulfide. Three additional urinary metabolites of furan were also characterized as follows: R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid, N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine, and its sulfoxide. It was postulated that these three metabolites are derived from degraded protein adducts. However, the possibility that these metabolites result from the reaction of BDA with free lysine and/or cysteine was not ruled out. In this latter case, one might predict that the reaction of thiol-BDA with free lysine would not occur exclusively on the epsilon-amino group. Reaction of BDA with N-acetylcysteine or GSH in the presence of lysine indicated that both the alpha- and the epsilon-amino groups of lysine can be modified by thiol-BDA. The N-acetylcysteine-BDA-N-acetyllysine urinary metabolites were solely linked through the epsilon-amino group of lysine. A GSH-BDA-lysine cross-link was a significant hepatocyte metabolite of furan. In this case, the major product resulted from reaction with the epsilon-amino group of lysine; however, small amounts of the alpha-amino reaction product were also

Degraded protein adducts of cis-2-butene-1,4-dial are urinary and hepatocyte metabolites of furan

PubMed Central

Lu, Ding; Sullivan, Mathilde M.; Phillips, Martin B.; Peterson, Lisa A.

2009-01-01

Furan is a liver toxicant and carcinogen in rodents. Based on these observations and the large potential for human exposure, furan has been classified as a possible human carcinogen. The mechanism of tumor induction by furan is unknown. However, the toxicity requires cytochrome P450 catalyzed oxidation of furan. The product of this oxidation, cis-2-butene-1,4-dial (BDA), reacts readily with glutathione, amino acids and DNA and is a bacterial mutagen in Ames assay strain TA104. Characterization of the urinary metabolites of furan is expected to provide information regarding the structure(s) of the reactive metabolite(s). Recently, several urinary metabolites have been identified. We reported the presence of a mono-glutathione-BDA reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine cyclic sulfide. Three additional urinary metabolites of furan were also characterized: R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid, N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-L-cysteine and its sulfoxide. It was postulated that these three metabolites are derived from degraded protein adducts. However, the possibility that these metabolites result from reaction of BDA with free lysine and/or cysteine was not ruled out. In this latter case, one might predict that the reaction of thiol-BDA with free lysine would not occur exclusively on the ε-amino group. Reaction of BDA with N-acetylcysteine or GSH in the presence of lysine indicated that both the α- and ε-amino groups of lysine can be modified by thiol-BDA. The N-acetylcysteine-BDA-N-acetyllysine urinary metabolites were solely linked through the ε-amino group of lysine. A GSH-BDA-lysine crosslink was a significant hepatocyte metabolite of furan. In this case, the major product resulted from reaction with the ε-amino group of lysine, however, small amounts of the α-amino reaction product were also observed. Western analysis of liver and hepatocyte

Helicobacter pylori modulates host cell responses by CagT4SS-dependent translocation of an intermediate metabolite of LPS inner core heptose biosynthesis

PubMed Central

Faber, Eugenia; Bats, Simon H.; Murillo, Tatiana; Speidel, Yvonne; Coombs, Nina

2017-01-01

Highly virulent Helicobacter pylori cause proinflammatory signaling inducing the transcriptional activation and secretion of cytokines such as IL-8 in epithelial cells. Responsible in part for this signaling is the cag pathogenicity island (cagPAI) that codetermines the risk for pathological sequelae of an H. pylori infection such as gastric cancer. The Cag type IV secretion system (CagT4SS), encoded on the cagPAI, can translocate various molecules into cells, the effector protein CagA, peptidoglycan metabolites and DNA. Although these transported molecules are known to contribute to cellular responses to some extent, a major part of the cagPAI-induced signaling leading to IL-8 secretion remains unexplained. We report here that biosynthesis of heptose-1,7-bisphosphate (HBP), an important intermediate metabolite of LPS inner heptose core, contributes in a major way to the H. pylori cagPAI-dependent induction of proinflammatory signaling and IL-8 secretion in human epithelial cells. Mutants defective in the genes required for synthesis of HBP exhibited a more than 95% reduction of IL-8 induction and impaired CagT4SS-dependent cellular signaling. The loss of HBP biosynthesis did not abolish the ability to translocate CagA. The human cellular adaptor TIFA, which was described before to mediate HBP-dependent activity in other Gram-negative bacteria, was crucial in the cagPAI- and HBP pathway-induced responses by H. pylori in different cell types. The active metabolite was present in H. pylori lysates but not enriched in bacterial supernatants. These novel results advance our mechanistic understanding of H. pylori cagPAI-dependent signaling mediated by intracellular pattern recognition receptors. They will also allow to better dissect immunomodulatory activities by H. pylori and to improve the possibilities of intervention in cagPAI- and inflammation-driven cancerogenesis. PMID:28715499

Pharmacokinetics of dietary kaempferol and its metabolite 4-hydroxyphenylacetic acid in rats.

PubMed

Zabela, Volha; Sampath, Chethan; Oufir, Mouhssin; Moradi-Afrapoli, Fahimeh; Butterweck, Veronika; Hamburger, Matthias

2016-12-01

Kaempferol is a major flavonoid in the human diet and in medicinal plants. The compound exerts anxiolytic activity when administered orally in mice, while no behavioural changes were observed upon intraperitoneal administration, or upon oral administration in gut sterilized animals. 4-Hydroxyphenylacetic acid (4-HPAA), which possesses anxiolytic effects when administered intraperitoneally, is a major intestinal metabolite of kaempferol. Pharmacokinetic properties of the compounds are currently not clear. UHPLC-MS/MS methods were validated to support pharmacokinetic studies of kaempferol and 4-HPAA in rats. Non-compartmental and compartmental analyses were performed. After intravenous administration, kaempferol followed a one-compartment model, with a rapid clearance (4.40-6.44l/h/kg) and an extremely short half-life of 2.93-3.79min. After oral gavage it was not possible to obtain full plasma concentration-time profiles of kaempferol. Pharmacokinetics of 4-HPAA was characterized by a two-compartment model, consisting of a quick distribution phase (half-life 3.04-6.20min) followed by a fast elimination phase (half-life 19.3-21.1min). Plasma exposure of kaempferol is limited by poor oral bioavailability and extensive metabolism. Both compounds are rapidly eliminated, so that effective concentrations at the site of action do not appear to be reached. At present, it is not clear how the anxiolytic-like effects reported for the compounds can be explained. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of Secondary Metabolites of Permafrost Bacillus sp. on Cytokine Synthesis by Human Peripheral Blood Mononuclear Cells.

PubMed

Kalenova, L F; Kolyvanova, S S; Bazhin, A S; Besedin, I M; Mel'nikov, V P

2017-06-01

We studied the effects of secondary metabolites of Bacillus sp. isolated from late Neogene permafrost on secretion of proinflammatory (TNF-α, IL-1β, IL-8, IL-2, and IFNγ) and antiinflammatory (IL-4 and IL-10) cytokines by human peripheral blood mononuclear cells. It was found that metabolites of Bacillus sp. produced more potent effect on cytokine secretion than mitogen phytohemagglutinin and metabolites of Bacillus cereus, medicinal strain IP5832. Activity of metabolites depended on the temperature of bacteria incubation. "Cold" metabolites of Bacillus sp. (isolated at -5°C) primarily induced Th1-mediated secretion of IFNγ, while "warm" metabolites (obtained at 37°C) induced Th2-mediated secretion of IL-4. The results suggest that Bacillus sp. metabolites are promising material for the development of immunomodulating drugs.

Impact of limited solvent capacity on metabolic rate, enzyme activities, and metabolite concentrations of S. cerevisiae glycolysis.

PubMed

Vazquez, Alexei; de Menezes, Marcio A; Barabási, Albert-László; Oltvai, Zoltan N

2008-10-01

The cell's cytoplasm is crowded by its various molecular components, resulting in a limited solvent capacity for the allocation of new proteins, thus constraining various cellular processes such as metabolism. Here we study the impact of the limited solvent capacity constraint on the metabolic rate, enzyme activities, and metabolite concentrations using a computational model of Saccharomyces cerevisiae glycolysis as a case study. We show that given the limited solvent capacity constraint, the optimal enzyme activities and the metabolite concentrations necessary to achieve a maximum rate of glycolysis are in agreement with their experimentally measured values. Furthermore, the predicted maximum glycolytic rate determined by the solvent capacity constraint is close to that measured in vivo. These results indicate that the limited solvent capacity is a relevant constraint acting on S. cerevisiae at physiological growth conditions, and that a full kinetic model together with the limited solvent capacity constraint can be used to predict both metabolite concentrations and enzyme activities in vivo.

Impact of Limited Solvent Capacity on Metabolic Rate, Enzyme Activities, and Metabolite Concentrations of S. cerevisiae Glycolysis

PubMed Central

Vazquez, Alexei; de Menezes, Marcio A.; Barabási, Albert-László; Oltvai, Zoltan N.

2008-01-01

The cell's cytoplasm is crowded by its various molecular components, resulting in a limited solvent capacity for the allocation of new proteins, thus constraining various cellular processes such as metabolism. Here we study the impact of the limited solvent capacity constraint on the metabolic rate, enzyme activities, and metabolite concentrations using a computational model of Saccharomyces cerevisiae glycolysis as a case study. We show that given the limited solvent capacity constraint, the optimal enzyme activities and the metabolite concentrations necessary to achieve a maximum rate of glycolysis are in agreement with their experimentally measured values. Furthermore, the predicted maximum glycolytic rate determined by the solvent capacity constraint is close to that measured in vivo. These results indicate that the limited solvent capacity is a relevant constraint acting on S. cerevisiae at physiological growth conditions, and that a full kinetic model together with the limited solvent capacity constraint can be used to predict both metabolite concentrations and enzyme activities in vivo. PMID:18846199

N-demethylation of N-methyl-4-aminoantipyrine, the main metabolite of metamizole.

PubMed

Bachmann, Fabio; Duthaler, Urs; Rudin, Deborah; Krähenbühl, Stephan; Haschke, Manuel

2018-05-08

Metamizole is an old analgesic used frequently in some countries. Active metabolites of metamizole are the non-enzymatically generated N-methyl-4-aminoantipyrine (4-MAA) and its demethylation product 4-aminoantipyrine (4-AA). Previous studies suggested that 4-MAA demethylation can be performed by hepatic cytochrome P450 (CYP) 3A4, but the possible contribution of other CYPs remains unclear. Using human liver microsomes (HLM), liver homogenate and HepaRG cells, we could confirm 4-MAA demethylation by CYPs. Based on CYP induction (HepaRG cells) and CYP inhibition (HLM) we could identify CYP2B6, 2C8, 2C9 and 3A4 as major contributors to 4-MAA demethylation. The 4-MAA demethylation rate by HLM was 280 pmol/mg protein/h, too low to account for in vivo 4-MAA demethylation in humans. Since peroxidases can perform N-demethylation, we investigated horseradish peroxidase and human myeloperoxidase (MPO). Horse radish peroxidase efficiently demethylated 4-MAA, depending on the hydrogen peroxide concentration. This was also true for MPO; this reaction was saturable with a K m of 22.5 μM and a maximal velocity of 14 nmol/min/mg protein. Calculation of the entire body MPO capacity revealed that the demethylation capacity by granulocyte/granulocyte precursors was approximately 600 times higher than the liver capacity and could account for 4-MAA demethylation in humans. 4-MAA demethylation could also be demonstrated in MPO-expressing granulocyte precursor cells (HL-60). In conclusion, 4-MAA can be demethylated in the liver by several CYPs, but hepatic metabolism cannot fully explain 4-MAA demethylation in humans. The current study suggests that the major part of 4-MAA is demethylated by circulating granulocytes and granulocyte precursors in bone marrow. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of Sulfated Metabolites of 4-Chlorobiphenyl (PCB3) in the Serum and Urine of Male Rats

PubMed Central

Dhakal, Kiran; He, Xianran; Lehmler, Hans-Joachim; Teesch, Lynn M.; Duffel, Michael W.; Robertson, Larry W.

2012-01-01

Polychlorinated biphenyls (PCBs) are legacy pollutants that exert toxicities through various mechanisms. In the recent years exposure to PCBs via inhalation has been recognized as a hazard. Those PCBs with lower numbers of chlorine atoms (LC-PCBs) are semi-volatile, and have been reported in the urban air, as well as in the indoor air of older buildings. LC-PCBs are bioactivated to phenols and further to quinone electrophiles with genotoxic/carcinogenic potential. We hypothesized that phenolic LC-PCBs are subject to conjugation and excretion in the urine. PCB3, often present in high concentrations in air, is a prototypical congener for the study of the metabolism and toxicity of LC-PCBs. Our objective was to identify metabolites of PCB3 in urine that could be potentially employed in the estimation of exposure to LC-PCBs. Male Sprague Dawley rats (150–175 g) were housed in metabolism cages and received a single intraperitoneal injection of 600 µmol/kg body weight of PCB3. Urine was collected every four hours; rats were euthanized at 36 h and serum was collected. LC-MS analysis of urine before and after incubation with β-glucuronidase and sulfatase showed that sulfate conjugates were in higher concentrations than glucuronide conjugates and free phenolic forms. At least two major metabolites, and two minor metabolites were identified in urine that could be attributed to mercapturic acid metabolites of PCB3. Quantitation by authentic standards confirmed that approximately 3% of the dose was excreted in the urine as sulfates over 36 hours; with peak excretion occurring at 10–20 h after exposure. The major metabolites were 4’PCB3 sulfate, 3’PCB3 sulfate, 2’PCB3 sulfate, and presumably a catechol sulfate. The serum concentration of 4’PCB3 sulfate was 6.18±2.16 µg/mL. This is the first report that sulfated metabolites of PCBs are formed in vivo. These findings suggest a prospective approach for exposure assessment of LC- PCBs by analysis of phase II

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens.

PubMed

Vinale, F; Marra, R; Scala, F; Ghisalberti, E L; Lorito, M; Sivasithamparam, K

2006-08-01

Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.

Antimicrobial activities of secondary metabolites and phylogenetic study of sponge endosymbiotic bacteria, Bacillus sp. at Agatti Island, Lakshadweep Archipelago.

PubMed

Mohan, Gopi; Thipparamalai Thangappanpillai, Ajith Kumar; Ramasamy, Balagurunathan

2016-09-01

Twenty-one species of sponges were recorded under the class of Demospongiae and Calcareous sponges of which 19 species were new to Agatti reef. A total of 113 Sponge endosymbiotic bacterial strains were isolated from twenty-one species of sponges and screened for antimicrobial activity. Five bacterial strains of sponge endosymbiotic bacteria (SEB) namely SEB32, SEB33, SEB36, SEB43 and SEB51 showed antimicrobial activity against virulent marine fish pathogens such as Vibrio alginolyticus , Vibrio vulnificus , Vibrio parahaemolyticus , Aeromonas salmonicida , Flavobacterium sp., Edwardsiella sp., Proteus mirabilis and Citrobacter brackii . The secondary metabolites produced by SEB32 from sponge Dysidea fragilis (Montagu, 1818) [48] was selected with broad range of antibacterial activity and subjected for production, characterization by series of chromatography techniques and spectroscopic methods. Based on the results of FT-IR and mass spectrometry, the active molecule was tentatively predicted as "Pyrrol" and the structure is Pyrrolo[1,2 -a ]pyrazine-1,4-dione, hexahydro- with molecular formula of C7H10N2O2. The LC 50 of active molecule was 31 μg/ml and molecular weight of the metabolites was 154. The potential strain SEB32 was identified by gene sequence (GenBank Accession number JX985748) and identified as Bacillus sp. from GenBank database.

The urinary metabolites of DINCH® have an impact on the activities of the human nuclear receptors ERα, ERβ, AR, PPARα and PPARγ.

PubMed

Engel, Anika; Buhrke, Thorsten; Kasper, Stefanie; Behr, Anne-Cathrin; Braeuning, Albert; Jessel, Sönke; Seidel, Albrecht; Völkel, Wolfgang; Lampen, Alfonso

2018-05-01

DINCH ® (di-isononyl cyclohexane-1,2-dicarboxylate) is a non-phthalate plasticizer that has been developed to replace phthalate plasticizers such as DEHP (di-2-ethylhexyl phthalate) or DINP (di-isononyl phthalate). DINCH ® is metabolized to its corresponding monoester and subsequently to oxidized monoester derivatives. These are conjugated to glucuronic acid and subject to urinary excretion. In contrast to DINCH ® , there are almost no toxicological data available regarding its primary and secondary metabolites. The present study aimed at the characterization of potential endocrine properties of DINCH ® and five DINCH ® metabolites by using reporter gene assays to monitor the activity of the human nuclear receptors ERα, ERβ, AR, PPARα and PPARγ in vitro. DINCH ® itself did not have any effect on the activity of these receptors whereas DINCH ® metabolites were shown to activate all these receptors. In the case of AR, DINCH ® metabolites predominantly enhanced dihydrotestosterone-stimulated AR activity. In the H295R steroidogenesis assay, neither DINCH ® nor any of its metabolites affected estradiol or testosterone synthesis. In conclusion, primary and secondary DINCH ® metabolites exert different effects at the molecular level compared to DINCH ® itself. All these in vitro effects of DINCH ® metabolites, however, were only observed at high concentrations such as 10 μM or above which is about three orders of magnitude above reported DINCH ® metabolite concentrations in human urine. Thus, the in vitro data do not support the notion that DINCH ® or any of the investigated metabolites may exert considerable endocrine effects in vivo at relevant human exposure levels. Copyright © 2018 Elsevier B.V. All rights reserved.

Mutagenic activity of austocystins - secondary metabolites of Aspergillus ustus

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

Kfir, R.; Johannsen, E.; Vleggaar, R.

1986-11-01

Mycotoxins constitute a group of toxic secondary fungal metabolites. Fungi that produce these toxins frequently contaminate food and feed, creating a potential threat to human and animal health. Biological activities of mycotoxins include, amongst others: toxicity, mutagenicity and carcinogenicity, which can be expressed with or without metabolic activation. Austocystins are similar in structure to aflatoxin B/sup 1/ and are probably synthesized in a similar manner. The Ames Salmonella test, a widely accepted method employed for the detection of mutagenic activity of various chemical compounds was used for testing the mutagenic activity of different mycotoxins. As aflatoxin B/sup 1/ was foundmore » by the Ames test to be highly mutagenic, the same test was applied for the study of possible mutagenicity of the austocystins. The mutagenic activity of these compounds was studied with and without metabolic activation using two tester strains of S. typhimurium, one capable of detecting frame shift mutation (strain TA98) and the other capable of detecting base pair substitution (strain TA100).« less

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

PubMed Central

Haque, Jamil A.; McDonald, Matthew G.; Kulman, John D.

2014-01-01

Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K–dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of γ-glutamyl carboxylation and its antagonism in live cells. We engineered a human embryonic kidney (HEK) 293–derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal γ-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular γ-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of γ-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo. PMID:24297869

LC-MS-MS analysis of 2,4-dinitrophenol and its phase I and II metabolites in a case of fatal poisoning.

PubMed

Politi, Lucia; Vignali, Claudia; Polettini, Aldo

2007-01-01

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis of biological fluids (blood, urine, gastric content, and bile) collected at autopsy in a case of suspected 2,4-dinitrophenol (DNP) fatal poisoning allowed the determination of DNP and its known metabolites (2-amino-4-nitrophenol and nitro-4-aminophenol). The tentative identification of three conjugated metabolites (DNP glucuronide, DNP sulfate, and 2-amino-4-nitrophenol glucuronide) could be made on the basis of their pseudomolecular ion, isotopic and fragmentation patterns, and retention characteristics. Another DNP metabolite reported in the literature, 2,4-diaminophenol, was not detected in the samples. Postmortem blood concentrations were 48.4 mg/L for DNP and 1.2 mg/L for 2-amino-4-nitrophenol. Gas chromatography-MS screening and quantification in postmortem blood revealed the presence of toxic concentrations of citalopram and its desmethylated metabolite (0.58 and 0.40 mg/L, respectively) and therapeutic or lower than therapeutic levels of olanzapine (0.04 mg/L), desalkylflurazepam (0.02 mg/L), and nordazepam (0.01 mg/L). Based on LC-MS-MS results and on available literature data on DNP poisonings, it was concluded that DNP poisoning played a contributing role, together with citalopram, in the cause of death.

Pharmacokinetics of isotretinoin and its major blood metabolite following a single oral dose to man.

PubMed

Colburn, W A; Vane, F M; Shorter, H J

1983-01-01

A pharmacokinetic profile of isotretinoin and its major dermatologically active blood metabolite, 4-oxo-isotretinoin, was developed following a single 80 mg oral suspension dose of isotretinoin to 15 normal male subjects. Blood samples were assayed for isotretinoin and 4-oxo-isotretinoin using a newly developed reverse-phase HPLC method. Following rapid absorption from the suspension formulation, isotretinoin is distributed and eliminated with harmonic mean half-lives of 1.3 and 17.4 h, respectively. Maximum concentrations of isotretinoin in blood were observed at 1 to 4 h after dosing. Maximum concentrations of the major blood metabolite of isotretinoin, 4-oxo-isotretinoin, are approximately one-half those of isotretinoin and occur at 6 to 16 h after isotretinoin dosing. The ratio of areas under the curve for metabolite and parent drug following the single dose suggests that average steady-state ratios of metabolite to parent drug during a dosing interval will be approximately 2.5. Both isotretinoin and its metabolite can be adequately described using a single linear pharmacokinetic model.

High-performance liquid chromatographic determination of loxoprofen and its diastereomeric alcohol metabolites in biological fluids by fluorescence labelling with 4-bromomethyl-6,7-methylenedioxycoumarin.

PubMed

Naganuma, H; Kawahara, Y

1990-09-14

A simple and sensitive high-performance liquid chromatographic procedure to determine loxoprofen and its diastereomeric alcohol metabolites in biological specimens is described. The analysis involves liquid-liquid extraction with benzene, pre-column derivatization with a highly fluorogenic reagent, 4-bromomethyl-6,7-methylenedioxycoumarin (BrMDC) and subsequent separation on a reversed-phase column. Loxoprofen, its pharmacologically active metabolite, trans-alcohol, and less active cis-alcohol were completely separated within 20 min with a mobile phase of 55% of aqueous acetonitrile containing acetic acid. Any endogenous substances do not interfere in the analysis of either plasma or urine samples. The quantitation limit was 0.01 micrograms/ml for human plasma and 0.05 micrograms/ml for urine. The method was applied to a pharmacokinetic study in healthy human subjects who had received 60 mg of loxoprofen sodium.

[The pharmacokinetics of the dipeptide analog of piracetam with nootropic activity GVS-111 and of its basic metabolites].

PubMed

Boĭko, S S; Zherdev, V P; Dvorianinov, A A; Gudasheva, T A; Ostrovskaia, R U; Voronina, T A; Rozantsev, G G; Seredenin, S B

1997-01-01

The pharmacokinetics of a new nootropic dipeptide analog of piracetam-N-phenylacetyl-L-prolylglycine (GWS-111) and its main metabolites were studied in rats by means of high performance liquid chromatography and gas-liquid chromatography. The compound under study showed a greater resistance to an enzymatic effect than natural neuropeptides. In addition to an unchanged compound three of its metabolites were found in the blood plasma of the rats. One of them, cyclo-Pro-Gly was an active metabolite of GWS-111.

Microbial secondary metabolites in homes in association with moisture damage and asthma.

PubMed

Kirjavainen, P V; Täubel, M; Karvonen, A M; Sulyok, M; Tiittanen, P; Krska, R; Hyvärinen, A; Pekkanen, J

2016-06-01

We aimed to characterize the presence of microbial secondary metabolites in homes and their association with moisture damage, mold, and asthma development. Living room floor dust was analyzed by LC-MS/MS for 333 secondary metabolites from 93 homes of 1-year-old children. Moisture damage was present in 15 living rooms. At 6 years, 8 children had active and 15 lifetime doctor-diagnosed asthma. The median number of different metabolites per house was 17 (range 8-29) and median sum load 65 (4-865) ng/m(2) . Overall 42 different metabolites were detected. The number of metabolites present tended to be higher in homes with mold odor or moisture damage. The higher sum loads and number of metabolites with loads over 10 ng/m(2) were associated with lower prevalence of active asthma at 6 years (aOR 0.06 (95% CI <0.001-0.96) and 0.05 (<0.001-0.56), respectively). None of the individual metabolites, which presence tended (P < 0.2) to be increased by moisture damage or mold, were associated with increased risk of asthma. Microbial secondary metabolites are ubiquitously present in home floor dust. Moisture damage and mold tend to increase their numbers and amount. There was no evidence indicating that the secondary metabolites determined would explain the association between moisture damage, mold, and the development of asthma. © 2015 The Authors. Indoor Air published by John Wiley & Sons Ltd.

Structural characterization of a therapeutic anti-methamphetamine antibody fragment: oligomerization and binding of active metabolites.

PubMed

Peterson, Eric C; Celikel, Reha; Gokulan, Kuppan; Varughese, Kottayil I

2013-01-01

Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, K(D) = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or "ecstasy"). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni(2+). Two of the histidine residues of each C-terminal His-tag interact with Ni(2+) in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy.

Influence of the urine flow rate on some caffeine metabolite ratios used to assess CYP1A2 activity.

PubMed

Sinués, Blanca; Fanlo, Ana; Bernal, María Luisa; Mayayo, Esteban; Soriano, María Antonia; Martínez-Ballarin, Enrique

2002-12-01

Five established metabolite ratios (MRs) to measure P450 CYP1A2 activity--MR1 (17X + 17U)/137X, MR2 (AFMU + 1X + 1U)/17U, MR3 (17X/137X), MR4 (AFMU + 1X + 1U + 17X + 17U)/137X, and MR5 (AFMU + 1X + 1U)/17X--were calculated in urine 4-5 hours after caffeine intake. First, to assess the potential of omeprazole to induce CYP1A2 activity, a caffeine test was performed in 27 subjects on two occasions: before and after 14 days on omeprazole (20 mg/day). Samples of urine were analyzed by high-performance liquid chromatography (HPLC) to quantify caffeine and metabolites used to calculate the different caffeine MRs. MR1, MR3, and MR4 were enhanced after treatment; the percentage of change was inversely associated with that of the urine flow, with r values of -0.48, -0.49, and -0.47, respectively. However, MR2 or MR5 were not modified. To determine the reason for these contradictory results, the authors analyzed data of metabolites, ratios, and their components (numerators and denominators) from 152 subjects (who underwent one caffeine test) and their relationship with the urinary flow. Caffeine concentration in urine was the only compound nondependent on the urine flow. Consistently, ratios containing caffeine (MR1, MR3, and MR4) were highly influenced by the rate of urine excretion, since the flow dependence of their numerators is not canceled out by that of caffeine in their denominators. The dependency of the caffeine excretion on renal factors may explain the opposite results found with the different ratios in the aforementioned prospective study of drug interaction, the absence of closer correlations of the five MRs to each other, the discrepancies about the type of frequency distribution of the different MRs (either normal or multimodal), and the higher sensitivity of MR2 to detect gender differences in CYP1A2 activity found in this study. In summary, the data clearly emphasize the need for a strict control of the liquid intake to avoid high urine flows when MRs

6,7,4'-Trihydroxyisoflavone, a major metabolite of daidzein, improves learning and memory via the cholinergic system and the p-CREB/BDNF signaling pathway in mice.

PubMed

Ko, Yong-Hyun; Kim, Sun Yeou; Lee, Seok-Yong; Jang, Choon-Gon

2018-05-05

Daidzein is one of the major isoflavfones found in soy food and plants. Following ingestion, daidzein is readily converted to hydroxylated metabolites in the human body. 6,7,4'-Trihydroxyisoflavone (THIF), one of the metabolites of daidzein, has several pharmacological activities, including anti-cancer and anti-obesity properties. However, no reports exist on the effects of 6,7,4'-THIF for cognitive function in mice. The present study aimed to investigate the effects of 6,7,4'-THIF against scopolamine-induced learning and memory impairments using the Y-maze and passive avoidance test. A single administration of 6,7,4'-THIF significantly improved scopolamine-induced cognitive dysfunction in these in vivo tests. Moreover, treatment with 6,7,4'-THIF alone enhanced learning and memory performance in the same behavioral tests. Molecular studies showed that 6,7,4'-THIF significantly inhibited acetylcholinesterase and thiobarbituric acid reactive substance (TBARS) activities in the hippocampus of scopolamine-induced mice. In addition, immunohistochemistry and Western blot results revealed that 6,7,4'-THIF significantly increased brain-derived neurotrophic factor (BDNF) and phosphor cAMP response element binding (CREB) in the hippocampus of mice. Taken together, these findings suggest that 6,7,4'-THIF improves cognitive dysfunction induced by scopolamine and enhances learning and memory by activation of the cholinergic system and the p-CREB/BDNF signaling pathway in mice. Copyright © 2018 Elsevier B.V. All rights reserved.

Antimicrobial and antiprotozoal activities of secondary metabolites from the fungus Eurotium repens

PubMed Central

Gao, Jiangtao; Radwan, Mohamed M.; León, Francisco; Wang, Xiaoning; Jacob, Melissa R.; Tekwani, Babu L.; Khan, Shabana I.; Lupien, Shari; Hill, Robert A.; Dugan, Frank M.; Cutler, Horace G.

2011-01-01

In this study, we examined in vitro antibacterial, antifungal, antimalarial, and antileishmanial activities of secondary metabolites (1–8) isolated from the fungus Eurotium repens. All compounds showed mild to moderate antibacterial or antifungal or both activities except 7. The activity of compound 6 was the best of the group tested. The in vitro antimalarial evaluation of these compounds revealed that compounds 1–3, 5, and 6 showed antimalarial activities against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum with IC50 values in the range of 1.1–3.0 μg/ml without showing any cytotoxicity to the mammalian cells. Compound 5 displayed the highest antimalarial activity. Antileishmanial activity against Leishmania donovani promastigotes was observed for compounds 1–6 with IC50 values ranging from 6.2 to 23 μg/ml. Antileishmanial activity of compounds 5 and 6 (IC50 values of 7.5 and 6.2 μg/ml, respectively) was more potent than 1–4 (IC50 values ranging from 19–23 μg/ml). Compounds 7 and 8 did not show any antiprotozoal effect. Preliminary structure and activity relationship studies indicated that antibacterial, antifungal, antimalarial, and antileishmanial activities associated with phenol derivates (1–6) seem to be dependent on the number of double bonds in the side chain, which would be important for lead optimization in the future. PMID:23024574

Antiestrogens and antiestrogen metabolites: preparation of tritium-labeled (+-)-cis-3-(p-(1,2,3,4-tetrahydro-6-methoxy-2-phenyl-1-naphthyl)-phenoxyl)-1,2-propanediol (U23469) and characterization and synthesis of a biologically important metabolite

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

Tatee, T.; Carlson, K.E.; Katzenellenbogen, J.A.

1979-12-01

The Upjohn antiestrogen (+-)-cis-3-(p-(1,2,3,4-tetrahydro-6-methoxy-2-phenyl-1-naphthyl)-phenoxyl)-1,2-propanediol (2b, U 23469) has been prepared in tritium-labeled form by reduction of an unsaturated dihydronaphthalene precursor with carrier-free tritium gas over a palladium catalyst followed by alkylation with 3-iodo-1,2-propanediol. After extensive chromatographic purification, the final material was obtained with a specific activity of 13 Ci/mmol and a radiochemical purity of 94%. In vivo studies with immature rats show that (3H)2b is slowly converted to a more polar metabolite that is selectively accumulated in the nuclear fraction of the uterus where it is bound to the estrogen receptor. Chromatographic comparisons indicate that this metabolite is the demethylatedmore » analogue 2c, a compound that has an affinity for estrogen receptor more than 300 times greater than that of 2b. These studies suggest that the demethylated analogue 2c may be a biologically important metabolite of 2b that is involved in the action of this antiestrogen.« less

Regulatory requirements for genotoxicity assessment of plant protection product active ingredients, impurities, and metabolites.

PubMed

Booth, Ewan D; Rawlinson, Paul J; Maria Fagundes, Priscila; Leiner, Kevin A

2017-06-01

Active ingredients in plant protection products are subject to rigorous safety assessment during their development, including assessment of genotoxicity. Plant protection products are used for agriculture in multiple regions and for the registration of active ingredients it is necessary to satisfy the data requirements of these different regions. There are no overarching global agreements on which genotoxicity studies need to be conducted to satisfy the majority of regulatory authorities. The implementation of new OECD guidelines for the in vitro micronucleus, transgenic rodent somatic and germ cell gene mutation and in vivo comet assays, as well as the revision of a number of other OECD test guidelines has resulted in some changes to data requirements. This review describes the genotoxicity data requirements for chemical active ingredients as well as biologicals, microbials, ground water metabolites, metabolites, and impurities in a number of regions. Similarities and differences are highlighted. Environ. Mol. Mutagen. 58:325-344, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Isolated and mixed effects of diuron and its metabolites on biotransformation enzymes and oxidative stress response of Nile tilapia (Oreochromis niloticus).

PubMed

Felício, Andréia Arantes; Freitas, Juliane Silberschmidt; Scarin, Jéssica Bolpeti; de Souza Ondei, Luciana; Teresa, Fabrício Barreto; Schlenk, Daniel; de Almeida, Eduardo Alves

2018-03-01

Diuron is one of the most used herbicide in the world, and its field application has been particularly increased in Brazil due to the expansion of sugarcane crops. Diuron has often been detected in freshwater ecosystems and it can be biodegraded into three main metabolites in the environment, the 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 3,4-dichlorophenyl-N-methylurea (DCPMU). Negative effects under aquatic biota are still not well established for diuron, especially when considering its presence in mixture with its different metabolites. In this study, we evaluated the effects of diuron alone or in combination with its metabolites, DCPMU, DCPU and 3,4-DCA on biochemical stress responses and biotransformation activity of the fish Oreochromis niloticus. Results showed that diuron and its metabolites caused significant but dispersed alterations in oxidative stress markers and biotransformation enzymes, except for ethoxyresorufin-O-deethylase (EROD) activity, that presented a dose-dependent increase after exposure to either diuron or its metabolites. Glutathione S-transferase (GST) activity was significant lower in gills after exposure to diuron metabolites, but not diuron. Diuron, DCPMU and DCA also decreased the multixenobiotic resistance (MXR) activity. Lipid peroxidation levels were increased in gill after exposure to all compounds, indicating that the original compound and diuron metabolites can induce oxidative stress in fish. The integration of all biochemical responses by the Integrated Biomarker Response (IBR) model indicated that all compounds caused significant alterations in O. niloticus, but DCPMU caused the higher alterations in both liver and gill. Our findings imply that diuron and its metabolites may impair the physiological response related to biotransformation and antioxidant activity in fish at field concentrations. Such alterations could interfere with the ability of aquatic animals to adapt to environments contaminated by

Microwave-assisted rapid synthesis of methyl 2,4,5-trimethoxyphenylpropionate, a metabolite of Cordia alliodora.

PubMed

Sinha, A K; Joshi, B P; Sharma, A; Kumar, J K; Kaul, V K

2003-12-01

Microwave assisted condensation of asaronaldehyde (2) with malonic acid in piperidine-AcOH provides 2,4,5-trimethoxycinnamic acid (3) in 87% yield within 4 min, which upon further reduction with PdCl2- HCOOH-aq. NaOH gives 3-(2,4,5-trimethoxy)phenyl propionic acid (4) in 88% yield within 3 min. Esterification of 4 with MeOH-H+ gives methyl 2,4,5-trimethoxyphenylpropionate (1), a metabolite of Cordia alliodora, in 94% yield within 3 min (overall 69% yield).

Comparison of pharmacokinetics of loxoprofen and its active metabolites after an intravenous, intramuscular, and oral administration of loxoprofen in rats: evidence for extrahepatic metabolism.

PubMed

Koo, Tae-Sung; Kim, Dae-Hyun; Ahn, Sung-Hoon; Kim, Kang-Pil; Kim, In-Wha; Seo, Seung-Yong; Suh, Young-Ger; Kim, Dae-Duk; Shim, Chang-Koo; Chung, Suk-Jae

2005-10-01

The objective of this study was to characterize the extent of the formation of the active (trans-alcohol form) and inactive (cis-alcohol) metabolites of loxoprofen and to compare the kinetics after its intragastric, intravenous, and intramuscular administrations in rats. After intravenous administration of the drug at doses of 5-20 mg/kg, the clearance and the volume of distribution for loxoprofen, and the ratios of the AUC for the metabolites to the parent drug were not statistically different with the dosage; the formation clearances were 1.08 and 0.87 mL/min/kg for the active and its isomeric metabolite, respectively. After the intragastric, intravenous, or intramuscular administration, AUC for loxoprofen and the metabolites at a dose of 10 mg/kg were not statistically different for the different routes of administration. The formation of the metabolites with the concomitant loss of loxoprofen was found in incubations with liver homogenates and blood but not with a muscle homogenate or plasma, indicating that the conversion of loxoprofen to the metabolites may occur both in the liver and extraheptic tissue(s). Thus, approximately 22% of the loxoprofen may have been converted to the active metabolite in the liver and the extraheptic tissue(s) and the pharmacokinetics of the active metabolite was independent of the route of administration. Copyright (c) 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Use of High-Pressure Liquid Chromatography to Determine Plasma Levels of Metronidazole and Metabolites After Intravenous Administration

PubMed Central

Wheeler, L. A.; De Meo, M.; Halula, M.; George, L.; Heseltine, P.

1978-01-01

A rapid and sensitive high-pressure liquid chromatography assay for metronidazole and its two principle metabolites, 1-(2-hydroxyethyl-2-hydroxymethyl)-5-nitro-imidazole [hydroxy metabolite] and 1-acetic acid-2-methyl-5-metronidazole [acid metabolite], was developed. The retention times observed were 5.7, 3.3, and 4.5 min, respectively. A reverse-phase μC18 Bondapak column using a solvent system of methanol, acetonitrile, and 0.005 M pH 4 potassium dihydrogen phosphate (4:3:93, vol/vol) was used to achieve separation of the three compounds. Patients receiving metronidazole therapy were given a loading dose of 13.6 mg of drug per kg intravenously over 1 h, followed by a maintenance dose of 1.43 mg/kg per h. The range of metronidazole concentrations observed was 6.8 to 47.5 μg/ml. These levels are well above the minimal inhibitory concentrations of most clinically significant anaerobic bacteria including Bacteroides fragilis. Little of the acid metabolite was observed in the plasma. The concentration of hydroxy metabolite ranged from 1.6 to 16 μg/ml. The latter may represent an additional source of antimicrobial activity since the hydroxy metabolite has approximately 30% the biological activity of metronidazole. PMID:646342

Characterization of macromolecular baseline of human brain using metabolite cycled semi-LASER at 9.4T.

PubMed

Giapitzakis, Ioannis-Angelos; Avdievich, Nikolai; Henning, Anke

2018-08-01

Macromolecular resonances (MM) arise mainly from cytosolic proteins and overlap with metabolites, influencing metabolite quantification. Macromolecules can serve as valuable biomarkers for diseases and pathologies. The objectives of this study were to characterize MM at 9.4T in the human brain (occipital and left parietal lobe) and to describe the RF coil setup used for MM acquisition in the two regions. An adiabatic inversion pulse was optimised for metabolite nulling at 9.4T using double inversion recovery and was combined for the first time with metabolite cycled (MC) semi-LASER and appropriate coil configuration. MM spectra (seven volunteers) from two brain locations were averaged and smoothed creating MM templates, which were then parametrized using simulated Voigt-shaped lines within LCModel. Quantification was performed on individual data sets, including corrections for different tissue composition and the T 1 and T 2 relaxation of water. Our coil configuration method resulted in efficient B1+ (>30 T/√kW) for both brain regions. The 15 MM components were detected and quantified in MM baselines of the two brain areas. No significant differences in concentration levels of MM between different regions were found. Two new MM peaks were reported (M7 & M8). Double inversion, which was combined with MC semi-LASER, enabled the acquisition of high spectral resolution MM spectra for both brain regions at 9.4T. The 15 MM components were detected and quantified. Two new MM peaks were reported for the first time (M7 & M8) and preliminarily assigned to β-methylene protons of aspartyl-groups. Magn Reson Med 80:462-473, 2018. © 2018 International Society for Magnetic Resonance in Medicine. © 2018 International Society for Magnetic Resonance in Medicine.

A validated HPLC-MS/MS assay for quantifying unstable pharmacologically active metabolites of clopidogrel in human plasma: application to a clinical pharmacokinetic study.

PubMed

Furlong, Michael T; Savant, Ishani; Yuan, Moucun; Scott, Laura; Mylott, William; Mariannino, Thomas; Kadiyala, Pathanjali; Roongta, Vikram; Arnold, Mark E

2013-05-01

Clopidogrel is prescribed for the treatment of Acute Coronary Syndrome and recent myocardial infarction, recent stroke, or established peripheral arterial disease. A sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed and validated to enable reliable quantification of four diastereomeric and chemically reactive thiol metabolites, two of which are pharmacologically active, in human plasma. The metabolites were stabilized by alkylation of their reactive thiol moieties with 2-bromo-3'-methoxyacetophenone (MPB). Following organic solvent mediated-protein precipitation in a 96-well plate format, chromatographic separation was achieved by gradient elution on an Ascentis Express RP-amide column. Chromatographic conditions were optimized to ensure separation of the four derivatized active metabolites. Derivatized metabolites and stable isotope-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The HPLC-MS/MS assay was validated over concentration ranges of 0.125-125 ng/mL for metabolites H1-H3 and 0.101-101 ng/mL for H4. Intra- and inter-assay precision values for replicate quality control samples were within 14.3% for all analytes during the assay validation. Mean quality control accuracy values were within ±6.3% of nominal values for all analytes. Assay recoveries were high (>79%). The four derivatized analytes were stable in human blood for at least 2 h at room temperature and on ice. The analytes were also stable in human plasma for at least 25 h at room temperature, 372 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of all four thiol metabolites in human plasma in support of a human pharmacokinetic study. Copyright © 2013 Elsevier B.V. All rights reserved.

Hydroquinone, a reactive metabolite of benzene, enhances interleukin-4 production in CD4+ T cells and increases immunoglobulin E levels in antigen-primed mice

PubMed Central

Lee, M H; Chung, S W; Kang, B Y; Kim, K-M; Kim, T S

2002-01-01

Exposure to cigarette smoke is known to increase the risk of the development of allergic disease. The mechanism is not well understood. In this study, we determined the effect of hydroquinone (HQ), a major metabolite of benzene present in large quantities in cigarette tar, on interleukin-4 (IL-4) production by CD4+ T cells. HQ significantly enhanced IL-4 production by keyhole limpet haemocyanin (KLH)-primed CD4+ T cells in a dose-dependent manner. The enhancing effect of HQ on IL-4 production was maximal at a concentration of 50 µm. It increased the level of IL-4 production approximately 10-fold. HQ enhanced IL-4 mRNA expression and also IL-4 gene promoter activity, suggesting that the enhancing effect of HQ on IL-4 production may occur at the transcriptional level. Furthermore, the injection of KLH-primed mice with HQ resulted in a significant increase in the levels of IL-4 and immunoglobulin E. These findings provide evidence that HQ, a major component of cigarette tar, may enhance allergic immune responses by inducing the production of IL-4 in CD4+ T cells. PMID:12153512

Anti-Adhesive Activity of Cranberry Phenolic Compounds and Their Microbial-Derived Metabolites against Uropathogenic Escherichia coli in Bladder Epithelial Cell Cultures.

PubMed

de Llano, Dolores González; Esteban-Fernández, Adelaida; Sánchez-Patán, Fernando; Martínlvarez, Pedro J; Moreno-Arribas, Maria Victoria; Bartolomé, Begoña

2015-05-27

Cranberry consumption has shown prophylactic effects against urinary tract infections (UTI), although the mechanisms involved are not completely understood. In this paper, cranberry phenolic compounds and their potential microbial-derived metabolites (such as simple phenols and benzoic, phenylacetic and phenylpropionic acids) were tested for their capacity to inhibit the adherence of uropathogenic Escherichia coli (UPEC) ATCC®53503™ to T24 epithelial bladder cells. Catechol, benzoic acid, vanillic acid, phenylacetic acid and 3,4-dihydroxyphenylacetic acid showed anti-adhesive activity against UPEC in a concentration-dependent manner from 100-500 µM, whereas procyanidin A2, widely reported as an inhibitor of UPEC adherence on uroepithelium, was only statistically significant (p < 0.05) at 500 µM (51.3% inhibition). The results proved for the first time the anti-adhesive activity of some cranberry-derived phenolic metabolites against UPEC in vitro, suggesting that their presence in the urine could reduce bacterial colonization and progression of UTI.

Structural Characterization of a Therapeutic Anti-Methamphetamine Antibody Fragment: Oligomerization and Binding of Active Metabolites

PubMed Central

Gokulan, Kuppan; Varughese, Kottayil I.

2013-01-01

Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, KD = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or “ecstasy”). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni2+. Two of the histidine residues of each C-terminal His-tag interact with Ni2+ in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy. PMID:24349338

Fungal secondary metabolites - strategies to activate silent gene clusters.

PubMed

Brakhage, Axel A; Schroeckh, Volker

2011-01-01

Filamentous fungi produce a multitude of low molecular weight bioactive compounds. The increasing number of fungal genome sequences impressively demonstrated that their biosynthetic potential is far from being exploited. In fungi, the genes required for the biosynthesis of a secondary metabolite are clustered. Many of these bioinformatically newly discovered secondary metabolism gene clusters are silent under standard laboratory conditions. Consequently, no product can be found. This review summarizes the current strategies that have been successfully applied during the last years to activate these silent gene clusters in filamentous fungi, especially in the genus Aspergillus. The techniques take advantage of genome mining, vary from the simple search for compounds with bioinformatically predicted physicochemical properties up to methods that exploit a probable interaction of microorganisms. Until now, the majority of successful approaches have been based on molecular biology like the generation of gene "knock outs", promoter exchange, overexpression of transcription factors or other pleiotropic regulators. Moreover, strategies based on epigenetics opened a new avenue for the elucidation of the regulation of secondary metabolite formation and will certainly continue to play a significant role for the elucidation of cryptic natural products. The conditions under which a given gene cluster is naturally expressed are largely unknown. One technique is to attempt to simulate the natural habitat by co-cultivation of microorganisms from the same ecosystem. This has already led to the activation of silent gene clusters and the identification of novel compounds in Aspergillus nidulans. These simulation strategies will help discover new natural products in the future, and may also provide fundamental new insights into microbial communication. Copyright © 2010 Elsevier Inc. All rights reserved.

Testosterone metabolism revisited: discovery of new metabolites.

PubMed

Pozo, Oscar J; Marcos, Josep; Ventura, Rosa; Fabregat, Andreu; Segura, Jordi

2010-10-01

The metabolism of testosterone is revisited. Four previously unreported metabolites were detected in urine after hydrolysis with KOH using a liquid chromatography-tandem mass spectrometry method and precursor ion scan mode. The metabolites were characterized by a product ion scan obtained with accurate mass measurements. Androsta-4,6-dien-3,17-dione, androsta-1,4-dien-3,17-dione, 17-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione were proposed as feasible structures for these metabolites on the basis of the mass spectrometry data. The proposed structures were confirmed by analysis of synthetic reference compounds. Only 15-androsten-3,17-dione could not be confirmed, owing to the lack of a commercially available standard. That all four compounds are testosterone metabolites was confirmed by the qualitative analysis of several urine samples collected before and after administration of testosterone undecanoate. The metabolite androsta-1,4-dien-3,17-dione has a structure analogous to that of the exogenous anabolic steroid boldenone. Specific transitions for boldenone and its metabolite 17β-hydroxy-5β-androst-1-en-3-one were also monitored. Both compounds were also detected after KOH treatment, suggesting that this metabolic pathway is involved in the endogenous detection of boldenone previously reported by several authors.

Secondary metabolites from the unique bamboo, Melocanna baccifera.

PubMed

Govindan, Balaji; Johnson, Anil John; Viswanathan, Gayathri; Ramaswamy, Venkataraman; Koshy, Konnath Chacko; Baby, Sabulal

2018-02-15

Phytochemistry of fruits and leaves of the unique bamboo Melocanna baccifera resulted in the isolation of 27 secondary metabolites, including 4-Oxabicyclo[3.2.2]nona-1(7),5,8-triene and Verbacine. Biological activity studies of Verbacine revealed it as an inhibitor of acetylcholinesterase and as cytotoxic against C6 cancer cells.

Identification of phase-I metabolites and chronic toxicity study of the Kv1.3 blocker PAP-1 (5-(4-phenoxybutoxy)psoralen) in the rat.

PubMed

Hao, B; Chen, Z-W; Zhou, X-J; Zimin, P I; Miljanich, G P; Wulff, H; Wang, Y-X

2011-03-01

1. PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in preclinical development for psoriasis. This study was undertaken to identify the major phase I metabolites of PAP-1 in Sprague-Dawley (SD) rats. 2. Five phase I metabolites, that is 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)butoxy]psoralen (M3), 5-[4-(3-hydroxyphenoxy)butoxy]psoralen (M4), and 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated from the bile of rats and identified by mass spectrometry and NMR spectroscopy. The last four metabolites are new compounds. 3. Incubation of PAP-1 with SD rat liver microsomes rendered the same five major metabolites in a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner suggesting that cytochrome P450 (CYP) enzymes are involved in PAP-1 metabolism. Inhibitors of rat CYP1A1/2 (alpha-naphthoflavone) and CYP3A (ketoconazole) but not CYP2D6 (quinidine), CYP2E (diethyldithiocarbamate), or CYP2C9 (sulphaphenazole) blocked the metabolism of PAP-1 in rat microsomes. 4. Of the five metabolites M3, M4, and M5 were found to inhibit Kv1.3 currents with nanomolar IC50s, while M1 and M2 were inactive. Our results identified the Kv1.3-inactive M1 as the major phase I metabolite, and suggest that hydroxylation and O-dealkylation are the major pathways of PAP-1 metabolism. 5. We further conducted a 6-month repeat-dose toxicity study with PAP-1 at 50 mg/kg in both male and female Lewis rats and did not observe any toxic effects.

Blockade of rat alpha3beta4 nicotinic receptor function by methadone, its metabolites, and structural analogs.

PubMed

Xiao, Y; Smith, R D; Caruso, F S; Kellar, K J

2001-10-01

The opioid agonist properties of (+/-)-methadone are ascribed almost entirely to the (-)-methadone enantiomer. To extend our knowledge of the pharmacological actions of methadone at ligand-gated ion channels, we investigated the effects of the two enantiomers of methadone and its metabolites R-(+)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium perchlorate (EDDP) and R-(+)-2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline hydrochloride (EMDP), as well as structural analogs of methadone, including (-)-alpha-acetylmethadol hydrochloride (LAAM) and (+)-alpha-propoxyphene, on rat alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs) stably expressed in a human embryonic kidney 293 cell line, designated KXalpha3beta4R2. (+/-)-methadone inhibited nicotine-stimulated 86Rb+ efflux from the cells in a concentration-dependent manner with an IC50 value of 1.9 +/- 0.2 microM, indicating that it is a potent nAChR antagonist. The (-)- and (+)-enantiomers of methadone have similar inhibitory potencies on nicotine-stimulated 86Rb+ efflux, with IC50 values of approximately 2 microM. EDDP, the major metabolite of methadone, is even more potent, with an IC50 value of approximately 0.5 microM, making it one of the most potent nicotinic receptor blockers reported. In the presence of (+/-)-methadone, EDDP, or LAAM, the maximum nicotine-stimulated 86Rb+ efflux was markedly decreased, but the EC50 value for nicotine stimulation was altered only slightly, if at all, indicating that these compounds block alpha3beta4 nicotinic receptor function by a noncompetitive mechanism. Consistent with a noncompetitive mechanism, (+/-)-methadone, its metabolites, and structural analogs have very low affinity for nicotinic receptor agonist binding sites in membrane homogenates from KXalpha3beta4R2 cells. We conclude that both enantiomers of methadone and its metabolites as well as LAAM and (+)-alpha-propoxyphene are potent noncompetitive antagonists of alpha3beta4 nAChRs.

Detection of metabolites of the new synthetic cannabinoid CUMYL-4CN-BINACA in authentic urine samples and human liver microsomes using high-resolution mass spectrometry.

PubMed

Öztürk, Yeter Erol; Yeter, Oya; Öztürk, Serkan; Karakus, Goksun; Ates, Ismail; Buyuk, Yalçın; Yurdun, Turkan

2018-03-01

CUMYL-4CN-BINACA(1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide) is a recently introduced indazole-3-carboxamide-type synthetic cannabinoid (SC) that was detected in herbal incense seized by of the Council of Forensic Medicine, Istanbul Narcotics Department, in May 2016 in Turkey. Recently introduced SCs are not detected in routine toxicological analysis; therefore, analytical methods to measure these compounds are in demand. The present study aims to identify urinary marker metabolites of CUMYL-4CN-BINACA by investigating its metabolism in human liver microsomes and to confirm the results in authentic urine samples (n = 80). In this study, 5 μM CUMYL-4CN-BINACA was incubated with human liver microsomes (HLMs) for up to 3 hours, and metabolites were identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Less than 21% of the CUMYL-4CN-BINACA parent compound remained after 3 hours of incubation. We identified 18 metabolites that were formed via monohydroxylation, dealkylation, oxidative decyanation to aldehyde, alcohol, and carboxylic acid formation, glucuronidation or reaction combinations. CUMYL-4CN-BINACA N-butanoic acid (M16) was found to be major metabolite in HLMs. In urine samples CUMYL-4CN-BINACA was not detected; CUMYL-4CN-BINACA N-butanoic acid (M16) was major metabolite after β-glucuronidase hydrolysis. Based on these findings, we recommend using M16 (CUMYL-4CN-BINACA N-butanoic acid), M8 and M11 (hydroxylcumyl CUMYL-4CN-BINACA) as urinary marker metabolites to confirm CUMYL-4CN-BINACA intake. Copyright © 2017 John Wiley & Sons, Ltd.

Biological responses of progestogen metabolites in normal and cancerous human breast.

PubMed

Pasqualini, Jorge R; Chetrite, Gérard S

2010-12-01

At present, more than 200 progestogen molecules are available, but their biological response is a function of various factors: affinity to progesterone or other receptors, their structure, the target tissues considered, biological response, experimental conditions, dose, method of administration and metabolic transformations. Metabolic transformation is of huge importance because in various biological processes the metabolic product(s) not only control the activity of the maternal hormone but also have an important activity of its own. In this regard, it was observed that the 20-dihydro derivative of the progestogen dydrogesterone (Duphaston®) is significantly more active than the parent compound in inhibiting sulfatase and 17β-hydroxysteroid dehydrogenase in human breast cancer cells. Estrone sulfatase activity is also inhibited by norelgestromin, a norgestimate metabolite. Interesting information was obtained with a similar progestogen, tibolone, which is rapidly metabolized into the active 3α/3β-hydroxy and 4-ene metabolites. All these metabolites can inhibit sulfatase and 17β-hydroxysteroid dehydrogenase and stimulate sulfotransferase in human breast cancer cells. Another attractive aspect is the metabolic transformation of progesterone itself in human breast tissues. In the normal breast progesterone is mainly converted to 4-ene derivatives, whereas in the tumor tissue it is converted mostly to 5α-pregnane derivatives. 20α-Dihydroprogesterone is found mainly in normal breast tissue and possesses antiproliferative properties as well as the ability to act as an anti-aromatase agent. Consequently, this progesterone metabolite could be involved in the control of estradiol production in the normal breast and therefore implicated in one of the multifactorial mechanisms of the breast carcinogenesis process. In conclusion, a better understanding of both natural and synthetic hormone metabolic transformations and their control could potentially provide

Neuropharmacological and neuroprotective activities of some metabolites produced by cell suspension culture of Waltheria americana Linn.

PubMed

Mundo, Jorge; Villeda-Hernández, Juana; Herrera-Ruiz, Maribel; Gutiérrez, María Del Carmen; Arellano-García, Jesús; León-Rivera, Ismael; Perea-Arango, Irene

2017-10-01

Waltheria americana is a plant used in Mexican traditional medicine to treat some nervous system disorders. The aims of the present study were to isolate and determine the neuropharmacological and neurprotective activities of metabolites produced by a cell suspension culture of Waltheria americana. Submerged cultivation of W. americana cells provided biomass. A methanol-soluble extract (WAsc) was obtained from biomass. WAsc was fractionated yielding the chromatographic fractions 4WAsc-H 2 O and WAsc-CH 2 Cl 2 . For the determination of anticonvulsant activity in vivo, seizures were induced in mice by pentylenetetrazol (PTZ). Neuropharmacological activities (release of gamma amino butyric acid (GABA) and neuroprotection) of chromatographic fractions were determined by in vitro histological analysis of brain sections of mice post mortem. Fraction 4WAsc-H 2 O (containing saccharides) did not produce neuronal damage, neurodegeneration, interstitial tissue edema, astrocytic activation, nor cell death. Pretreatment of animals with 4WAsc-H 2 O and WAsc-CH 2 Cl 2 from W. americana cell suspensions induced an increase in: GABA release, seizure latency, survival time, neuroprotection, and a decrease in the degree of severity of tonic/tonic-clonic convulsions, preventing PTZ-induced death of up to 100% of animals of study. Bioactive compounds produced in suspension cell culture of W. americana produce neuroprotective and neuropharmacological activities associated with the GABAergic neurotransmission system. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Tandem mass spectrometric analysis of cyclophosphamide, ifosfamide and their metabolites.

PubMed

Liu, Zhongfa; Chan, Kenneth K; Wang, Jeffrey J

2005-01-01

A detailed multi-stage (MSn) fragmentation study of cyclophosphamide (CP), ifosfamide (IF) and their major metabolites, using an ion-trap mass spectrometer and a Q-TOF mass spectrometer, was performed with the aid of specifically deuterium-labeled analogs. The analytes showed good responses in positive-ion electrospray mass spectrometry as [MH]+ ions. Tandem mass spectra revealed a wealth of structurally specific ions, allowing characterization of the fragmentation pathways of these analytes. The major fragmentation pathways of the protonated CP and IF are elimination of ethylene from C5 and C6 of 1,3,2-oxazaphosphorine-2-oxide via a McLafferty rearrangement, and cleavage of the P-N bond. However, their activated 4-OOH and 4-OH metabolites primarily underwent hydrogen peroxide elimination and dehydration, respectively, followed by fragmentation pathways similar to those of CP and IF. These results should prove useful in structural elucidation of future analogs of CP and IF, and/or of their metabolites. Copyright (c) 2005 John Wiley & Sons, Ltd.

Effects of Bisphenol A Metabolite 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene on Lung Function and Type 2 Pulmonary Alveolar Epithelial Cell Growth

PubMed Central

Liu, Shing-Hwa; Su, Chin-Chuan; Lee, Kuan-I; Chen, Ya-Wen

2016-01-01

Bisphenol A (BPA) is recognized as a major pollutant worldwide. 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is a major active metabolite of BPA. The epidemiological and animal studies have reported that BPA is harmful to lung function. The role of MBP in lung dysfunction after BPA exposure still remains unclear. This study investigated whether MBP would induce lung alveolar cell damage and evaluated the role of MBP in the BPA exposure-induced lung dysfunction. An in vitro type 2 alveolar epithelial cell (L2) model and an ex vivo isolated reperfused rat lung model were used to determine the effects of BPA or MBP on cell growth and lung function. MBP, but not BPA, dose-dependently increased the mean artery pressure (Pa), pulmonary capillary pressure (Pc), pulmonary capillary filtration coefficient (Kfc), and wet/dry weight ratio in isolated reperfused rat lungs. MBP significantly reduced cell viability and induced caspases-3/7 cleavage and apoptosis and increased AMP-activated protein kinas (AMPK) phosphorylation and endoplasmic reticulum (ER) stress-related molecules expression in L2 cells, which could be reversed by AMPK-siRNA transfection. These findings demonstrated for the first time that MBP exposure induced type 2 alveolar cell apoptosis and lung dysfunction through an AMPK-regulated ER stress signaling pathway. PMID:27982077

Effects of Bisphenol A Metabolite 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene on Lung Function and Type 2 Pulmonary Alveolar Epithelial Cell Growth.

PubMed

Liu, Shing-Hwa; Su, Chin-Chuan; Lee, Kuan-I; Chen, Ya-Wen

2016-12-16

Bisphenol A (BPA) is recognized as a major pollutant worldwide. 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is a major active metabolite of BPA. The epidemiological and animal studies have reported that BPA is harmful to lung function. The role of MBP in lung dysfunction after BPA exposure still remains unclear. This study investigated whether MBP would induce lung alveolar cell damage and evaluated the role of MBP in the BPA exposure-induced lung dysfunction. An in vitro type 2 alveolar epithelial cell (L2) model and an ex vivo isolated reperfused rat lung model were used to determine the effects of BPA or MBP on cell growth and lung function. MBP, but not BPA, dose-dependently increased the mean artery pressure (Pa), pulmonary capillary pressure (Pc), pulmonary capillary filtration coefficient (K fc ), and wet/dry weight ratio in isolated reperfused rat lungs. MBP significantly reduced cell viability and induced caspases-3/7 cleavage and apoptosis and increased AMP-activated protein kinas (AMPK) phosphorylation and endoplasmic reticulum (ER) stress-related molecules expression in L2 cells, which could be reversed by AMPK-siRNA transfection. These findings demonstrated for the first time that MBP exposure induced type 2 alveolar cell apoptosis and lung dysfunction through an AMPK-regulated ER stress signaling pathway.

Solid-Phase Extraction of Sulfur Mustard Metabolites Using an Activated Carbon Fiber Sorbent.

PubMed

Lee, Jin Young; Lee, Yong Han

2016-01-01

A novel solid-phase extraction method using activated carbon fiber (ACF) was developed and validated. ACF has a vast network of pores of varying sizes and microporous structures that result in rapid adsorption and selective extraction of sulfur mustard metabolites according to the pH of eluting solvents. ACF could not only selectively extract thiodiglycol and 1-methylsulfinyl-2-[2-(methylthio)-ethylsulfonyl]ethane eluting a 9:1 ratio of dichloromethane to acetone, and 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] and 1,1'-sulfonylbis- [2-S-(N-acetylcysteinyl)ethane] eluting 3% hydrogen chloride in methanol, but could also eliminate most interference without loss of analytes during the loading and washing steps. A sample preparation method has been optimized for the extraction of sulfur mustard metabolites from human urine using an ACF sorbent. The newly developed extraction method was applied to the trace analysis of metabolites of sulfur mustard in human urine matrices in a confidence-building exercise for the analysis of biomedical samples provided by the Organisation for the Prohibition of Chemical Weapons. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Total synthesis of 4-acetyl-1,3-dihydroimidazo[4,5-c]pyridin-2-one, a new microbial metabolite from a Streptomyces species.

PubMed

Bender, Tobias; von Zezschwitz, Paultheo

2009-07-01

The structure of a new secondary metabolite from Streptomyces sp. was determined as 4-acetyl-1,3-dihydroimidazo[4,5-c]pyridin-2-one by synthesis of the natural product itself and of the regioisomeric 7-acetylimidazo[4,5-b]pyridine derivative. The former compound was prepared, in 28% overall yield, in a sequence of nitration, reduction, condensation, and Stille reaction of 4-aminopyridine, while the regioisomer was obtained in 5% overall yield by amination, nitration, reduction, condensation, and oxidation of 4-ethylpyridine.

LC-MS/MS determination of alectinib and its major human metabolite M4 in human urine: prevention of nonspecific binding.

PubMed

Heinig, Katja; Herzog, Denis; Ferrari, Luca; Fraier, Daniela; Miya, Kazuhiro; Morcos, Peter N

2017-03-01

Alectinib (Alecensa ® ) is an anaplastic lymphoma kinase inhibitor for the treatment of anaplastic lymphoma kinase positive non-small-cell lung cancer, and M4 is its major pharmacologically active metabolite. To characterize the pharmacokinetics and excretion of alectinib and M4 in human urine, a bioanalytical method was required. An LC-MS/MS method using supported liquid extraction was developed for the determination of alectinib and M4 in human urine over the concentration range 0.5-500 ng/ml. Accuracy ranged from 92.0 to 112.2% and precision (CV) was below 9.6%. The method was successfully employed to determine alectinib and M4 concentrations in urine samples from a clinical mass balance study. Addition of the surfactant Tween-20 to urine prevented nonspecific binding of the analytes.

Effects of atorvastatin metabolites on induction of drug-metabolizing enzymes and membrane transporters through human pregnane X receptor

PubMed Central

Hoffart, E; Ghebreghiorghis, L; Nussler, AK; Thasler, WE; Weiss, TS; Schwab, M; Burk, O

2012-01-01

BACKGROUND AND PURPOSE Atorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). EXPERIMENTAL APPROACH Ligand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation. KEY RESULTS All atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors. CONCLUSIONS AND IMPLICATIONS Atorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound. PMID:21913896

3D-QSAR Studies on a Series of Dihydroorotate Dehydrogenase Inhibitors: Analogues of the Active Metabolite of Leflunomide

PubMed Central

Li, Shun-Lai; He, Mao-Yu; Du, Hong-Guang

2011-01-01

The active metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. Self-organizing molecular field analysis (SOMFA), a simple three-dimensional quantitative structure-activity relationship (3D-QSAR) method is used to study the correlation between the molecular properties and the biological activities of a series of analogues of the active metabolite. The statistical results, cross-validated rCV2 (0.664) and non cross-validated r2 (0.687), show a good predictive ability. The final SOMFA model provides a better understanding of DHODH inhibitor-enzyme interactions, and may be useful for further modification and improvement of inhibitors of this important enzyme. PMID:21686163

Fatty Acid Amide Hydrolase-Dependent Generation of Antinociceptive Drug Metabolites Acting on TRPV1 in the Brain

PubMed Central

Blomgren, Anders; Simonsen, Charlotte; Daulhac, Laurence; Libert, Frédéric; Chapuy, Eric; Etienne, Monique; Högestätt, Edward D.; Zygmunt, Peter M.; Eschalier, Alain

2013-01-01

The discovery that paracetamol is metabolized to the potent TRPV1 activator N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) and that this metabolite contributes to paracetamol’s antinociceptive effect in rodents via activation of TRPV1 in the central nervous system (CNS) has provided a potential strategy for developing novel analgesics. Here we validated this strategy by examining the metabolism and antinociceptive activity of the de-acetylated paracetamol metabolite 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA), both of which may undergo a fatty acid amide hydrolase (FAAH)-dependent biotransformation to potent TRPV1 activators in the brain. Systemic administration of 4-aminophenol and HMBA led to a dose-dependent formation of AM404 plus N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) and arvanil plus olvanil in the mouse brain, respectively. The order of potency of these lipid metabolites as TRPV1 activators was arvanil = olvanil>>AM404> HPODA. Both 4-aminophenol and HMBA displayed antinociceptive activity in various rodent pain tests. The formation of AM404, arvanil and olvanil, but not HPODA, and the antinociceptive effects of 4-aminophenol and HMBA were substantially reduced or disappeared in FAAH null mice. The activity of 4-aminophenol in the mouse formalin, von Frey and tail immersion tests was also lost in TRPV1 null mice. Intracerebroventricular injection of the TRPV1 blocker capsazepine eliminated the antinociceptive effects of 4-aminophenol and HMBA in the mouse formalin test. In the rat, pharmacological inhibition of FAAH, TRPV1, cannabinoid CB1 receptors and spinal 5-HT3 or 5-HT1A receptors, and chemical deletion of bulbospinal serotonergic pathways prevented the antinociceptive action of 4-aminophenol. Thus, the pharmacological profile of 4-aminophenol was identical to that previously reported for paracetamol, supporting our suggestion that this drug metabolite contributes to paracetamol’s analgesic activity via activation

The effect of varying halogen substituent patterns on the cytochrome P450 catalysed dehalogenation of 4-halogenated anilines to 4-aminophenol metabolites.

PubMed

Cnubben, N H; Vervoort, J; Boersma, M G; Rietjens, I M

1995-05-11

The cytochrome P450 catalysed biotransformation of 4-halogenated anilines was studied in vitro with special emphasis on the dehalogenation to 4-aminophenol metabolites. The results demonstrated that a fluorine substituent at the C4 position was more easily eliminated from the aromatic ring than a chloro-, bromo- or iodo-substituent. HPLC analysis of in vitro biotransformation patterns revealed that the dehalogenation of the C4-position was accompanied by formation of non-halogenated 4-aminophenol, without formation of NIH-shifted metabolites. Changes in the apparent Vmax for the microsomal oxidative dehalogenation appeared to correlate with the electronegativity of the halogen substituent at C4, the fluorine substituent being the one most easily eliminated. A similar decrease in the rate of dehalogenation from a fluoro- to a chloro- to a bromo- to an iodo-substituent was observed in a system with purified reconstituted cytochrome P450 IIB1, in a tertiair butyl hydroperoxide supported microsomal cytochrome P450 system as well as in a system with microperoxidase 8. This microperoxidase 8 is a haem-based mini-enzyme without a substrate binding site, capable of catalysing cytochrome P450-like reaction chemistry. Together, these results excluded the possibility that the difference in the rate of dehalogenation with a varying C4-halogen substituent arose from a change in the contribution of cytochrome P450 enzymes involved in oxidative dehalogenation with a change in the halogen substituent. Rather, they strongly suggested that the difference was indeed due to an intrinsic electronic parameter of the various C4 halogenated anilines dependent on the type of halogen substituent. Additional in vitro experiments with polyfluorinated anilines demonstrated that elimination of the C4-fluorine substituent became more difficult upon the introduction of additional electron withdrawing fluorine substituents in the aniline-ring. 19F-NMR analysis of the metabolite patterns showed

Bioconversion of 7-hydroxyflavanone: isolation, characterization and bioactivity evaluation of twenty-one phase I and phase II microbial metabolites.

PubMed

Mikell, Julie Rakel; Khan, Ikhlas Ahmad

2012-01-01

Microbial metabolism of 7-hydroxyflavanone (1) with fungal culture Cunninghamella blakesleeana (ATCC 8688a), yielded flavanone 7-sulfate (2), 7,4'-dihydroxyflavanone (3), 6,7-dihydroxyflavanone (4), 6-hydroxyflavanone 7-sulfate (5), and 7-hydroxyflavanone 6-sulfate (6). Mortierella zonata (ATCC 13309) also transformed 1 to metabolites 2 and 3 as well as 4'-hydroxyflavanone 7-sulfate (7), flavan-4-cis-ol 7-sulfate (8), 2',4'-dihydroxychalcone (9), 7,8-dihydroxyflavanone (10), 8-hydroxyflavanone 7-sulfate (11), and 8-methoxy-7-hydroxyflavanone (12). Beauveria bassiana (ATCC 7159) metabolized 1 to 2, 3, and 8, flavanone 7-O-β-D-O-4-methoxyglucopyranoside (13), and 8-hydroxyflavanone 7-O-β-D-O-4-methoxyglucopyranoside (14). Chaetomium cochlioides (ATCC 10195) also transformed 1 to 2, 3, 9, together with 7-hydroxy-4-cis-ol (15). Mucor ramannianus (ATCC 9628) metabolized 1 in addition to 7, to also 4,2',4'-trihydroxychalcone (16), 7,3',4'-trihydroxyflavanone (17), 4'-hydroxyflavanone 7-O-α-L-rhamnopyranoside (18), and 7,3',4'-trihydroxy-6-methoxyflavanone (19). The organism Aspergillus alliaceus (ATCC 10060) transformed 1 to metabolites 3, 16, 7,8,4'-trihydroxyflavanone (20), and 7-hydroxyflavanone 4'-sulfate (21). A metabolite of 1, flavanone 7-O-β-D-O-glucopyranoside (22) was produced by Rhizopus oryzae (ATCC 11145). Structures of the metabolic products were elucidated by means of spectroscopic data. None of the metabolites tested showed antibacterial, antifungal and antimalarial activities against selected organisms. Metabolites 4 and 16 showed weak antileishmanial activity.

Two new metabolites from a soil fungus Curvularia affinis strain HS-FG-196.

PubMed

Zhang, Hui; Mao, Liang-Liang; Qian, Ping-Ting; Shan, Wei-Guang; Wang, Ji-Dong; Bai, Hua

2012-01-01

Two new metabolites, pyrenocine J (1) and pyrenochaetic acid D (2), together with two known metabolites, pyrenocine A (3) and pyrenochaetic acid A (4), were isolated from a soil fungus, Curvularia affinis strain HS-FG-196. Their structures were established by extensive spectroscopic analysis. Compound 1 showed cytotoxic activity against the human hepatic cancer cell line HepG2 with an IC(50) value of 28.5 μg/ml.

Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K.

PubMed

Cheng, Z; Surichan, S; Ruparelia, K; Arroo, R; Boarder, M R

2011-04-01

The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4'-hydroxy-5,6,7,8-tetramethoxyflavone (4'-OH-TMF) in this effect. We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [(3)H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade. Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4'-OH-TMF. Low micromolar concentrations of tangeretin or 4'-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4'-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR. Tangeretin and 4'-OH-TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4'-OH-TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid-rich diet. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

High and ultra-high resolution metabolite mapping of the human brain using 1H FID MRSI at 9.4T.

PubMed

Nassirpour, Sahar; Chang, Paul; Henning, Anke

2018-03-01

Magnetic resonance spectroscopic imaging (MRSI) is a promising technique for mapping the spatial distribution of multiple metabolites in the human brain. These metabolite maps can be used as a diagnostic tool to gain insight into several biochemical processes and diseases in the brain. In comparison to lower field strengths, MRSI at ultra-high field strengths benefits from a higher signal to noise ratio (SNR) as well as higher chemical shift dispersion, and hence spectral resolution. This study combines the benefits of an ultra-high field magnet with the advantages of an ultra-short TE and TR single-slice FID-MRSI sequence (such as negligible J-evolution and loss of SNR due to T 2 relaxation effects) and presents the first metabolite maps acquired at 9.4T in the healthy human brain at both high (voxel size of 97.6µL) and ultra-high (voxel size of 24.4µL) spatial resolutions in a scan time of 11 and 46min respectively. In comparison to lower field strengths, more anatomically-detailed maps with higher SNR from a larger number of metabolites are shown. A total of 12 metabolites including glutamate (Glu), glutamine (Gln), N-acetyl-aspartyl-glutamate (NAAG), Gamma-aminobutyric acid (GABA) and glutathione (GSH) are reliably mapped. Comprehensive description of the methodology behind these maps is provided. Copyright © 2016 Elsevier Inc. All rights reserved.

The structures of three metabolites of the algal hepatotoxin okadaic acid produced by oxidation with human cytochrome P450

PubMed Central

Liu, Li; Guoa, Fujiang; Crain, Sheila; Quilliam, Michael A.; Wang, Xiaotang; Rein, Kathleen S.

2012-01-01

Four metabolites of okadaic acid were generated by incubation with human recombinant cytochrome P450 3A4. The structures of two of the four metabolites have been determined by MS/MS experiments and 1D and 2D NMR methods using 94 and 133 μg of each metabolite. The structure of a third metabolite was determined by oxidation to a metabolite of known structure. Like okadaic acid, the metabolites are inhibitors of protein phosphatase PP2A. Although one of the metabolites does have an α,β unsaturated carbonyl with the potential to form adducts with an active site cysteine, all of the metabolites are reversible inhibitors of PP2A. PMID:22608922

Linear glandular trichomes of Helianthus (Asteraceae): morphology, localization, metabolite activity and occurrence

PubMed Central

Aschenbrenner, Anna-Katharina; Horakh, Silke; Spring, Otmar

2013-01-01

Capitate glandular trichomes of sunflower are well investigated, but detailed studies are lacking for the linear glandular trichomes (LGT), a second type of physiologically active plant hair present on the surface of sunflowers. Light, fluorescence and scanning electron microscopy as well as histochemical staining were used to investigate the structure and metabolite deposition of LGT. Consisting of 6–11 linearly arranged cells, LGT were found on the surface of most plant organs of Helianthus annuus. They were associated with the leaf vascular system, and also occurred along petioles, stems and the abaxial surface of chaffy bracts, ray and disc florets. The highest density was found on the abaxial surface of phyllaries. Phenotypically similar LGT were common in all species of the genus, but also occurred in most other genera of the Helianthinae so far screened. Brownish and fluorescent metabolites of an as yet unknown chemical structure, together with terpenoids, were produced and stored in apical cells of LGT. The deposition of compounds gradually progressed from the tip cell to the basal cells of older trichomes. This process was accompanied by nucleus degradation in metabolite-accumulating cells. The localization of these trichomes on prominent plant parts of the apical bud and the capitulum combined with the accumulation of terpenoids and other as yet unknown compounds suggests a chemo-ecological function of the LGT in plant–insect or plant–herbivore interaction.

Different effects of clopidogrel and clarithromycin on the enantioselective pharmacokinetics of sibutramine and its active metabolites in healthy subjects.

PubMed

Shinde, Dhananjay D; Kim, Ho-Sook; Choi, Jae-Seok; Pan, Wei; Bae, Soo Kyung; Yeo, Chang-Woo; Shon, Ji-Hong; Kim, Dong-Hyun; Shin, Jae Gook

2013-05-01

In this study, we assessed the effects of clopidogrel and clarithromycin, known CYP2B6 and CYP3A inhibitors, respectively, on the enantioselective disposition of racemic sibutramine in conjunction with CYP2B6 polymorphisms in humans. Sibutramine showed enantioselective plasma profiles with consistently higher concentrations of R-enantiomers. Clopidogrel and clarithromycin significantly increased the sibutramine plasma concentration, but their effects differed between enantiomers; a 2.2-fold versus 4.1-fold increase in the AUC in S-enantiomer and 1.8-fold versus 2.0-fold for the R-enantiomer, respectively. The AUCs of S- and R-desmethyl metabolites changed significantly during the clopidogrel phase (P < .001 and P < .001, respectively) but not during the clarithromycin phase (P = .099 and P = .090, respectively). Exposure to sibutramine was higher in subjects with the CYP2B6*6/*6 genotype, but no statistical difference was observed among the CYP2B6 genotypes. These results suggest that the enantioselective disposition of sibutramine and its active metabolites are influenced by the altered genetic and environmental factors of CYP2B6 and CYP3A activity in vivo. © The Author(s) 2013.

[Monograph for 3-(4-methylbenzylidene)camphor (4-MBC)--HBM values for the sum of metabolites 3-(4-carboxybenzylidene)camphor (3-4CBC) and 3-(4-carboxybenzylidene)-6-hydroxycamphor (3-4 CBHC) in the urine of adults and children. Statement of the HBM Commission of the German Federal Environment Agency].

PubMed

2016-01-01

The substance 3-(4-methylbenzylidene)camphor (4-MBC, CAS-No. 36861-47-9 as well as 38102-62-4) is used as UV-filter in cosmetics, mainly in sunscreen lotions. National as well as European evaluations are available for the substance, especially from the Scientific Committee on Consumer Products (SCCP). The SCCP did not derive a TDI-value, but used for a MoS assessment a NOAEL of 25 mg/(kg bw · d) based on effects on the thyroid gland of rats in a subchronic study with oral administration. Newer studies, however, indicate lower NOAEL values, leading to tolerable daily intakes of 0,01 mg/kg bw. The HBM Commission established for the metabolite 3-(4-carboxybenzylidene)camphor (3-4CBC) HBM-I values of 0,09 mg/l urine for adults and 0,06 mg/l urine for children. HBM-I values for the metabolite 3-(4-carboxybenzylidene)-6-hydroxycamphor (3-4CBHC) were set at 0,38 mg/l urine for adults and 0,25 mg/l urine for children. The rounded HBM-I value for the sum of metabolites 3-4CBC und 3-4CBHC is accordingly 0,5 mg/l urine for adults and 0,3 mg/l urine for children.

Effect of CYP2B6 genotype on the pharmacokinetics of sibutramine and active metabolites in healthy subjects.

PubMed

Chung, Jae Yong; Jang, Seong Bok; Lee, Yoon Jung; Park, Min Soo; Park, Kyungsoo

2011-01-01

Sibutramine is a pharmacologic intervention for the treatment of obesity. The effect of CYP2B6 genotypes on the pharmacokinetics of sibutramine and its active metabolites (desmethylsibutramine [M1] and didesmethylsibutramine [M2]) was evaluated in 57 healthy subjects. Each subject received a single oral dose of 10 or 15 mg sibutramine, and blood samples were collected up to 72 hours after dosing. The relationship between the genotypes and the pharmacokinetics of sibutramine, M1, and M2 was examined. A statistically significant difference in the elimination half-life (t(1/2)) of sibutramine M1 was found among the 3 genotype groups (P = .0006), between the *1/*1 and *1/*6 groups (P = .0001), and between the *1/*4 and *1/*6 groups (P = .012). The mean value of M1 t(1/2) in *1/*6 (33.3 ± 10.5 hours) was about 58% and 61% greater than that of the *1/*1 group (21.0 ± 7.4 hours) and the *1/*4 group (20.7 ± 9.8 hours), respectively. No significant differences in area under the concentration-time curve or maximum plasma drug concentration were observed between the groups. The CYP2B6*6 allele may be associated with a lower metabolic clearance of the M1 metabolite of sibutramine in human subjects.

Effects of two metabolites of ochratoxin A, (4R)-4-hydroxyochratoxin A and ochratoxin alpha, on immune response in mice.

PubMed Central

Creppy, E E; Størmer, F C; Röschenthaler, R; Dirheimer, G

1983-01-01

The metabolites of ochratoxin A, (4R)-4-hydroxyochratoxin A and ochratoxin alpha, were investigated for immunosuppressive properties in BALB/c mice. The standard plaque-counting technique for the estimation of antibody-producing spleen lymphocytes was used. (4R)-4-hydroxyochratoxin A was found to be an immunosuppressor almost as highly effective as ochratoxin A. Doses of 1 microgram of (4R)-4-hydroxyochratoxin A per kg administered to mice caused an 80% reduction in the number of cells producing immunoglobulin M (90% with ochratoxin A) and a 93% reduction in cells synthesizing immunoglobulin G (92% with ochratoxin A). Ochratoxin alpha, however, was ineffective. A possible mode of action is discussed. PMID:6341224

Reversible modulation of SIRT1 activity in a mouse strain

PubMed Central

Clark-Knowles, Katherine V.; He, Xiaohong; Jardine, Karen; Coulombe, Josée; Dewar-Darch, Danielle; Caron, Annabelle Z.

2017-01-01

The SIRT1 protein deacetylase is reported to have a remarkably wide spectrum of biological functions affecting such varied processes as aging, cancer, metabolism, neurodegeneration and immunity. However, the SIRT1 literature is also full of contradictions. To help establish the role(s) of SIRT1 in these and other biological processes, we set out to create a mouse in which the SIRT1 activity could be toggled between on and off states by fusing the estrogen receptor ligand-binding domain (ER) to the C terminus of the SIRT1 protein. We found that the catalytic activity of the SIRT1-ER fusion protein increased 4–5 fold in cells treated with its ligand, 4-hydroxy-tamoxifen (4OHT). The 4OHT-induced activation of SIRT1-ER was due in large part to a 2 to 4-fold increase in abundance of the SIRT1-ER protein in cells in culture and in tissues in vivo. This increase is reversible and is a consequence of 4OHT-induced stabilization of the SIRT1-ER protein. Since changes in SIRT1 level or activity of 2–4 fold are frequently reported to be sufficient to affect its biological functions, this mouse should be helpful in establishing the causal relationships between SIRT1 and the diseases and processes it affects. PMID:28273169

Plasma pharmacokinetics of catechin metabolite 4'-O-Me-EGC in healthy humans.

PubMed

Renouf, Mathieu; Redeuil, Karine; Longet, Karin; Marmet, Cynthia; Dionisi, Fabiola; Kussmann, Martin; Williamson, Gary; Nagy, Kornél

2011-10-01

Tea is an infusion of the leaves of the Camellia sinensis plant and is the most widely consumed beverage in the world after water. Green tea contains significant amounts of polyphenol catechins and represents a promising dietary component to maintain health and well-being. Epidemiological studies indicate that polyphenol intake may have potential health benefits, such as, reducing the incidence of coronary heart disease, diabetes and cancer. While bioavailability of green tea bioactives is fairly well understood, some gaps still remain to be filled, especially the identification and quantification of conjugated metabolites in plasma, such as, sulphated, glucuronidated or methylated compounds. In the present study, we aimed to quantify the appearance of green tea catechins in plasma with particular emphasis on their methylated forms. After feeding 400 mL of green tea, 1.25% infusion to 9 healthy subjects, we found significant amounts of EC, EGC and EGCg in plasma as expected. EGC was the most bioavailable catechin, and its methylated form (4'-O-Me-EGC) was also present in quantifiable amounts. Its kinetics followed that of its parent compound. However, the relative amount of the methylated form of EGC was lower than that of the parent compound, an important aspect which, in the literature, has been controversial so far. The quantitative results presented in our study were confirmed by co-chromatography and accurate mass analysis of the respective standards. We show that the relative abundance of 4'-O-Me-EGC is ~40% compared to the parent EGC. 4'-O-Me-EGC is an important metabolite derived from catechin metabolism. Its presence in significant amounts should not be overlooked when assessing human bioavailability of green tea.

Actions of incretin metabolites on locomotor activity, cognitive function and in vivo hippocampal synaptic plasticity in high fat fed mice.

PubMed

Porter, David; Faivre, Emilie; Flatt, Peter R; Hölscher, Christian; Gault, Victor A

2012-05-01

The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) improve markers of cognitive function in obesity-diabetes, however, both are rapidly degraded to their major metabolites, GLP-1(9-36)amide and GIP(3-42), respectively. Therefore, the present study investigated effects of GLP-1(9-36)amide and GIP(3-42) on locomotor activity, cognitive function and hippocampal synaptic plasticity in mice with diet-induced obesity and insulin resistance. High-fat fed Swiss TO mice treated with GLP-1(9-36)amide, GIP(3-42) or exendin(9-39)amide (twice-daily for 60 days) did not exhibit any changes in bodyweight, non-fasting plasma glucose and plasma insulin concentrations or glucose tolerance compared with high-fat saline controls. Similarly, locomotor and feeding activity, O(2) consumption, CO(2) production, respiratory exchange ratio and energy expenditure were not altered by chronic treatment with incretin metabolites. Administration of the truncated metabolites did not alter general behavior in an open field test or learning and memory ability as recorded during an object recognition test. High-fat mice exhibited a significant impairment in hippocampal long-term potentiation (LTP) which was not affected by treatment with incretin metabolites. These data indicate that incretin metabolites do not influence locomotor activity, cognitive function and hippocampal synaptic plasticity when administered at pharmacological doses to mice fed a high-fat diet. Copyright © 2012 Elsevier Inc. All rights reserved.

Bioavailability of Bergamot (Citrus bergamia) Flavanones and Biological Activity of Their Circulating Metabolites in Human Pro-Angiogenic Cells

PubMed Central

Spigoni, Valentina; Fantuzzi, Federica; Tassotti, Michele; Brighenti, Furio; Bonadonna, Riccardo C.; Dei Cas, Alessandra

2017-01-01

Myeloid angiogenic cells (MACs) play a key role in endothelial repairing processes and functionality but their activity may be impaired by the lipotoxic effects of some molecules like stearic acid (SA). Among the dietary components potentially able to modulate endothelial function in vivo, (poly)phenolic compounds represent serious candidates. Here, we apply a comprehensive multidisciplinary approach to shed light on the prospects of Bergamot (Citrus bergamia), a citrus fruit rich in flavanones and other phenolic compounds, in the framework of lipotoxicity-induced MACs impairment. The flavanone profile of bergamot juice was characterized and 16 compounds were identified, with a new 3-hydroxy-3-methylglutaryl (HMG) flavanone, isosakuranetin-7-O-neohesperidoside-6″-O-HMG, described for the first time. Then, a pilot bioavailability study was conducted in healthy volunteers to assess the circulating flavanone metabolites in plasma and urine after consumption of bergamot juice. Up to 12 flavanone phase II conjugates (sulfates and glucuronides of hesperetin, naringenin and eriodyctiol) were detected and quantified. Finally, the effect of some of the metabolites identified in vivo, namely hesperetin-7-O-glucuronide, hesperetin-3′-O-glucuronide, naringenin-7-O-glucuronide and naringenin-4′-O-glucuronide, was tested, at physiological concentrations, on gene expression of inflammatory markers and apoptosis in MACs exposed to SA. Under these conditions, naringenin-4′-O-glucuronide and hesperetin-7-O-glucuronide were able to modulate inflammation, while no flavanone glucuronide was effective in curbing stearate-induced lipoapoptosis. These results demonstrate that some flavanone metabolites, derived from the in vivo transformation of bergamot juice phenolics in humans, may mitigate stearate-induced inflammation in MACs. PMID:29211032

Bioavailability of Bergamot (Citrus bergamia) Flavanones and Biological Activity of Their Circulating Metabolites in Human Pro-Angiogenic Cells.

PubMed

Spigoni, Valentina; Mena, Pedro; Fantuzzi, Federica; Tassotti, Michele; Brighenti, Furio; Bonadonna, Riccardo C; Del Rio, Daniele; Dei Cas, Alessandra

2017-12-06

Myeloid angiogenic cells (MACs) play a key role in endothelial repairing processes and functionality but their activity may be impaired by the lipotoxic effects of some molecules like stearic acid (SA). Among the dietary components potentially able to modulate endothelial function in vivo, (poly)phenolic compounds represent serious candidates. Here, we apply a comprehensive multidisciplinary approach to shed light on the prospects of Bergamot ( Citrus bergamia ), a citrus fruit rich in flavanones and other phenolic compounds, in the framework of lipotoxicity-induced MACs impairment. The flavanone profile of bergamot juice was characterized and 16 compounds were identified, with a new 3-hydroxy-3-methylglutaryl (HMG) flavanone, isosakuranetin-7- O -neohesperidoside-6″- O -HMG, described for the first time. Then, a pilot bioavailability study was conducted in healthy volunteers to assess the circulating flavanone metabolites in plasma and urine after consumption of bergamot juice. Up to 12 flavanone phase II conjugates (sulfates and glucuronides of hesperetin, naringenin and eriodyctiol) were detected and quantified. Finally, the effect of some of the metabolites identified in vivo, namely hesperetin-7- O -glucuronide, hesperetin-3'- O -glucuronide, naringenin-7- O -glucuronide and naringenin-4'- O -glucuronide, was tested, at physiological concentrations, on gene expression of inflammatory markers and apoptosis in MACs exposed to SA. Under these conditions, naringenin-4'- O -glucuronide and hesperetin-7- O -glucuronide were able to modulate inflammation, while no flavanone glucuronide was effective in curbing stearate-induced lipoapoptosis. These results demonstrate that some flavanone metabolites, derived from the in vivo transformation of bergamot juice phenolics in humans, may mitigate stearate-induced inflammation in MACs.

Pseudomonas fluorescens N21.4 metabolites enhance secondary metabolism isoflavones in soybean (Glycine max) calli cultures.

PubMed

Algar, Elena; Gutierrez-Mañero, Francisco Javier; Bonilla, Alfonso; Lucas, Jose Antonio; Radzki, Wojtek; Ramos-Solano, Beatriz

2012-11-07

Phytopharmaceuticals are plant secondary metabolites that are strongly inducible and especially sensitive to biotic changes. Plant cell cultures are a good alternative to obtain secondary metabolites, in case effective stimulation can be achieved. In this study, metabolic elicitors from two rhizobacteria able to enhance isoflavone content in soybean seedlings were tested on three different soybean calli cell lines. Results show that metabolic elicitors from Chryseobacterium balustinum Aur9 were not effective. However, there are at least two different metabolic elicitors from Pseudomonas fluorescens N21.4, one under 10 kDa and another over 10 kDa, that trigger isoflavone metabolism in the three cell lines with different isoflavone content. Elicitors from N21.4 achieved total isoflavone increases up to 29.7% (0.205 mg/g), 64.5% (0.487 mg/g), and 23.4% (0.726 mg/g) in the low-, intermediate-, and high-yield lines, respectively. Therefore, these elicitors have a great potential to enhance isoflavone production in cell cultures for development of functional ingredients.

In vitro antiprogestational/antiglucocorticoid activity and progestin and glucocorticoid receptor binding of the putative metabolites and synthetic derivatives of CDB-2914, CDB-4124, and mifepristone.

PubMed

Attardi, Barbara J; Burgenson, Janet; Hild, Sheri A; Reel, Jerry R

2004-03-01

In determining the biological profiles of various antiprogestins, it is important to assess the hormonal and antihormonal activity, selectivity, and potency of their proximal metabolites. The early metabolism of mifepristone is characterized by rapid demethylation and hydroxylation. Similar initial metabolic pathways have been proposed for CDB-2914 (CDB: Contraceptive Development Branch of NICHD) and CDB-4124, and their putative metabolites have been synthesized. We have examined the functional activities and potencies, in various cell-based assays, and relative binding affinities (RBAs) for progesterone receptors (PR) and glucocorticoid receptors (GR) of the putative mono- and didemethylated metabolites of CDB-2914, CDB-4124, and mifepristone and of the 17alpha-hydroxy and aromatic A-ring derivatives of CDB-2914 and CDB-4124. The binding affinities of the monodemethylated metabolites for rabbit uterine PR and human PR-A and PR-B were similar to those of the parent compounds. Monodemethylated mifepristone bound to rabbit thymic GR with higher affinity than monodemethylated CDB-2914 or CDB-4124. T47D-CO cells were used to assess inhibition of R5020-stimulated endogenous alkaline phosphatase activity and transactivation of the PRE(2)-thymidine kinase (tk)-luciferase (LUC) reporter plasmid in transient transfections. The antiprogestational potency was as follows: mifepristone/CDB-2914/CDB-4124/monodemethylated metabolites (IC(50)'s approximately 10(-9)M) > aromatic A-ring derivatives (IC(50)'s approximately 10(-8)M) > didemethylated/17alpha-hydroxy derivatives (IC(50)'s approximately 10(-7)M). Antiglucocorticoid activity was determined by inhibition of dexamethasone-stimulated transcriptional activity in HepG2 cells. The mono- and didemethylated metabolites of CDB-2914 and CDB-4124 had less antiglucocorticoid activity (IC(50)'s approximately 10(-6)M) than monodemethylated mifepristone (IC(50) approximately 10(-8)M) or the other test compounds. At 10(-6)M in

Stereoselective Inhibition of CYP2C19 and CYP3A4 by Fluoxetine and Its Metabolite: Implications for Risk Assessment of Multiple Time-Dependent Inhibitor Systems

PubMed Central

Lutz, Justin D.; VandenBrink, Brooke M.; Babu, Katipudi N.; Nelson, Wendel L.; Kunze, Kent L.

2013-01-01

Recent guidance on drug-drug interaction (DDI) testing recommends evaluation of circulating metabolites. However, there is little consensus on how to quantitatively predict and/or assess the risk of in vivo DDIs by multiple time-dependent inhibitors (TDIs) including metabolites from in vitro data. Fluoxetine was chosen as the model drug to evaluate the role of TDI metabolites in DDI prediction because it is a TDI of both CYP3A4 and CYP2C19 with a circulating N-dealkylated inhibitory metabolite, norfluoxetine. In pooled human liver microsomes, both enantiomers of fluoxetine and norfluoxetine were TDIs of CYP2C19, (S)-norfluoxetine was the most potent inhibitor with time-dependent inhibition affinity constant (KI) of 7 μM, and apparent maximum time-dependent inhibition rate (kinact,app) of 0.059 min−1. Only (S)-fluoxetine and (R)-norfluoxetine were TDIs of CYP3A4, with (R)-norfluoxetine being the most potent (KI = 8 μM, and kinact,app = 0.011 min−1). Based on in-vitro-to-in-vivo predictions, (S)-norfluoxetine plays the most important role in in vivo CYP2C19 DDIs, whereas (R)-norfluoxetine is most important in CYP3A4 DDIs. Comparison of two multiple TDI prediction models demonstrated significant differences between them in in-vitro-to-in-vitro predictions but not in in-vitro-to-in-vivo predictions. Inclusion of all four inhibitors predicted an in vivo decrease in CYP2C19 (95%) and CYP3A4 (60–62%) activity. The results of this study suggest that adequate worst-case risk assessment for in vivo DDIs by multiple TDI systems can be achieved by incorporating time-dependent inhibition by both parent and metabolite via simple addition of the in vivo time-dependent inhibition rate/cytochrome P450 degradation rate constant (λ/kdeg) values, but quantitative DDI predictions will require a more thorough understanding of TDI mechanisms. PMID:23785064

[Secondary metabolites, lethality and antimicrobial activity of extracts from three corals and three marine mollusks from Sucre, Venezuela].

PubMed

Ordaz, Gabriel; D'Armas, Haydelba; Yáñez, Dayanis; Hernández, Juan; Camacho, Angel

2010-06-01

The study of biochemical activity of extracts obtained from marine organisms is gaining interest as some have proved to have efficient health or industrial applications. To evaluate lethality and antimicrobial activities, some chemical tests were performed on crude extracts of the octocorals Eunicea sp., Muricea sp. and Pseudopterogorgia acerosa and the mollusks Pteria colymbus, Phyllonotus pomum and Chicoreus brevifrons, collected in Venezuelan waters. The presence of secondary metabolites like alkaloids, unsaturated sterols and pentacyclic triterpenes in all invertebrates, was evidenced. Additionally, sesquiterpenlactones, saponins, tannins, cyanogenic and cardiotonic glycosides were also detected in some octocoral extracts, suggesting that biosynthesis of these metabolites is typical in this group. From the lethality bioassays, all extracts resulted lethal to Artemia salina (LC50<1000 microg/ml) with an increased of lethal activity with exposition time. P. pomum extract showed the highest lethality rate (LC50=46.8 microg/ml). Compared to the octocorals, mollusks extracts displayed more activity and a greater action spectrum against different bacterial strains, whereas octocorals also inhibited some fungi strains growth. Staphylococcus aureus was the most susceptible to the antimicrobial power of the extracts (66.7%), whereas Pseudomonas aeruginosa, Candida albicans and Aspergillus niger were not affected. The antibiosis shown by marine organisms extracts indicates that some of their biosynthesized metabolites are physiologically active, and may have possible cytotoxic potential or as a source of antibiotic components.

Environmentally relevant mixing ratios in cumulative assessments: a study of the kinetics of pyrethroids and their ester cleavage metabolites in blood and brain; and the effect of a pyrethroid mixture on the motor activity of rats.

PubMed

Starr, James M; Graham, Stephen E; Ross, David G; Tornero-Velez, Rogelio; Scollon, Edward J; Devito, Michael J; Crofton, Kevin M; Wolansky, Marcelo J; Hughes, Michael F

2014-06-05

National surveys of United States households and child care centers have demonstrated that pyrethroids are widely distributed in indoor habited dwellings and this suggests that co-exposure to multiple pyrethroids occurs in nonoccupational settings. The purpose of this research was to use an environmentally relevant mixture of pyrethroids to assess their cumulative effect on motor activity and develop kinetic profiles for these pyrethroids and their hydrolytic metabolites in brain and blood of rats. Rats were dosed orally at one of two levels (1.5× or 5.0× the calculated dose that decreases rat motor activity by 30%) with a mixture of cypermethrin, deltamethrin, esfenvalerate, cis-/trans-permethrin, and β-cyfluthrin in corn oil. At 1, 2, 4, 8, or 24h after dosing, the motor activity of each animal was assessed and the animals sacrificed. Concentrations of pyrethroids in brain and blood, and the following metabolites: cis-/trans-dichlorovinyl-dimethylcyclopropane-carboxylic acid, 3-phenoxybenzoic acid, 3-phenoxybenzyl alcohol, 4-fluoro-3-phenoxybenzoic acid, and cis-dibromovinyl-dimethylcyclopropane-carboxylic acid were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Using this pyrethroid mixture in rats, the results suggest there is greater metabolism of trans-permethrin prior to entering the systemic circulatory system. All pyrethroids had tissue half-lives (t1/2) of less than 5h, excepting esfenvalerate in brain. At early time points, relative pyrethroid brain concentrations approximated their dose mixture proportions and a sigmoidal Emax model described the relationship between motor activity decrease and total pyrethroid brain concentration. In blood, the t1/2's of the cyclopropane metabolites were longer than the phenoxybenzoic metabolites. However, relative to their respective precursors, concentrations of the phenoxybenzoic acids were much higher than concentrations of the cyclopropane metabolites. Brain concentrations of all

Biosynthesis of human diazepam and clonazepam metabolites.

PubMed

de Paula, Núbia C; Araujo Cordeiro, Kelly C F; de Melo Souza, Paula L; Nogueira, Diogo F; da Silva e Sousa, Diego B; Costa, Maísa B; Noël, François; de Oliveira, Valéria

2015-03-01

A screening of fungal and microbial strains allowed to select the best microorganisms to produce in high yields some of the human metabolites of two benzodiazepine drugs, diazepam and clonazepam, in order to study new pharmacological activities and for chemical standard proposes. Among the microorganisms tested, Cunninghamella echinulata ATCC 9244 and Rhizopus arrhizus ATCC 11145 strains, were the most active producers of the mains metabolites of diazepam which included demethylated, hydroxylated derivatives. Beauveria bassiana ATCC 7159 and Chaetomium indicum LCP 984200 produced the 7 amino-clonazepam metabolite and a product of acid hydrolysis of this benzodiazepine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antitrypanosomal Activity of Fexinidazole Metabolites, Potential New Drug Candidates for Chagas Disease

PubMed Central

Nascimento, Alvaro F. S.; Mazzeti, Ana Lia; Marques, Luiz F.; Gonçalves, Karolina R.; Mota, Ludmilla W. R.; Diniz, Lívia de F.; Caldas, Ivo S.; Talvani, André; Shackleford, David M.; Koltun, Maria; Saunders, Jessica; White, Karen L.; Scandale, Ivan; Charman, Susan A.; Chatelain, Eric

2014-01-01

This study was designed to verify the in vivo efficacy of sulfoxide and sulfone fexinidazole metabolites following oral administration in a murine model of Chagas disease. Female Swiss mice infected with the Y strain of Trypanosoma cruzi were treated orally once per day with each metabolite at doses of 10 to 100 mg/kg of body weight for a period of 20 days. Parasitemia was monitored throughout, and cures were detected by parasitological and PCR assays. The results were compared with those achieved with benznidazole treatment at the same doses. Fexinidazole metabolites were effective in reducing the numbers of circulating parasites and protecting mice against death, compared with untreated mice, but without providing cures at daily doses of 10 and 25 mg/kg. Both metabolites were effective in curing mice at 50 mg/kg/day (30% to 40%) and 100 mg/kg/day (100%). In the benznidazole-treated group, parasitological cure was detected only in animals treated with the higher dose of 100 mg/kg/day (80%). Single-dose pharmacokinetic parameters for each metabolite were obtained from a parallel group of uninfected mice and were used to estimate the profiles following repeated doses. Pharmacokinetic data suggested that biological efficacy most likely resides with the sulfone metabolite (or subsequent reactive metabolites formed following reduction of the nitro group) following administration of either the sulfoxide or the sulfone and that prolonged plasma exposure over the 24-h dosing window is required to achieve high cure rates. Fexinidazole metabolites were effective in treating T. cruzi in a mouse model of acute infection, with cure rates superior to those achieved with either fexinidazole itself or benznidazole. PMID:24841257

Central activation, metabolites, and calcium handling during fatigue with repeated maximal isometric contractions in human muscle.

PubMed

Cairns, Simeon P; Inman, Luke A G; MacManus, Caroline P; van de Port, Ingrid G L; Ruell, Patricia A; Thom, Jeanette M; Thompson, Martin W

2017-08-01

To determine the roles of calcium (Ca 2+ ) handling by sarcoplasmic reticulum (SR) and central activation impairment (i.e., central fatigue) during fatigue with repeated maximal voluntary isometric contractions (MVC) in human muscles. Contractile performance was assessed during 3 min of repeated MVCs (7-s contraction, 3-s rest, n = 17). In ten participants, in vitro SR Ca 2+ -handling, metabolites, and fibre-type composition were quantified in biopsy samples from quadriceps muscle, along with plasma venous [K + ]. In 11 participants, central fatigue was compared using tetanic stimulation superimposed on MVC in quadriceps and adductor pollicis muscles. The decline of peak MVC force with fatigue was similar for both muscles. Fatigue resistance correlated directly with % type I fibre area in quadriceps (r = 0.77, P = 0.009). The maximal rate of ryanodine-induced Ca 2+ -release and Ca 2+ -uptake fell by 31 ± 26 and 28 ± 13%, respectively. The tetanic force depression was correlated with the combined reduction of ATP and PCr, and increase of lactate (r = 0.77, P = 0.009). Plasma venous [K + ] increased from 4.0 ± 0.3 to 5.4 ± 0.8 mM over 1-3-min exercise. Central fatigue occurred during the early contractions in the quadriceps in 7 out of 17 participants (central activation ratio fell from 0.98 ± 0.05 to 0.86 ± 0.11 at 1 min), but dwindled at exercise cessation. Central fatigue was seldom apparent in adductor pollicis. Fatigue with repeated MVC in human limb muscles mainly involves peripheral aspects which include impaired SR Ca 2+ -handling and we speculate that anaerobic metabolite changes are involved. A faster early force loss in quadriceps muscle with some participants is attributed to central fatigue.

Evidence that Formation of Protoanemonin from Metabolites of 4-Chlorobiphenyl Degradation Negatively Affects the Survival of 4-Chlorobiphenyl-Cometabolizing Microorganisms

PubMed Central

Blasco, R.; Mallavarapu, M.; Wittich, R.; Timmis, K. N.; Pieper, D. H.

1997-01-01

A rapid decline in cell viability of different PCB-metabolizing organisms was observed in soil microcosms amended with 4-chlorobiphenyl. The toxic effect could not be attributed to 4-chlorobiphenyl but was due to a compound formed from the transformation of 4-chlorobiphenyl by the natural microflora. Potential metabolites of 4-chlorobiphenyl, 4-chlorobenzoate and 4-chlorocatechol, caused similar toxic effects. We tested the hypothesis that the toxic effects are due to the formation of protoanemonin, a plant-derived antibiotic, which is toxic to microorganisms and which has been shown to be formed from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway. Consistent with our hypothesis, addition to soil microcosms of strains able to reroute intermediary 4-chlorocatechol from the 3-oxoadipate pathway and into the meta-cleavage pathway or able to mineralize 4-chlorocatechol by a modified ortho-cleavage pathway resulted in reversal of this toxic effect. Surprisingly, while direct addition of protoanemonin influenced both the viability of fungi and the microbial activity of the soil microcosm, there was little effect on bacterial viability due to its rapid degradation. This rapid degradation accounts for our inability to detect this compound in soils amended with 4-chlorocatechol. However, significant accumulation of protoanemonin was observed by a mixed bacterial community enriched with benzoate or a mixture of benzoate and 4-methylbenzoate, providing the metabolic potential of the soil to form protoanemonin. The effects of soil heterogeneity and microcosm interactions are discussed in relation to the different effects of protoanemonin when applied as a shock load and when it is produced in small amounts from precursors over long periods. PMID:16535507

In vitro biosynthesis, isolation, and identification of predominant metabolites of 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one (RVX-208).

PubMed

Khmelnitsky, Yuri L; Mozhaev, Vadim V; Cotterill, Ian C; Michels, Peter C; Boudjabi, Sihem; Khlebnikov, Vladimir; Madhava Reddy, M; Wagner, Gregory S; Hansen, Henrik C

2013-06-01

The structures of the two predominant metabolites (M4 and M5) of RVX-208, observed both in in vitro human and animal liver microsomal incubations, as well as in plasma from animal in vivo studies, were determined. A panel of biocatalytic systems was tested to identify biocatalysts suitable for milligram scale production of metabolite M4 from RVX-208. Rabbit liver S9 fraction was selected as the most suitable system, primarily based on pragmatic metrics such as catalyst cost and estimated yield of M4 (∼55%). Glucuronidation of RVX-208 catalyzed by rabbit liver S9 fraction was optimized to produce M4 in amounts sufficient for structural characterization. Structural studies using LC/MS/MS analysis and (1)H NMR spectroscopy showed the formation of a glycosidic bond between the primary hydroxyl group of RVX-208 and glucuronic acid. NMR results suggested that the glycosidic bond has the β-anomeric configuration. A synthetic sample of M4 confirmed the proposed structure. Metabolite M5, hypothesized to be the carboxylate of RVX-208, was prepared using human liver microsomes, purified by HPLC, and characterized by LC/MS/MS and (1)H NMR. The structure was confirmed by comparison to a synthetic sample. Both samples confirmed M5 as a product of oxidation of primary hydroxyl group of RVX-208 to carboxylic acid. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

DRD4 dopamine receptor genotype and CSF monoamine metabolites in Finnish alcoholics and controls

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

Adamson, M.D.; Dean, M.; Goldman, D.

1995-06-19

The DRD4 dopamine receptor is thus far unique among neurotransmitter receptors in having a highly polymorphic gene structure that has been reported to produce altered receptor functioning. These allelic variations are caused by a 48-bp segment in exon III of the coding region which may be repeated from 2-10 times. Varying the numbers of repeated segments changes the length, structure, and, possibly, the functional efficiency of the receptor, which makes this gene an intriguing candidate for variations in dopamine-related behaviors, such as alcoholism and drug abuse. Thus far, these DRD4 alleles have been investigated for association with schizophrenia, bipolar disorder,more » Parkinson`s disease, and chronic alcoholism, and all have been largely negative for a direct association. We evaluated the DRD4 genotype in 226 Finish adult males, 113 of whom were alcoholics, many of the early onset type with features of impulsivity and antisocial traits. Genotype frequencies were compared to 113 Finnish controls who were free of alcohol abuse, substance abuse, and major mental illness. In 70 alcoholics and 20 controls, we measured CSF homovanillic acid (HVA), the major metabolite of dopamine, and 5-hydroxyindoleacetic acid (5-HIAA). No association was found between a particular DRD4 dopamine receptor allele and alcoholism. CSF concentrations of the monoamine metabolites showed no significant difference among the DRD4 genotypes. This study of the DRD4 dopamine receptor in alcoholics is the first to be conducted in a clinically and ethnically homogeneous population and to relate the DRD4 genotype to CSF monoamine concentrations. The results indicate that there is no association of the DRD4 receptor with alcoholism. 52 refs., 3 figs., 1 tab.« less

Antimalarial benzoheterocyclic 4-aminoquinolines: Structure-activity relationship, in vivo evaluation, mechanistic and bioactivation studies.

PubMed

Ongarora, Dennis S B; Strydom, Natasha; Wicht, Kathryn; Njoroge, Mathew; Wiesner, Lubbe; Egan, Timothy J; Wittlin, Sergio; Jurva, Ulrik; Masimirembwa, Collen M; Chibale, Kelly

2015-09-01

A novel class of benzoheterocyclic analogues of amodiaquine designed to avoid toxic reactive metabolite formation was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant) and NF54 (sensitive) strains of the malaria parasite Plasmodium falciparum. Structure-activity relationship studies led to the identification of highly promising analogues, the most potent of which had IC50s in the nanomolar range against both strains. The compounds further demonstrated good in vitro microsomal metabolic stability while those subjected to in vivo pharmacokinetic studies had desirable pharmacokinetic profiles. In vivo antimalarial efficacy in Plasmodium berghei infected mice was evaluated for four compounds, all of which showed good activity following oral administration. In particular, compound 19 completely cured treated mice at a low multiple dose of 4×10mg/kg. Mechanistic and bioactivation studies suggest hemozoin formation inhibition and a low likelihood of forming quinone-imine reactive metabolites, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

Microsomal metabolism of trenbolone acetate metabolites ...

EPA Pesticide Factsheets

Trenbolone acetate (TBA) is a synthetic growth promoter widely used in animal agriculture, and its metabolites are suspected endocrine disrupting compounds in agriculturally impacted receiving waters. However, beyond the three widely recognized TBA metabolites (17-trenbolone, 17-trenbolone and trendione), little is known about other metabolites formed in vivo and subsequently discharged into the environment, with some evidence suggesting these unknown metabolites comprise a majority of the TBA mass dosed to the animal. Here, we explored the metabolism of the three known TBA metabolites using rat liver microsome studies. All TBA metabolites are transformed into a complex mixture of monohydroxylated products. Based on product characterization, the majority are more polar than the parent metabolites but maintain their characteristic trienone backbone. A minor degree of interconversion between known metabolites was also observed, as were higher order hydroxylated products with a greater extent of reaction. Notably, the distribution and yield of products were generally comparable across a series of variably induced rat liver microsomes, as well as during additional studies with human and bovine liver microsomes. Bioassays conducted with mixtures of these transformation products suggest that androgen receptor (AR) binding activity is diminished as a result of the microsomal treatment, suggesting that the transformation products are generally less potent than

Farmed and wild sea bass (Dicentrarchus labrax) volatile metabolites: a comparative study by SPME-GC/MS.

PubMed

Vidal, Natalia P; Manzanos, María J; Goicoechea, Encarnación; Guillén, María D

2016-03-15

Farmed and wild European sea bass (Dicentrarchus labrax) could be distinguished by its volatile metabolites, an issue not addressed until now. The aim of this work was to study these metabolites by solid-phase microextraction followed by gas chromatography/mass spectrometry (SPME-GC/MS). Both farmed and wild sea bass have a great number of volatile metabolites, most of them being in low concentrations. These include alcohols, aldehydes, ketones, alkylfurans, acids, aliphatic and aromatic hydrocarbons, terpenes, sulfur and nitrogen derivatives, 2,6-di-tert-butyl-4-methylphenol and one derived compound, as well as 2,4,7,9-tetramethyl-5-decyne-4,7-diol, this latter compound presumably resulting from environmental contamination. Important differences have been detected between both types of sea bass, and also among individuals inside each group. Farmed specimens are richer in volatile metabolites than the wild counterparts; however, these latter, in general, contain a high number and abundance of metabolites resulting from microbial and enzymatic non-oxidative activity than the former. Clear differences in the volatile metabolites of wild and farmed sea bass have been found. A great deal of valuable information on sea bass volatile metabolites has been obtained, which can be useful in understanding certain aspects of the quality and safety of raw and processed sea bass. © 2015 Society of Chemical Industry.

Identification, Synthesis, and Biological Evaluation of the Major Human Metabolite of NLRP3 Inflammasome Inhibitor MCC950

PubMed Central

2016-01-01

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC–MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro-s-indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC–MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC–MS/MS showed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide (2a). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a, with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety. PMID:27994733

Identification, Synthesis, and Biological Evaluation of the Major Human Metabolite of NLRP3 Inflammasome Inhibitor MCC950.

PubMed

Salla, Manohar; Butler, Mark S; Pelingon, Ruby; Kaeslin, Geraldine; Croker, Daniel E; Reid, Janet C; Baek, Jong Min; Bernhardt, Paul V; Gillam, Elizabeth M J; Cooper, Matthew A; Robertson, Avril A B

2016-12-08

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC-MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro- s -indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC-MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC-MS/MS showed the metabolite was R -(+)- N -((1-hydroxy-1,2,3,5,6,7-hexahydro- s -indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide ( 2a ). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a , with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety.

Exploring the chemodiversity and biological activities of the secondary metabolites from the marine fungus Neosartorya pseudofischeri.

PubMed

Liang, Wan-Ling; Le, Xiu; Li, Hou-Jin; Yang, Xiang-Ling; Chen, Jun-Xiong; Xu, Jun; Liu, Huan-Liang; Wang, Lai-You; Wang, Kun-Teng; Hu, Kun-Chao; Yang, De-Po; Lan, Wen-Jian

2014-11-24

The production of fungal metabolites can be remarkably influenced by various cultivation parameters. To explore the biosynthetic potentials of the marine fungus, Neosartorya pseudofischeri, which was isolated from the inner tissue of starfish Acanthaster planci, glycerol-peptone-yeast extract (GlyPY) and glucose-peptone-yeast extract (GluPY) media were used to culture this fungus. When cultured in GlyPY medium, this fungus produced two novel diketopiperazines, neosartins A and B (1 and 2), together with six biogenetically-related known diketopiperazines,1,2,3,4-tetrahydro-2, 3-dimethyl-1,4-dioxopyrazino[1,2-a]indole (3), 1,2,3,4-tetrahydro-2-methyl-3-methylen e-1,4-dioxopyrazino[1,2-a]indole (4), 1,2,3,4-tetrahydro-2-methyl-1,3,4-trioxopyrazino[1,2-a] indole (5), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio)gliotoxin (11), didehydrobisdethiobis(methylthio)gliotoxin (12) and N-methyl-1H-indole-2-carboxamide (6). However, a novel tetracyclic-fused alkaloid, neosartin C (14), a meroterpenoid, pyripyropene A (15), gliotoxin (7) and five known gliotoxin analogues, acetylgliotoxin (8), reduced gliotoxin (9), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio) gliotoxin (11) and bis-N-norgliovictin (13), were obtained when grown in glucose-containing medium (GluPY medium). This is the first report of compounds 3, 4, 6, 9, 10 and 12 as naturally occurring. Their structures were determined mainly by MS, 1D and 2D NMR data. The possible biosynthetic pathways of gliotoxin-related analogues and neosartin C were proposed. The antibacterial activity of compounds 2-14 and the cytotoxic activity of compounds 4, 5 and 7-13 were evaluated. Their structure-activity relationships are also preliminarily discussed.

A pooled analysis of CYP2D6 genotype in breast cancer prevention trials of low-dose tamoxifen.

PubMed

Johansson, Harriet; Gandini, Sara; Serrano, Davide; Gjerde, Jennifer; Lattanzi, Monia; Macis, Debora; Guerrieri-Gonzaga, Aliana; Aristarco, Valentina; Mellgren, Gunnar; Lien, Ernst; DeCensi, Andrea; Bonanni, Bernardo

2016-08-01

Decreased CYP2D6 activity is associated with lower levels of active tamoxifen metabolites. We examined the impact of CYP2D6 genotype on tamoxifen pharmacokinetics, biomarker activity, and efficacy in a pooled analysis of low-dose tamoxifen. Four randomized breast cancer prevention trials of very-low-dose (1 mg/day, n = 52 or 10 mg/week, n = 152) or low-dose tamoxifen (5 mg/day, n = 171) were pooled. DNA from 367 subjects was genotyped for CYP2D6 alleles associated with absent (PM allele: *3, *4, *5, *6, *7, *8, *12, and *14), reduced (IM allele: *9, *10, *17, *29, *41), normal (EM allele), or increased (UM: *XN) enzyme activity. Associations of tamoxifen, metabolites, activity biomarkers, and event-free survival with rapid (UM/EM, UM/IM, EM/EM, EM/IM, or EM/PM alleles) versus slow metabolizers (PM/IM or PM/PM) were investigated through random effects models, with 'study' as the random factor, and Cox regression models, adjusting for confounders. Rapid metabolizers had higher endoxifen levels than slow metabolizers: 15.3 versus 12.2 ng/mL (P = 0.018) with 5 mg/day, and 3.8 versus 2.8 ng/mL (P = 0.004) with 1 mg/day or 10 mg/week tamoxifen. The IGF-I decrease correlated with endoxifen (P = 0.002) and 4-hydroxytamoxifen levels, demonstrating steeper decreases at higher metabolite levels (P = 0.001). After a median follow-up of 12 years, rapid metabolizers with prior history of breast neoplasms allocated to tamoxifen 5 mg/day had a 60 % reduction of risk of recurrences (HR = 0.40, 95 % CI: 0.16-0.99) compared to slow metabolizers. CYP2D6 genotype may have an impact on tamoxifen efficacy at low doses. Trials investigating tamoxifen dose adjustments based on the woman's hormonal context and CYP2D6 genotype are warranted.

Relationship between NH+4 Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity 1

PubMed Central

Vanlerberghe, Greg C.; Schuller, Kathryn A.; Smith, Ronald G.; Feil, Regina; Plaxton, William C.; Turpin, David H.

1990-01-01

The rate of NH4+ assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH4+ assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H14CO−3 into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C3 organism. PMID:16667699

GC-MS-based metabolite profiling of Cosmos caudatus leaves possessing alpha-glucosidase inhibitory activity.

PubMed

Javadi, Neda; Abas, Faridah; Abd Hamid, Azizah; Simoh, Sanimah; Shaari, Khozirah; Ismail, Intan Safinar; Mediani, Ahmed; Khatib, Alfi

2014-06-01

Cosmos caudatus, which is known as "Ulam Raja," is an herbal plant used in Malaysia to enhance vitality. This study focused on the evaluation of the α-glucosidase inhibitory activity of different ethanolic extracts of C. caudatus. Six series of samples extracted with water, 20%, 40%, 60%, 80%, and 100% ethanol (EtOH) were employed. Gas chromatography-mass spectrometry (GC-MS) and orthogonal partial least-squares (OPLS) analysis was used to correlate bioactivity of different extracts to different metabolite profiles of C. caudatus. The obtained OPLS scores indicated a distinct and remarkable separation into 6 clusters, which were indicative of the 6 different ethanol concentrations. GC-MS can be integrated with multivariate data analysis to identify compounds that inhibit α-glucosidase activity. In addition, catechin, α-linolenic acid, α-D-glucopyranoside, and vitamin E compounds were identified and indicate the potential α-glucosidase inhibitory activity of this herb. GC-MS and multivariate data analysis was applied to discriminate Cosmos caudatus samples extracted with water and different ratio of ethanol. Orthogonal partial least-squares (OPLS) model developed was used to determine the major metabolites contributed to α-glucosidase inhibitory activity. This approach also has the ability to predict the bioactivity of a new set of extracts based on a developed validated regression model that is important for quality control of the herb preparation. © 2014 Institute of Food Technologists®

Production of Secondary Metabolites in Extreme Environments: Food- and Airborne Wallemia spp. Produce Toxic Metabolites at Hypersaline Conditions

PubMed Central

Frisvad, Jens C.; Kocev, Dragi; Džeroski, Sašo; Gunde-Cimerman, Nina

2016-01-01

The food- and airborne fungal genus Wallemia comprises seven xerophilic and halophilic species: W. sebi, W. mellicola, W. canadensis, W. tropicalis, W. muriae, W. hederae and W. ichthyophaga. All listed species are adapted to low water activity and can contaminate food preserved with high amounts of salt or sugar. In relation to food safety, the effect of high salt and sugar concentrations on the production of secondary metabolites by this toxigenic fungus was investigated. The secondary metabolite profiles of 30 strains of the listed species were examined using general growth media, known to support the production of secondary metabolites, supplemented with different concentrations of NaCl, glucose and MgCl2. In more than two hundred extracts approximately one hundred different compounds were detected using high-performance liquid chromatography-diode array detection (HPLC-DAD). Although the genome data analysis of W. mellicola (previously W. sebi sensu lato) and W. ichthyophaga revealed a low number of secondary metabolites clusters, a substantial number of secondary metabolites were detected at different conditions. Machine learning analysis of the obtained dataset showed that NaCl has higher influence on the production of secondary metabolites than other tested solutes. Mass spectrometric analysis of selected extracts revealed that NaCl in the medium affects the production of some compounds with substantial biological activities (wallimidione, walleminol, walleminone, UCA 1064-A and UCA 1064-B). In particular an increase in NaCl concentration from 5% to 15% in the growth media increased the production of the toxic metabolites wallimidione, walleminol and walleminone. PMID:28036382

Disposition of Phenolic and Sulfated Metabolites after Inhalation Exposure to 4-Chlorobiphenyl (PCB3) in Female Rats

PubMed Central

2015-01-01

PCBs, such as PCB3, are air contaminants in buildings and outdoors. Metabolites of PCB3 are potential endocrine disrupting chemicals and genotoxic agents. We studied the disposition of phenolic and sulfated metabolites after acute nose-only inhalation exposure to airborne PCB3 for 2 h in female rats. Inhalation exposure was carried out in three groups. In the first group, rats exposed to an estimated dose of 26 μg/rat were euthanized at 0, 1, 2, and 4 h after exposure. Highest concentrations of phenols and sulfates were observed at 0 h, and the values were 7 ± 1 and 560 ± 60 ng/mL in serum, 213 ± 120 and 842 ± 80 ng/g in liver, 31 ± 27 and 22 ± 7 ng/g in lung, and 27 ± 6 and 3 ± 0 ng/g in brain, respectively. First-order serum clearance half-lives of 0.5 h for phenols and 1 h for sulfates were estimated. In the second group, rats exposed to an estimated dose of 35 μg/rat were transferred to metabolism cages immediately after exposure for the collection of urine and feces over 24 h. Approximately 45 ± 5% of the dose was recovered from urine and consisted mostly of sulfates; the 18 ± 5% of the dose recovered from feces was exclusively phenols. Unchanged PCB3 was detected in both urine and feces but accounted for only 5 ± 3% of the dose. Peak excretion of metabolites in both urine and feces occurred within 18 h postexposure. In the third group, three bile-cannulated rats exposed to an estimated dose of 277 μg/rat were used for bile collection. Bile was collected for 4 h immediately after 2 h exposure. Biliary metabolites consisted mostly of sulfates, some glucuronides, and lower amounts of the free phenols. Control rats in each group were exposed to clean air. Clinical serum chemistry values, serum T4 level, and urinary 8-hydroxy-2′-deoxyguanosine were similar in treated and control rats. These data show that PCB3 is rapidly metabolized to phenols and conjugated to sulfates after inhalation and that both of these metabolites are distributed to liver

4-Pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?

PubMed

Synesiou, Elena; Fairbanks, Lynnette D; Simmonds, H Anne; Slominska, Ewa M; Smolenski, Ryszard T; Carrey, Elizabeth A

2011-06-01

We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD(+) metabolites (nicotinamide, N(1)-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N(1)-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD(+) from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD(+) analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD(+) analogue that inhibits IMP dehydrogenase in other cells.

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is Enantioselectively Oxidized to Hydroxylated Metabolites by Rat Liver Microsomes

PubMed Central

Wu, Xianai; Pramanik, Ananya; Duffel, Michael W.; Hrycay, Eugene G.; Bandiera, Stelvio M.; Lehmler, Hans-Joachim; Kania-Korwel, Izabela

2011-01-01

Developmental exposure to multiple-ortho substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB-), dexamethasone (DEX-) and corn oil (VEH-)treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzymes. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer didn't affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of

Trichoderma secondary metabolites that affect plant metabolism.

PubMed

Vinale, Francesco; Sivasithamparam, Krishnapillai; Ghisalberti, Emilio L; Ruocco, Michelina; Wood, Sheridan; Lorito, Matteo

2012-11-01

Recently, there have been many exciting new developments relating to the use of Trichoderma spp. as agents for biocontrol of pathogens and as plant growth promoters. Several mechanisms have been proposed to explain the positive effects of these microorganisms on the plant host. One factor that contributes to their beneficial biological activities is related to the wide variety of metabolites that they produce. These metabolites have been found not only to directly inhibit the growth and pathogenic activities of the parasites, but also to increase disease resistance by triggering the system of defence in the plant host. In addition, these metabolites are also capable of enhancing plant growth, which enables the plant to counteract the disease with compensatory vegetative growth by the augmented production of root and shoot systems. This review takes into account the Trichoderma secondary metabolites that affect plant metabolism and that may play an important role in the complex interactions of this biocontrol agent with the plant and pathogens.

Flavone Biotransformation by Aspergillus niger and the Characterization of Two Newly Formed Metabolites

PubMed Central

Assawah, Suzan W.; El-Sharkawy, Saleh H.; Abdel-Salam, Amal

2008-01-01

Aspergillus niger isolated from Allium sativum was used at large scale fermentation (150 mg flavone/200 ml medium) to obtain suitable amounts of the products, efficient for identification. Then spectral analysis (UV, IR, 1H-NMR, 13C-NMR) and mass spectrometry were performed for the two products, which contributed to the identification process. The metabolite (1) was identified as 2'-hydroxydihydrochalcone, and the metabolite (2) was identified as 2'-hydroxyphenylmethylketone, which were more active than flavone itself. Antioxidant activities of the two isolated metabolites were tested compared with ascorbic acid. Antioxidant activity of metabolite (1) was recorded 64.58% which represented 79% of the antioxidant activity of ascorbic acid, and metabolite (2) was recorded 54.16% (67% of ascorbic acid activity). However, the antioxidant activity of flavone was recorded 37.50% which represented 46% of ascorbic acid activity. The transformed products of flavone have antimicrobial activity against Pseudomonas aeruginosa, Aspergillus flavus and Candida albicans, with MIC was recorded 250 µg/ml for metabolite (2) against all three organism and 500, 300, and 300 µg/ml for metabolite (1) against tested microorganisms (P. aeruginosa, Escherichia coli, Bacillus subtilis, and Klebsiella pneumonia, Fusarium moniliforme, A. flavus, Saccharomyces cerviceae, Kluveromyces lactis and C. albicans) at this order. PMID:23990746

Kynurenine pathway metabolites are associated with hippocampal activity during autobiographical memory recall in patients with depression.

PubMed

Young, Kymberly D; Drevets, Wayne C; Dantzer, Robert; Teague, T Kent; Bodurka, Jerzy; Savitz, Jonathan

2016-08-01

Inflammation-related changes in the concentrations of inflammatory mediators such as c-reactive protein (CRP), interleukin 1β (IL-1), and IL-6 as well as kynurenine metabolites are associated with major depressive disorder (MDD) and affect depressive behavior, cognition, and hippocampal plasticity in animal models. We previously reported that the ratios of kynurenic acid (KynA) to the neurotoxic metabolites, 3-hydroxykynurenine (3HK) and quinolinic acid (QA), were positively correlated with hippocampal volume in depression. The hippocampus is critical for autobiographical memory (AM) recall which is impaired in MDD. Here we tested whether the ratios, KynA/3HK and KynA/QA were associated with AM recall performance as well as hippocampal activity during AM recall. Thirty-five unmedicated depressed participants and 25 healthy controls (HCs) underwent fMRI scanning while recalling emotionally-valenced AMs and provided serum samples for the quantification of kynurenine metabolites, CRP, and cytokines (IL-1 receptor antagonist - IL-1RA; IL-6, tumor necrosis factor alpha - TNF, interferon gamma -IFN-γ, IL-10). KynA/3HK and KynA/QA were lower in the MDD group relative to the HCs. The concentrations of the CRP and the cytokines did not differ significantly between the HCs and the MDD group. Depressed individuals recalled fewer specific AMs and displayed increased left hippocampal activity during the recall of positive and negative memories. KynA/3HK was inversely associated with left hippocampal activity during specific AM recall in the MDD group. Further, KynA/QA was positively correlated with percent negative specific memories recalled in the MDD group and showed a non-significant trend toward a positive correlation with percent positive specific memories recalled in HCs. In contrast, neither CRP nor the cytokines were significantly associated with AM recall or activity of the hippocampus during AM recall. Conceivably, an imbalance in levels of KynA versus QA

Baicalin and its metabolites suppresses gluconeogenesis through activation of AMPK or AKT in insulin resistant HepG-2 cells.

PubMed

Wang, Tao; Jiang, Hongmei; Cao, Shijie; Chen, Qian; Cui, Mingyuan; Wang, Zhijie; Li, Dandan; Zhou, Jing; Wang, Tao; Qiu, Feng; Kang, Ning

2017-12-01

Scutellaria baicalensis Georgi (S. baicalensis), as a traditional Chinese herbal medicine, is an important component of several famous Chinese medicinal formulas for treating patients with diabetes mellitus. Baicalin (BG), a main bioactive component of S. baicalensis, has been reported to have antidiabetic effects. However, pharmacokinetic studies have indicated that BG has poor oral bioavailability. Therefore, it is hard to explain the pharmacological effects of BG in vivo. Interestingly, several reports show that BG is extensively metabolized in rats and humans. Therefore, we speculate that the BG metabolites might be responsible for the pharmacological effects. In this study, BG and its three metabolites (M1-M3) were examined their effects on glucose consumption in insulin resistant HepG-2 cells with a commercial glucose assay kit. Real-time PCR and western blot assay were used to confirm genes and proteins of interest, respectively. The results demonstrate that BG and its metabolites (except for M3) enhanced the glucose consumption which might be associated with inhibiting the expression of the key gluconeogenic genes, including glucose-6-phosphatase (G6Pase), phosphoenolypyruvate carboxykinase (PEPCK) and glucose transporter 2 (GLUT2). Further study found that BG and M1 could suppress hepatic gluconeogenesis via activation of the AMPK pathway, while M2 could suppress hepatic gluconeogenesis via activation of the PI3K/AKT signaling pathway. Taken together, our findings suggest that both BG and its metabolites have antihyperglycemic activities, and might be the active forms of oral doses of BG in vivo. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Effect of Food on the Single-dose Pharmacokinetics and Tolerability of Subutinib and its Active Metabolite in Chinese Healthy Volunteers.

PubMed

Ding, L-K; Jia, N; Yang, L; Li, J-K; Song, W; Wang, M-H; Wang, C; Gao, X-H; Wen, A-D

2016-03-01

The aim of this study is to investigate a food effect on the single-dose pharmacokinetics and tolerability of subutinib maleate capsules in healthy Chinese volunteers. The author evaluated the effect of being under a fasting or fed state at the time of drug intake on the single-dose of subutinib maleate capsules in a randomized, balanced, single-dose, 2-treatment (fasting and fed), 2-period design with a 3-week washout period. The end points were the maximum plasma drug concentration (Cmax) and areas under the plasma-concentration curve (AUC) for 336 h exposure (AUC0-336) and total exposure (AUC0-∞). All volunteers completed the whole study without side effects being observed. For subutinib, Cmax were 6.13 and 5.04 ng·mL(-1), and AUC0-336 were 278.4 and 304.5 h·ng·mL(-1) in the fasting and the fed state, respectively. For active metabolite, Cmax were 0.90 and 0.61 ng·mL(-1), and AUC0-336 were 65.5 and 56.4 h·ng·mL(-1) in the fasting and the fed state, respectively. The authors showed that food intake was associated with a slight increase in AUC values but decrease in Cmax of subutinib, and it was associated with a decrease both in AUC and Cmax of active metabolite. © Georg Thieme Verlag KG Stuttgart · New York.

EFFECTS OF METHOPRENE, ITS METABOLITES, AND BREAKDOWN PRODUCTS ON RETINOID-ACTIVATED PATHWAYS IN TRANSFECTED CELL LINES

EPA Science Inventory

Methoprene is a terpene-based insecticide designed to act as an agonist of insect juvenile hormone, which is essential for the transition from larval to adult forms in some metamorphic insects. Recent evidence suggests that a methoprene metabolite, methoprene acid, activates a ve...

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

PubMed

Lechuga, Carmen G; Simón-Carrasco, Lucía; Jacob, Harrys K C; Drosten, Matthias

2017-01-01

Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Ras lox (H-Ras -/- , N-Ras -/- , K-Ras lox/lox , RERT ert/ert ) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

Concentrations of isoflavones and their metabolites in the blood of pregnant and non-pregnant heifers fed soy bean.

PubMed

Woclawek-Potocka, Izabela; Piskula, Mariusz Krzysztof; Bah, Mamadou; Siemieniuch, Marta Jolanta; Korzekwa, Anna; Brzezicka, Edyta; Skarzynski, Dariusz Jan

2008-10-01

The present study compared the changes in isoflavones (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cyclic and pregnant heifers after feeding with soy bean. Twelve healthy heifers were divided into three groups: cyclic heifers (days 8-12 of the estrous cycle; control group; n=4), an early pregnancy group (2 months pregnant; n=4) and a late pregnancy group (8 months pregnant; n=4). All heifers were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on an HPLC system. In the blood plasma of the early- and late-pregnant heifers, we found lower concentrations and time-dependent decreases in daidzein and genistein in comparison to cyclic heifers (P<0.05). Moreover, we noticed significant increases of equol and para-ethyl-phenol in the blood plasma of the early-pregnant heifers (P<0.05). In contrast, in the blood plasma of the late-pregnant heifers, we did not find an increase in the isoflavone metabolite concentrations compared with the early-pregnant heifers (P>0.05). In conclusion, physiological status (cyclicity or pregnancy) of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the heifers. The stage of pregnancy affects isoflavone absorption, biotransformation and metabolism differently and results in higher concentrations of active metabolites of isoflavones during early pregnancy in comparison to their lower concentrations during late pregnancy. Therefore, we surmise that cows are more sensitive to active isoflavone metabolite actions during early pregnancy than cyclic heifers and heifers in late pregnancy.

Depsides: Lichen Metabolites Active against Hepatitis C Virus

PubMed Central

Vu, Thi Huyen; Le Lamer, Anne-Cécile; Lalli, Claudia; Boustie, Joël; Samson, Michel

2015-01-01

A thorough phytochemical study of Stereocaulon evolutum was conducted, for the isolation of structurally related atranorin derivatives. Indeed, pilot experiments suggested that atranorin (1), the main metabolite of this lichen, would interfere with the lifecycle of hepatitis C virus (HCV). Eight compounds, including one reported for the first time (2), were isolated and characterized. Two analogs (5, 6) were also synthesized, to enlarge the panel of atranorin-related structures. Most of these compounds were active against HCV, with a half-maximal inhibitory concentration of about 10 to 70 µM, with depsides more potent than monoaromatic phenols. The most effective inhibitors (1, 5 and 6) were then added at different steps of the HCV lifecycle. Interestingly, atranorin (1), bearing an aldehyde function at C-3, inhibited only viral entry, whereas the synthetic compounds 5 and 6, bearing a hydroxymethyl and a methyl function, respectively, at C-3 interfered with viral replication. PMID:25793970

The Significance of Lichens and Their Metabolites

NASA Astrophysics Data System (ADS)

Huneck, S.

Lichens, symbiontic organisms of fungi and algae, synthesize numerous metabolites, the "lichen substances," which comprise aliphatic, cycloaliphatic, aromatic, and terpenic compounds. Lichens and their metabolites have a manifold biological activity: antiviral, antibiotic, antitumor, allergenic, plant growth inhibitory, antiherbivore, and enzyme inhibitory. Usnic acid, a very active lichen substance is used in pharmaceutical preparations. Large amounts of Pseudevernia furfuracea and Evernia prunastri are processed in the perfume industry, and some lichens are sensitive reagents for the evaluation of air pollution.

Insights into the mechanisms of Promysalin, a secondary metabolite with genus-specific antibacterial activity against Pseudomonas

USDA-ARS?s Scientific Manuscript database

Promysalin, a secondary metabolite produced by Pseudomonas putida RW10S1, has antibacterial activity against a wide variety of Pseudomonas sp., including both human and plant pathogens. Promysalin induces swarming and biofilm formation in the producing species, and inhibits growth of susceptible sp...

Absorption rates and free radical scavenging values of vitamin C-lipid metabolites in human lymphoblastic cells.

PubMed

Weeks, Benjamin S; Perez, Pedro P

2007-10-01

In this study we investigated the cellular absorption rates, antioxidant and free radical scavenging activity of vitamin C-lipid metabolites. The absorption was measured in a human lymphoblastic cell line using a spectrophotometric technique. Cellular vitamin C levels in the human lymphoblastic H9 cell line were measured using the 2,4-dinitrophenylhydrazine spectrophotometric technique. Free radical scavenging activity of vitamin C-lipid metabolites was measured by the reduction of 1,1-diphenyl-2-picryl hydrazyl (DPPH) to 1,1-diphenyl-2-picryl hydrazine. Vitamin C-lipid metabolite scavenging of peroxyl radical oxygen reactive species (ORAC) was determined by fluorescence spectrophotometry. Compared to ascorbic acid (AA), calcium ascorbate (CaA), and calcium ascorbate-calcium threonate-dehydroascorbate (Ester-C), vitamin C-lipid metabolites (PureWay-C) were more rapidly absorbed by the H9 human T-lymphocytes. The vitamin C-lipid metabolites (PureWay-C) also reduced pesticide-induced T-lymphocyte aggregation by 84%, while calcium ascorbate-calcium threonate-dehydroascorbate (Ester-C) reduced aggregation by only 34%. The vitamin C-lipid metabolites (PureWay-C) demonstrated free radical scavenging activity of nearly 100% reduction of DPPH at 20 microg/ml and oxygen radical scavenging of over 1200 micro Trolox equivalents per gram. These data demonstrate that the vitamin C-lipid metabolites (PureWay-C) are more rapidly taken-up and absorbed by cells than other forms of vitamin C, including Ester-C. This increased rate of absorption correlates with an increased protection of the T-lymphocytes from pesticide toxicities. Further, vitamin C-lipid metabolites (PureWay-C) are a potent antioxidant and have significant free radical scavenging capabilities.

α-Glucosidase Inhibition and Antibacterial Activity of Secondary Metabolites from the Ecuadorian Species Clinopodium taxifolium (Kunth) Govaerts.

PubMed

Morocho, Vladimir; Valle, Andrea; García, Jessica; Gilardoni, Gianluca; Cartuche, Luis; Suárez, Alírica I

2018-01-11

The phytochemical investigation of both volatile and fixed metabolites of Clinopodium taxifolium (Kunth) Govaerts (Lamiaceae) was performed for the first time. It allowed the isolation and characterization of the essential oil and six known compounds: carvacrol ( 1 ), squalane ( 2 ), uvaol ( 3 ), erythrodiol ( 4 ), ursolic acid ( 5 ), and salvigenin ( 6 ). Their structures were identified and characterized by Nuclear Magnetic Resonance (NMR) and Gas Chromatography coupled to Mass Spectroscopy (GC-MS), and corroborated by literature. The essential oil of the leaves was obtained by hydrodistillation in two different periods and analyzed by GC-MS and GC coupled to Flame Ionization Detector (GC-FID). A total of 54 compounds were detected, of which 42 were identified (including trace constituents). The major constituents were carvacrol methyl ether (18.9-23.2%), carvacrol (13.8-16.3%) and, carvacryl acetate (11.4-4.8%). The antibacterial activities were determined as Minimum Inhibition Concentration (MIC) against Staphylococcus aureus , Enterococcus faecalis , Escherichia coli , Klebsiella pneumoniae , Proteus vulgaris , Pseudomonas aeruginosa and Micrococcus luteus . The hexane and methanol extracts exhibited activity only against Klebsiella pneumoniae (250 and 500 μg/mL respectively), while the ethyl acetate extract was inactive. The hypoglycemic activity was evaluated by the in vitro inhibition of α-glucosidase. The ethyl acetate (EtOAc) extract showed strong inhibitory activity with IC 50 = 24.88 µg/mL, however methanolic and hexanic extracts showed weak activity. As a pure compound, only ursolic acid showed a strong inhibitory activity, with IC 50 = 72.71 μM.

Determination of the 4-monohydroxy metabolites of perhexiline in human plasma, urine and liver microsomes by liquid chromatography.

PubMed

Davies, Benjamin J; Herbert, Megan K; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

2006-11-07

The use of perhexiline (PHX) is limited by hepatic and neurological toxicity associated with elevated concentrations in plasma that are the result of polymorphism of the cytochrome P450 2D6 isoform (CYP2D6). PHX is cleared by hepatic oxidation that produces three 4-monohydroxy metabolites: cis-OH-PHX, trans1-OH-PHX and trans2-OH-PHX. The current study describes an HPLC-fluorescent method utilising pre-column derivatization with dansyl chloride. Following derivatization, the metabolites were resolved on a C18 column with a gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of the metabolites in human plasma and urine following clinical doses and for kinetic studies using human liver microsomes. The method demonstrates sufficient sensitivity, accuracy and precision between 5.0 and 0.01, 50.0 and 0.2 and 1.0 and 0.005 mg/l in human plasma, urine and liver microsomes, respectively, with intra-assay coefficients of variation and bias <15%, except at the lowest limit of quantification (<20%). The inter-assay coefficients of variation and bias were <15%. The application of this method to plasma and urine samples of five CYP2D6 extensive metaboliser (EM) patients at steady state with respect to PHX dosing determined that the mean (+/-S.D.) renal clearances of trans1-OH-PHX and cis-OH-PHX were 1.58+/-0.35 and 0.16+/-0.06l/h, respectively. The mean (+/-S.D.) dose recovered in urine as free and glucuronidated 4-monohydroxy PHX metabolites was 20.6+/-11.6%.

New Pioglitazone Metabolites and Absence of Opened-Ring Metabolites in New N-Substituted Thiazolidinedione.

PubMed

Campos, Michel Leandro; Cerqueira, Letícia Bonancio; Silva, Bruna Cristina Ulian; Franchin, Taísa Busaranho; Galdino-Pitta, Marina Rocha; Pitta, Ivan Rocha; Peccinini, Rosângela Gonçalves; Pontarolo, Roberto

2018-06-01

Thiazolidinediones (TZDs) are drugs used to treat type 2 diabetes mellitus; however, several safety concerns remain regarding the available drugs in this class. Therefore, the search for new TZD candidates is ongoing; metabolism studies play a crucial step in the development of new candidates. Pioglitazone, one of the most commonly used TZDs, and GQ-11, a new N -substituted TZD, were investigated in terms of their metabolic activity in rat and human liver microsomes to assess their metabolic stability and investigate their metabolites. Methods for preparation of samples were based on liquid-liquid extraction and protein precipitation. Quantitation was performed using liquid chromatography (LC)-tandem mass spectrometry, and the metabolite investigation was performed using ultraperformance LC coupled to a hybrid quadrupole-time of flight mass spectrometer. The predicted intrinsic clearance of GQ-11 was 70.3 and 46.1 ml/kg per minute for rats and humans, respectively. The predicted intrinsic clearance of pioglitazone was 24.1 and 15.9 ml/kg per minute for rats and humans, respectively. The pioglitazone metabolite investigation revealed two unpublished metabolites (M-D and M-A). M-A is a hydration product and may be related to the mechanism of ring opening and the toxicity of pioglitazone. The metabolites of GQ-11 are products of oxidation; no ring-opening metabolite was observed for GQ-11. In conclusion, under the same experimental conditions, a ring-opening metabolite was observed only for pioglitazone. The resistance of GQ-11 to the ring opening is probably related to N -substitution in the TZD ring. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Identification of minor secondary metabolites from the latex of Croton lechleri (Muell-Arg) and evaluation of their antioxidant activity.

PubMed

De Marino, Simona; Gala, Fulvio; Zollo, Franco; Vitalini, Sara; Fico, Gelsomina; Visioli, Francesco; Iorizzi, Maria

2008-06-01

Dragon's blood (Sangre de drago), a viscous red sap derived from Croton lechleri Muell-Arg (Euphorbiaceae), is extensively used by indigenous cultures of the Amazonian basin for its wound healing properties. The aim of this study was to identify the minor secondary metabolites and test the antioxidant activity of this sustance. A bioguided fractionation of the n-hexane, chloroform, n-butanol, and aqueous extracts led to the isolation of 15 compounds: three megastigmanes, four flavan-3-ols, three phenylpropanoids, three lignans, a clerodane, and the alkaloid taspine. In addition to these known molecules, six compounds were isolated and identified for the first time in the latex: blumenol B, blumenol C, 4,5-dihydroblumenol A, erythro-guaiacyl-glyceryl-beta-O-4'- dihydroconiferyl ether, 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol and floribundic acid glucoside. Combinations of spectroscopic methods ((1)H-, (13)C- NMR and 2D-NMR experiments), ESI-MS, and literature comparisons were used for compound identification. In vitro antioxidant activities were assessed by DPPH, total antioxidant capacity and lipid peroxidation assays. Flavan-3-ols derivatives (as major phenolic compounds in the latex) exhibited the highest antioxidant activity.

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

Ribeiro, Mariana P.C.; Laboratory of Biochemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra; Nunes-Correia, Isabel

Recent reports suggest that N-methyl-D-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. Inmore » contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination. - Highlights: • MK-801 and memantine decrease melanoma cell proliferation. • The combination of MK-801 with antiestrogens inhibits melanoma cell proliferation. • These combinations greatly enhance the effects of the compounds individually. • MK-801 combined with tamoxifen active metabolites induces cell cycle arrest in G1. • The combination of MK-801 and antiestrogens is an innovative strategy for melanoma.« less

Pharmacokinetics of intravenous and oral amitriptyline and its active metabolite nortriptyline in Greyhound dogs.

PubMed

Norkus, Christopher; Rankin, David; KuKanich, Butch

2015-11-01

To evaluate the pharmacokinetics of amitriptyline and its active metabolite nortriptyline after intravenous (IV) and oral amitriptyline administration in healthy dogs. Prospective randomized experiment. Five healthy Greyhound dogs (three males and two females) aged 2-4 years and weighing 32.5-39.7 kg. After jugular vein catheterization, dogs were administered a single oral or IV dose of amitriptyline (4 mg kg(-1)). Blood samples were collected at predetermined time points from baseline (0 hours) to 32 hours after administration and plasma concentrations of amitriptyline and nortriptyline were measured by liquid chromatography triple quadrupole mass spectrometry. Non-compartmental pharmacokinetic analyses were performed. Orally administered amitriptyline was well tolerated, but adverse effects were noted after IV administration. The mean maximum plasma concentration (CMAX) of amitriptyline was 27.4 ng mL(-1) at 1 hour and its mean terminal half-life was 4.33 hours following oral amitriptyline. Bioavailability of oral amitriptyline was 6%. The mean CMAX of nortriptyline was 14.4 ng mL(-1) at 2.05 hours and its mean terminal half-life was 6.20 hours following oral amitriptyline. Amitriptyline at 4 mg kg(-1) administered orally produced low amitriptyline and nortriptyline plasma concentrations. This brings into question whether the currently recommended oral dose of amitriptyline (1-4 mg kg(-1)) is appropriate in dogs. © 2015 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia.

Drug Metabolism in Human Brain: High Levels of Cytochrome P4503A43 in Brain and Metabolism of Anti-Anxiety Drug Alprazolam to Its Active Metabolite

PubMed Central

Agarwal, Varsha; Kommaddi, Reddy P.; Valli, Khader; Ryder, Daniel; Hyde, Thomas M.; Kleinman, Joel E.; Strobel, Henry W.; Ravindranath, Vijayalakshmi

2008-01-01

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more α-hydroxy alprazolam (α-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both α-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of α-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of α-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action. PMID:18545703

Enzyme inhibitory metabolites from endophytic Penicillium citrinum isolated from Boswellia sacra.

PubMed

Ali, Sajid; Khan, Abdul Latif; Ali, Liaqat; Rizvi, Tania Shamim; Khan, Sumera Afzal; Hussain, Javid; Hamayun, Muhammad; Al-Harrasi, Ahmed

2017-07-01

Fungal endophytes establish an important niche within the host plant through the secretion of chemical constituents. Isolation of bioactive metabolites could be a vital source for inhibiting the function of enzymes such as α-glucosidase and urease. The present study aimed to elucidate the potential of endophytes associated with Boswellia sacra through bioassay-guided isolation and identification of secondary metabolites with enzyme inhibitory ability. Endophytic fungal strains viz. Penicillium citrinum, P. spinulosum, Fusarium oxysporum, Alternaria alternata and Aspergillus caespitosus were identified through genomic DNA extraction, PCR amplification, sequencing and phylogenetic analysis. The enzymes inhibition analysis of the ethyl acetate extract from pure cultures suggested that P. citrinum possess significantly higher enzyme inhibitory activities compared to other strains. The active strain was subjected to chromatographic isolation and nuclear magnetic resonance methods to identify bioactive compounds. The bioactive extracts resulted in the isolation of 11-oxoursonic acid benzyl ester (1), n-nonane (2), 3-decene-1-ol (3), 2-Hydroxyphenyl acetic acid (4), and Glochidacuminosides A (5). Among pure compound, 11-oxoursonic acid benzyl ester (1) showed significantly higher enzyme inhibition activity compared to other metabolites. Our results suggest that the endophytic microorganism associated with the arid-land tree can offer a rich source of biologically active chemical constituents that could help discover lead drugs for enzyme inhibition.

Prediction of Losartan-Active Carboxylic Acid Metabolite Exposure Following Losartan Administration Using Static and Physiologically Based Pharmacokinetic Models.

PubMed

Nguyen, Hoa Q; Lin, Jian; Kimoto, Emi; Callegari, Ernesto; Tse, Susanna; Obach, R Scott

2017-09-01

The aim of this study was to evaluate a strategy based on static and dynamic physiologically based pharmacokinetic (PBPK) modeling for the prediction of metabolite and parent drug area under the time-concentration curve ratio (AUC m /AUC p ) and their PK profiles in humans using in vitro data when active transport processes are involved in disposition. The strategy was applied to losartan and its pharmacologically active metabolite carboxylosartan as test compounds. Hepatobiliary transport including transport-mediated uptake, canilicular and basolateral efflux, and metabolic clearance estimates were obtained from in vitro studies using human liver microsomes and sandwich-cultured hepatocytes. Human renal clearance of carboxylosartan was estimated from dog renal clearance using allometric scaling approach. All clearance mechanisms were mechanistically incorporated in a static model to predict the relative exposure of carboxylosartan versus losartan (AUC m /AUC p ). The predicted AUC m /AUC p were consistent with the observed data following intravenous and oral administration of losartan. Moreover, the in vitro parameters were used as initial parameters in PBPK permeability-limited disposition models to predict the concentration-time profiles for both parent and its active metabolite after oral administration of losartan. The PBPK model was able to recover the plasma profiles of both losartan and carboxylosartan, further substantiating the validity of this approach. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Influence of Different Drying Treatments and Extraction Solvents on the Metabolite Profile and Nitric Oxide Inhibitory Activity of Ajwa Dates.

PubMed

Abdul-Hamid, Nur Ashikin; Abas, Faridah; Ismail, Intan Safinar; Shaari, Khozirah; Lajis, Nordin H

2015-11-01

This study aimed to examine the variation in the metabolite profiles and nitric oxide (NO) inhibitory activity of Ajwa dates that were subjected to 2 drying treatments and different extraction solvents. (1)H NMR coupled with multivariate data analysis was employed. A Griess assay was used to determine the inhibition of the production of NO in RAW 264.7 cells treated with LPS and interferon-γ. The oven dried (OD) samples demonstrated the absence of asparagine and ascorbic acid as compared to the freeze dried (FD) dates. The principal component analysis showed distinct clusters between the OD and FD dates by the second principal component. In respect of extraction solvents, chloroform extracts can be distinguished by the absence of arginine, glycine and asparagine compared to the methanol and 50% methanol extracts. The chloroform extracts can be clearly distinguished from the methanol and 50% methanol extracts by first principal component. Meanwhile, the loading score plot of partial least squares analysis suggested that beta glucose, alpha glucose, choline, ascorbic acid and glycine were among the metabolites that were contributing to higher biological activity displayed by FD and methanol extracts of Ajwa. The results highlight an alternative method of metabolomics approach for determination of the metabolites that contribute to NO inhibitory activity. The association between metabolite profiles and nitric oxide (NO) inhibitory activity of the various extracts of Ajwa dates was evaluated by utilizing partial least squares (PLS) model. The validated PLS model can be employed to predict the NO inhibitory activity of new samples of date fruits based on their NMR spectra which was important for assessing fruit quality. The information gained might be used as guidance for quality control, nutritional values and as a basis for the preparation of any food supplements for human health that employs date palm fruit as the raw material. © 2015 Institute of Food

Endophytic Streptomyces in the traditional medicinal plant Arnica montana L.: secondary metabolites and biological activity.

PubMed

Wardecki, Tina; Brötz, Elke; De Ford, Christian; von Loewenich, Friederike D; Rebets, Yuriy; Tokovenko, Bogdan; Luzhetskyy, Andriy; Merfort, Irmgard

2015-08-01

Arnica montana L. is a medical plant of the Asteraceae family and grows preferably on nutrient poor soils in mountainous environments. Such surroundings are known to make plants dependent on symbiosis with other organisms. Up to now only arbuscular mycorrhizal fungi were found to act as endophytic symbiosis partners for A. montana. Here we identified five Streptomyces strains, microorganisms also known to occur as endophytes in plants and to produce a huge variety of active secondary metabolites, as inhabitants of A. montana. The secondary metabolite spectrum of these strains does not contain sesquiterpene lactones, but consists of the glutarimide antibiotics cycloheximide and actiphenol as well as the diketopiperazines cyclo-prolyl-valyl, cyclo-prolyl-isoleucyl, cyclo-prolyl-leucyl and cyclo-prolyl-phenylalanyl. Notably, genome analysis of one strain was performed and indicated a huge genome size with a high number of natural products gene clusters among which genes for cycloheximide production were detected. Only weak activity against the Gram-positive bacterium Staphylococcus aureus was revealed, but the extracts showed a marked cytotoxic activity as well as an antifungal activity against Candida parapsilosis and Fusarium verticillioides. Altogether, our results provide evidence that A. montana and its endophytic Streptomyces benefit from each other by completing their protection against competitors and pathogens and by exchanging plant growth promoting signals with nutrients.

Solving the Jigsaw Puzzle of Wound-Healing Potato Cultivars: Metabolite Profiling and Antioxidant Activity of Polar Extracts

PubMed Central

2015-01-01

Potato (Solanum tuberosum L.) is a worldwide food staple, but substantial waste accompanies the cultivation of this crop due to wounding of the outer skin and subsequent unfavorable healing conditions. Motivated by both economic and nutritional considerations, this metabolite profiling study aims to improve understanding of closing layer and wound periderm formation and guide the development of new methods to ensure faster and more complete healing after skin breakage. The polar metabolites of wound-healing tissues from four potato cultivars with differing patterns of tuber skin russeting (Norkotah Russet, Atlantic, Chipeta, and Yukon Gold) were analyzed at three and seven days after wounding, during suberized closing layer formation and nascent wound periderm development, respectively. The polar extracts were assessed using LC-MS and NMR spectroscopic methods, including multivariate analysis and tentative identification of 22 of the 24 biomarkers that discriminate among the cultivars at a given wound-healing time point or between developmental stages. Differences among the metabolites that could be identified from NMR- and MS-derived biomarkers highlight the strengths and limitations of each method, also demonstrating the complementarity of these approaches in terms of assembling a complete molecular picture of the tissue extracts. Both methods revealed that differences among the cultivar metabolite profiles diminish as healing proceeds during the period following wounding. The biomarkers included polyphenolic amines, flavonoid glycosides, phenolic acids and glycoalkaloids. Because wound healing is associated with oxidative stress, the free radical scavenging activities of the extracts from different cultivars were measured at each wounding time point, revealing significantly higher scavenging activity of the Yukon Gold periderm especially after 7 days of wounding. PMID:24998264

Association of environmental chemicals & estrogen metabolites in children.

PubMed

Ihde, Erin Speiser; Loh, Ji Meng; Rosen, Lawrence

2015-12-17

The prevalence of pediatric hormonal disorders and hormonally-sensitive cancers are rising. Chemicals including bisphenol A (BPA), phthalates, parabens, 4-nonylphenol (4NP) and triclosan have been linked to disruption of endocrine pathways and altered hormonal status in both animal and human studies. Additionally, changes in estrogen metabolism have been associated with pediatric endocrine disorders and linked to estrogen-dependent cancers. The main objective of the study was to measure the presence of these environmental chemicals in prepubescent children and assess the relationship between chemical metabolites and estrogen metabolism. 50 subjects (25 male, 25 female) were recruited from the principal investigator's existing patient population at his pediatric primary care office. The first 5 boys and 5 girls in each age group (4 through 8 years old inclusive) who presented for annual examinations were included, as long as they were Tanner Stage I (prepubertal) on physical exam, without diagnosis of hormonally-related condition and/or cancer and able to give a urine sample. Urine samples were collected in glass containers for analysis of chemical and estrogen metabolites. Study kits and lab analysis were provided by Genova Diagnostics (Duluth, GA). Summary statistics for the concentrations of each chemical metabolite as well as estrogen metabolites were computed (minimum, maximum, median and inter-quartile range) for males only, for females only and for all subjects. Comparisons between groups (e.g. males v. females) were assessed using the nonparametric Wilcoxon test, since the data was skewed. The correlation between concentrations of chemical metabolites and estrogen metabolites in prepubescent children were examined by the Spearman's correlation coefficient (ρ). 100 % of subjects had detectable levels of at least five chemicals [corrected] in their urine, and 74 % had detectable levels of eight or more chemicals. 28 % of subjects had measurable levels of 4NP

Biodegradation of clofibric acid and identification of its metabolites.

PubMed

Salgado, R; Oehmen, A; Carvalho, G; Noronha, J P; Reis, M A M

2012-11-30

Clofibric acid (CLF) is the pharmaceutically active metabolite of lipid regulators clofibrate, etofibrate and etofyllinclofibrate, and it is considered both environmentally persistent and refractory. This work studied the biotransformation of CLF in aerobic sequencing batch reactors (SBRs) with mixed microbial cultures, monitoring the efficiency of biotransformation of CLF and the production of metabolites. The maximum removal achieved was 51% biodegradation (initial CLF concentration=2 mg L(-1)), where adsorption and abiotic removal mechanisms were shown to be negligible, showing that CLF is indeed biodegradable. Tests showed that the observed CLF biodegradation was mainly carried out by heterotrophic bacteria. Three main metabolites were identified, including α-hydroxyisobutyric acid, lactic acid and 4-chlorophenol. The latter is known to exhibit higher toxicity than the parent compound, but it did not accumulate in the SBRs. α-Hydroxyisobutyric acid and lactic acid accumulated for a period, where nitrite accumulation may have been responsible for inhibiting their degradation. A metabolic pathway for the biodegradation of CLF is proposed in this study. Copyright © 2012 Elsevier B.V. All rights reserved.

Synthesis and Evaluation of Vitamin D Receptor-Mediated Activities of Cholesterol and Vitamin D Metabolites

PubMed Central

Teske, Kelly A.; Bogart, Jonathan W.; Sanchez, Luis M.; Yu, Olivia B.; Preston, Joshua V.; Cook, James M.; Silvaggi, Nicholas R.; Bikle, Daniel D.; Arnold, Leggy A.

2016-01-01

A systematic study with phase 1 and phase 2 metabolites of cholesterol and vitamin D was conducted to determine whether their biological activity is mediated by the vitamin D receptor (VDR). The investigation necessitated the development of novel synthetic routes for lithocholic acid (LCA) glucuronides (Gluc). Biochemical and cell-based assays were used to demonstrate that hydroxylated LCA analogs were not able to bind VDR. This excludes VDR from mediating their biological and pharmacological activities. Among the synthesized LCA conjugates a novel VDR agonist was identified. LCA Gluc II increased the expression of CYP24A1 in DU145 cancer cells especially in the presence of the endogenous VDR ligand 1,25(OH)2D3. Furthermore, the methyl ester of LCA was identified as novel VDR antagonist. For the first time, we showed that calcitroic acid, the assumed inactive final metabolite of vitamin D, was able to activate VDR-mediated transcription to a higher magnitude than bile acid LCA. Due to a higher metabolic stability in comparison to vitamin D, a very low toxicity, and high concentration in bile and intestine, calcitroic acid is likely to be an important mediator of the protective vitamin D properties against colon cancer. PMID:26774929

The glycolytic metabolite methylglyoxal activates Pap1 and Sty1 stress responses in Schizosaccharomyces pombe.

PubMed

Zuin, Alice; Vivancos, Ana P; Sansó, Miriam; Takatsume, Yoshifumi; Ayté, José; Inoue, Yoshiharu; Hidalgo, Elena

2005-11-04

Methylglyoxal, a toxic metabolite synthesized in vivo during glycolysis, inhibits cell growth. One of the mechanisms protecting eukaryotic cells against its toxicity is the glyoxalase system, composed of glyoxalase I and II (glo1 and glo2), which converts methylglyoxal into d-lactic acid in the presence of glutathione. Here we have shown that the two principal oxidative stress response pathways of Schizosaccharomyces pombe, Sty1 and Pap1, are involved in the response to methylglyoxal toxicity. The mitogen-activated protein kinase Sty1 is phosphorylated and accumulates in the nucleus following methylglyoxal treatment. Moreover, glo2 expression is induced by methylglyoxal and environmental stresses in a Sty1-dependent manner. The transcription factor Pap1 also accumulates in the nucleus, activating the expression of its target genes following methylglyoxal treatment. Our studies showed that the C-terminal cysteine-rich domain of Pap1 is sufficient for methylglyoxal sensing. Furthermore, the redox status of Pap1 is not changed by methylglyoxal. We propose that methylglyoxal treatment triggers Pap1 and Sty1 nuclear accumulation, and we describe the molecular basis of such activation mechanisms. In addition, we discuss the potential physiological significance of these responses to a natural toxic metabolite.

Biosynthesis of the active compounds of Isatis indigotica based on transcriptome sequencing and metabolites profiling

PubMed Central

2013-01-01

Backgroud Isatis indigotica is a widely used herb for the clinical treatment of colds, fever, and influenza in Traditional Chinese Medicine (TCM). Various structural classes of compounds have been identified as effective ingredients. However, little is known at genetics level about these active metabolites. In the present study, we performed de novo transcriptome sequencing for the first time to produce a comprehensive dataset of I. indigotica. Results A database of 36,367 unigenes (average length = 1,115.67 bases) was generated by performing transcriptome sequencing. Based on the gene annotation of the transcriptome, 104 unigenes were identified covering most of the catalytic steps in the general biosynthetic pathways of indole, terpenoid, and phenylpropanoid. Subsequently, the organ-specific expression patterns of the genes involved in these pathways, and their responses to methyl jasmonate (MeJA) induction, were investigated. Metabolites profile of effective phenylpropanoid showed accumulation pattern of secondary metabolites were mostly correlated with the transcription of their biosynthetic genes. According to the analysis of UDP-dependent glycosyltransferases (UGT) family, several flavonoids were indicated to exist in I. indigotica and further identified by metabolic profile using UPLC/Q-TOF. Moreover, applying transcriptome co-expression analysis, nine new, putative UGTs were suggested as flavonol glycosyltransferases and lignan glycosyltransferases. Conclusions This database provides a pool of candidate genes involved in biosynthesis of effective metabolites in I. indigotica. Furthermore, the comprehensive analysis and characterization of the significant pathways are expected to give a better insight regarding the diversity of chemical composition, synthetic characteristics, and the regulatory mechanism which operate in this medical herb. PMID:24308360

Identification of sinensetin metabolites in rat urine by an isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry.

PubMed

Wei, Guor-Jien; Sheen, Jenn-Feng; Lu, Wen-Chien; Hwang, Lucy Sun; Ho, Chi-Tang; Lin, Ching-I

2013-05-29

Sinensetin (SIN), one of the major polymethoxyflavones (PMFs) contained mainly in the citrus peels, has been reported to possess various bioactivities, including antifungal, antimutagenic, anticancer, and anti-inflammatory activities. Although the biotransformation of SIN in fungi and insects has been reported, the information about the metabolism of SIN in mammals is still unclear. In this study, formation of SIN metabolites in rats was investigated. Four isotope-labeled SINs ([4'-D3]SIN, [3'-D3]SIN, [5-D3]SIN, and [6-D3]SIN) were synthesized and administered to rat. The urine samples were collected and main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry. The administered compound and four SIN metabolites were detected in rat urine. These metabolites were identified as 4'-hydroxy-5,6,7,3'-tetramethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 6-hydroxy-5,7,3',4'-tetramethoxyflavone, and 7-hydroxy-5,6,3',4'-tetramethoxyflavone sulfate.

Characterization of proflavine metabolites in rainbow trout.

PubMed

Yu, Z; Hayton, W L; Chan, K K

1997-04-01

Proflavine (3,6-diaminoacridine) has potential for use as an antiinfective in fish, and its metabolism by rainbow trout was therefore studied. Fourteen hours after intraarterial bolus administration of 10 mg/kg of proflavine, three metabolites were found in liver and bile, and one metabolite was found in plasma using reversed-phase HPLC with UV detection at 262 nm. Treatment with hydrochloric acid converted the three metabolites to proflavine, which suggested that the metabolites were proflavine conjugates. Treatment with beta-glucuronidase and saccharic acid 1,4-lactone, a specific beta-glucuronidase inhibitor, revealed that two metabolites were proflavine glucuronides. For determination of UV-VIS absorption and mass spectra, HPLC-purified metabolites were isolated from liver. Data from these experiments suggested that the proflavine metabolites were 3-N-glucuronosyl proflavine (PG), 3-N-glucuronosyl,6-N-acetyl proflavine (APG), and 3-N-acetylproflavine (AP). The identities of the metabolites were verified by chemical synthesis. When synthetic PG and AP were compared with the two metabolites isolated from trout, they had the same molecular weight as determined by matrix-assisted, laser desorption ionization, time-of-flight MS. In addition, they coeluted on HPLC under different mobile phase conditions. Finally, the in vitro incubation with liver subcellular preparations confirmed this characterization and provided the evidence that APG can be formed by glucuronidation of AP or acetylation of PG.

Metabolite-cycled STEAM and semi-LASER localization for MR spectroscopy of the human brain at 9.4T.

PubMed

Giapitzakis, Ioannis-Angelos; Shao, Tingting; Avdievich, Nikolai; Mekle, Ralf; Kreis, Roland; Henning, Anke

2018-04-01

Metabolite cycling (MC) is an MRS technique for the simultaneous acquisition of water and metabolite spectra that avoids chemical exchange saturation transfer effects and for which water may serve as a reference signal or contain additional information in functional or diffusion studies. Here, MC was developed for human investigations at ultrahigh field. MC-STEAM and MC-semi-LASER are introduced at 9.4T with an optimized inversion pulse and elaborate coil setup. Experimental and simulation results are given for the implementation of adiabatic inversion pulses for MC. The two techniques are compared, and the effect of frequency and phase correction based on the MC water spectra is evaluated. Finally, absolute quantification of metabolites is performed. The proposed coil configuration results in a maximum B1 + of 48 μΤ in a voxel within the occipital lobe. Frequency and phase correction of single acquisitions improve signal-to-noise ratio (SNR) and linewidth, leading to high-resolution spectra. The improvement of SNR of N-acetylaspartate (SNR NAA ) for frequency aligned data, acquired with MC-STEAM and MC-semi-LASER, are 37% and 30%, respectively (P < 0.05). Moreover, a doubling of the SNR NAA for MC-semi-LASER in comparison with MC-STEAM is observed (P < 0.05). Concentration levels for 18 metabolites from the human occipital lobe are reported, as acquired with both MC-STEAM and MC-semi-LASER. This work introduces a novel methodology for single-voxel MRS on a 9.4T whole-body scanner and highlights the advantages of semi-LASER compared to STEAM in terms of excitation profile. In comparison with MC-STEAM, MC-semi-LASER yields spectra with higher SNR. Magn Reson Med 79:1841-1850, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Determination of some L-3,4-dihydroxyphenylalanine and dopamine metabolites in urine by means of mass fragmentography.

PubMed

Muskiet, F A; Fremouw-Ottevangers, D C; van der Meulen, J; Wolthers, B G; de Vries, J A

1978-01-01

We describe a mass-fragmentographic method for determination in urine of the following metabolites of L-3,4-dihydroxyphenylalanine and dopamine: vanillactic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylethanol, and 3,4-dihydroxyphenylethanol. Deuterated analogs were used as internal standards. The method is fast, reproducible, sensitive, and selective, and does not require the use of time-consuming clean-up procedures. Normal excretion values in terms of creatinine, expressed as a function of age, as well as values obtained for patients with neurogenic tumors, a patient during therapy with L-3,4-dihydroxyphenylalanine, and a patient receiving dopamine are presented and discussed.

Environmental monitoring and assessment of antibacterial metabolite producing actinobacteria screened from marine sediments in south coastal regions of Karnataka, India.

PubMed

Skariyachan, Sinosh; Garka, Shruthi; Puttaswamy, Sushmitha; Shanbhogue, Shobitha; Devaraju, Raksha; Narayanappa, Rajeswari

2017-06-01

Assessment of the therapeutic potential of secondary metabolite producing microorganisms from the marine coastal areas imparts scope and application in the field of environmental monitoring. The present study aims to screen metabolites with antibacterial potential from actionbacteria associated with marine sediments collected from south coastal regions of Karnataka, India. The actinobacteria were isolated and characterized from marine sediments by standard protocol. The metabolites were extracted, and antibacterial potential was analyzed against eight hospital associated bacteria. The selected metabolites were partially characterized by proximate analysis, SDS-PAGE, and FTIR-spectroscopy. The antibiogram of the test clinical isolates revealed that they were emerged as multidrug-resistant strains (P ≤ 0.05). Among six actinobacteria (IS1-1S6) screened, 100 μl -1 metabolite from IS1 showed significant antibacterial activities against all the clinical isolates except Pseudomonas aeruginosa. IS2 demonstrated antimicrobial potential towards Proteus mirabilis, Streptococcus pyogenes, and Escherichia coli. The metabolite from IS3 showed activity against Strep. pyogenes and E. coli. The metabolites from IS4, IS5, and IS6 exhibited antimicrobial activities against Ps. aeruginosa (P ≤ 0.05). The two metabolites that depicted highest antibacterial activities against the test strains were suggested to be antimicrobial peptides with low molecular weight. These isolates were characterized and designated as Streptomyces sp. strain mangaluru01 and Streptomyces sp. mangaloreK01 by 16S ribosomal DNA (rDNA) sequencing. This study suggests that south coastal regions of Karnataka, India, are one of the richest sources of antibacterial metabolites producing actinobacteria and monitoring of these regions for therapeutic intervention plays profound role in healthcare management.

Chemical and bioactive diversities of the genus Chaetomium secondary metabolites.

PubMed

Zhang, Q; Li, H-Q; Zong, S-C; Gao, J-M; Zhang, A-L

2012-02-01

The genus Chaetomium fungi are considered to be a rich source of novel and bioactive secondary metabolites of great importance. Up till now, a variety of more than 200 secondary metabolites belonging to diverse structural types of chaetoglobosins, epipolythiodioxopiperazines, azaphilones, xanthones, anthraquinones, chromones, depsidones, terpenoids, and steroids have been discovered. Most of these fungal metabolites exhibited antitumor, cytotoxic, antimalarial, enzyme inhibitory, antibiotic, and other activities. This review covers the extraction, structure elucidation, structural diversity, and biological activities of natural products isolated from about 30 fungi associated with marine- and terrestrial- origins, and highlights some bioactive compounds as well as their mechanisms of action and structure-activity relationships.

Effect of Cadmium and Copper Exposure on Growth, Secondary Metabolites and Antioxidant Activity in the Medicinal Plant Sambung Nyawa (Gynura procumbens (Lour.) Merr).

PubMed

Ibrahim, Mohd Hafiz; Chee Kong, Yap; Mohd Zain, Nurul Amalina

2017-10-12

A randomized complete block (RCBD) study was designed to investigate the effects of cadmium (Cd) and copper (Cu) on the growth, bioaccumulation of the two heavy metals, metabolite content and antibacterial activities in Gyanura procumbens (Lour.) Merr. Nine treatments including (1) control (no Cd and Cu); (2) Cd 2 = cadmium 2 mg/L; (3) Cd 4 = cadmium 4 mg/L; (4) Cu 70 = copper 70 mg/L; (5) Cu 140 = copper 140 mg/L); (6) Cd 2 + Cu 70 = cadmium 2 mg/L + copper 70 mg/L); (7) Cd 2 + Cu 140 = cadmium 2 mg/L + copper 70 mg/L); (8) Cd 4 + Cu 70 = cadmium 4 mg/L+ copper 70 mg/L and (9) Cd 4 + Cu 140 = cadmium 4 mg/L + copper 140 mg/L) were evaluated in this experiment. It was found that the growth parameters (plant dry weight, total leaf area and basal diameter) were reduced with the exposure to increased concentrations of Cd and Cu and further decreased under interaction between Cd and Cu. Production of total phenolics, flavonoids and saponin was observed to be reduced under combined Cd and Cu treatment. The reduction in the production of plant secondary metabolites might be due to lower phenyl alanine lyase (PAL) activity under these conditions. Due to that, the 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant potential (FRAP) and antibacterial activities was also found to be reduced by the combined treatments. The current experiments show that the medicinal properties of G. procumbens are reduced by cadmium and copper contamination. The accumulation of heavy metal also was found to be higher than the safety level recommended by the WHO in the single and combined treatments of Cd and Cu. These results indicate that exposure of G. procumbens to Cd and Cu contaminated soil may potentially harm consumers due to bioaccumulation of metals and reduced efficacy of the herbal product.

Light-induced biochemical variations in secondary metabolite production and antioxidant activity in callus cultures of Stevia rebaudiana (Bert).

PubMed

Ahmad, Naveed; Rab, Abdur; Ahmad, Nisar

2016-01-01

Stevia rebaudiana (S. rebaudiana) is a very important species with worldwide medicinal and commercial uses. Light is one of the major elicitors that fluctuate morphogenic potential and biochemical responses. In the present study, we investigated the effect of various spectral lights on biomass accumulation and secondary metabolite production in callus cultures of S. rebaudiana. Leaf explants were placed on Murashige and Skoog (MS) medium and exposed to various spectral lights. 6-Benzyle adenine (BA) and 2, 4-dichlorophenoxy acetic acid (2, 4-D; 2.0 mgl(-1)) were used for callus induction. The control light (16/8h) produced optimum callogenic response (92.73%) than other colored lights. Compared to other colored lights, control grown cultures displayed maximum biomass accumulation (5.78 gl(-1)) during a prolonged log phase at the 18th day of growth kinetics. Cultures grown under blue light enhanced total phenolic content (TPC; 102.32 μg/g DW), total flavonoid content (TFC; 22.07 μg/g DW) and total antioxidant capacity (TAC; 11.63 μg/g DW). On the contrary, green and red lights improved reducing power assay (RPA; 0.71Fe(II)g(-1) DW) and DPPH-radical scavenging activity (DRSA; 80%). Herein, we concluded that the utilization of colored lights is a promising strategy for enhanced production of antioxidant secondary metabolites in callus cultures of S. rebaudiana. Copyright © 2015 Elsevier B.V. All rights reserved.

Chemical epigenetics alters the secondary metabolite composition of guttate excreted by an atlantic-forest-soil-derived Penicillium citreonigrum.

PubMed

Wang, Xiaoru; Sena Filho, José G; Hoover, Ashley R; King, Jarrod B; Ellis, Trevor K; Powell, Douglas R; Cichewicz, Robert H

2010-05-28

Chemical epigenetic manipulation of Penicillium citreonigrum led to profound changes in the secondary metabolite profile of its guttate. While guttate from control cultures exhibited a relatively simple assemblage of secondary metabolites, the guttate collected from cultures treated with 50 muM 5-azacytidine (a DNA methyltransferase inhibitor) was highly enriched in compounds representing at least three distinct biosynthetic families. The metabolites obtained from the fungus included six azaphilones (sclerotiorin (1), sclerotioramine (6), ochrephilone (2), dechloroisochromophilone III (3), dechloroisochromophilone IV (4), and 6-((3E,5E)-5,7-dimethyl-2-methylenenona-3,5-dienyl)-2,4-dihydroxy-3-methylbenzaldehyde (5)), pencolide (7), and two new meroterpenes (atlantinones A and B (9 and 10, respectively)). While pencolide was detected in the exudates of both control and 5-azacytidine-treated cultures, all of the other natural products were found exclusively in the guttates of the epigenetically modified fungus. All of the metabolites from the P. citreonigrum guttate were tested for antimicrobial activity in a disk diffusion assay. Both sclerotiorin and sclerotioramine caused modest inhibition of Staphylococcus epidermidis growth; however, only sclerotioramine was active against a panel of Candida strains.

Differences in Butadiene Adduct Formation between Rats and Mice Not Due to Selective Inhibition of CYP2E1 by Butadiene Metabolites

PubMed Central

Pianalto, Kaila M.; Hartman, Jessica H.; Boysen, Gunnar; Miller, Grover P.

2013-01-01

CYP2E1 metabolizes 1,3-butadiene (BD) into genotoxic and possibly carcinogenic 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EB-diol). The dose response of DNA and protein adducts derived from BD metabolites increase linearly at low BD exposures and then saturate at higher exposures in rats, but not mice. It was hypothesized that differences in adduct formation between rodents reflect more efficient BD oxidation in mice than rats. Herein, we assessed whether BD-derived metabolites selectively inhibit rat but not mouse CYP2E1 activity using B6C3F1 mouse and Fisher 344 rat liver microsomes. Basal CYP2E1 activities toward 4-nitrophenol were similar between rodents. Through IC50 studies, EB was the strongest inhibitor (IC50 54 μM, mouse; 98 μM, rat), BD-diol considerably weaker (IC50 1200 μM, mouse; 1000 μM, rat), and DEB inhibition nonexistent (IC50 >25 mM). Kinetic studies showed that in both species EB and BD-diol inhibited 4-nitrophenol oxidation through two-site mechanisms in which inhibition constants reflected trends observed in IC50 studies. None of the reactive epoxide metabolites inactivated CYP2E1 irreversibly. Thus, there was no selective inhibition or inactivation of rat CYP2E1 by BD metabolites relative to mouse Cyp2e1, and it can be inferred that CYP2E1 activity toward BD between rodent species would similarly not be impacted by the presence of BD metabolites. Inhibition of CYP2E1 by BD metabolites is then not responsible for the reported species difference in BD metabolism, formation of BD-derived DNA and protein adducts, mutagenicity and tumorigenesis. PMID:24021170

Differences in butadiene adduct formation between rats and mice not due to selective inhibition of CYP2E1 by butadiene metabolites.

PubMed

Pianalto, Kaila M; Hartman, Jessica H; Boysen, Gunnar; Miller, Grover P

2013-11-25

CYP2E1 metabolizes 1,3-butadiene (BD) into genotoxic and possibly carcinogenic 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EB-diol). The dose response of DNA and protein adducts derived from BD metabolites increases linearly at low BD exposures and then saturates at higher exposures in rats, but not mice. It was hypothesized that differences in adduct formation between rodents reflect more efficient BD oxidation in mice than rats. Herein, we assessed whether BD-derived metabolites selectively inhibit rat but not mouse CYP2E1 activity using B6C3F1 mouse and Fisher 344 rat liver microsomes. Basal CYP2E1 activities toward 4-nitrophenol were similar between rodents. Through IC50 studies, EB was the strongest inhibitor (IC50 54μM, mouse; 98μM, rat), BD-diol considerably weaker (IC50 1200μM, mouse; 1000μM, rat), and DEB inhibition nonexistent (IC50>25mM). Kinetic studies showed that in both species EB and BD-diol inhibited 4-nitrophenol oxidation through two-site mechanisms in which inhibition constants reflected trends observed in IC50 studies. None of the reactive epoxide metabolites inactivated CYP2E1 irreversibly. Thus, there was no selective inhibition or inactivation of rat CYP2E1 by BD metabolites relative to mouse Cyp2e1, and it can be inferred that CYP2E1 activity toward BD between rodent species would similarly not be impacted by the presence of BD metabolites. Inhibition of CYP2E1 by BD metabolites is then not responsible for the reported species difference in BD metabolism, formation of BD-derived DNA and protein adducts, mutagenicity and tumorigenesis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Combined mass spectrometry-based metabolite profiling of different pigmented rice (Oryza sativa L.) seeds and correlation with antioxidant activities.

PubMed

Kim, Ga Ryun; Jung, Eun Sung; Lee, Sarah; Lim, Sun-Hyung; Ha, Sun-Hwa; Lee, Choong Hwan

2014-09-29

Nine varieties of pigmented rice (Oryza sativa L.) seeds that were black, red, or white were used to perform metabolite profiling by using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and gas chromatography (GC) TOF-MS, to measure antioxidant activities. Clear grouping patterns determined by the color of the rice seeds were identified in principle component analysis (PCA) derived from UPLC-Q-TOF-MS. Cyanidin-3-glucoside, peonidin-3-glucoside, proanthocyanidin dimer, proanthocyanidin trimer, apigenin-6-C-glugosyl-8-C-arabiboside, tricin-O-rhamnoside-O-hexoside, and lipids were identified as significantly different secondary metabolites. In PCA score plots derived from GC-TOF-MS, Jakwangdo (JKD) and Ilpoom (IP) species were discriminated from the other rice seeds by PC1 and PC2. Valine, phenylalanine, adenosine, pyruvate, nicotinic acid, succinic acid, maleic acid, malonic acid, gluconic acid, xylose, fructose, glucose, maltose, and myo-inositol were significantly different primary metabolites in JKD species, while GABA, asparagine, xylitol, and sucrose were significantly distributed in IP species. Analysis of antioxidant activities revealed that black and red rice seeds had higher activity than white rice seeds. Cyanidin-3-glucoside, peonidin-3-glucoside, proanthocyanidin dimers, proanthocyanidin trimers, and catechin were highly correlated with antioxidant activities, and were more plentiful in black and red rice seeds. These results are expected to provide valuable information that could help improve and develop rice-breeding techniques.

Secondary metabolites of cyanobacteria Nostoc sp.

NASA Astrophysics Data System (ADS)

Kobayashi, Akio; Kajiyama, Shin-Ichiro

1998-03-01

Cyanobacteria attracted much attention recently because of their secondary metabolites with potent biological activities and unusual structures. This paper reviews some recent studies on the isolation, structural, elucidation and biological activities of the bioactive compounds from cyanobacteria Nostoc species.

Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

PubMed Central

Lu, Jia; Zhao, Chen; Zhao, Yingze; Zhang, Jingfang; Zhang, Yue; Chen, Li; Han, Qiyuan; Ying, Yue; Peng, Shuai; Ai, Runna; Wang, Yu

2018-01-01

Abstract Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) has emerged as a versatile tool for targeted gene editing and transcriptional programming. Here, we designed and vigorously optimized a series of Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) for transcriptional activation by grafting a mutated human estrogen receptor (ERT2) to multiple CRISPR/Cas9 systems, which renders them 4-hydroxytamoxifen (4-OHT) inducible for the access of genome. Further, extra functionality of simultaneous genome editing was achieved with one device we named HIT2. Optimized terminal devices herein delivered advantageous performances in comparison with several existing designs. They exerted selective, titratable, rapid and reversible response to drug induction. In addition, these designs were successfully adapted to an orthogonal Cas9. HIT systems developed in this study can be applied for controlled modulation of potentially any genomic loci in multiple modes. PMID:29237052

Characterization of new metabolites from in vivo biotransformation of 2-amino-3-methylimidazo[4,5-f]quinoline in mouse by mass spectrometry

PubMed Central

Hsu, Fong-Fu; Lakshmi, Vijaya M.; Zenser, Terry V.

2010-01-01

In studying the metabolic pathways underlying the mechanism of carcinogenesis of the heterocyclic amine of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), we recently found a new metabolite, which gave a [M + H]+ ion of m/z 217 when subjected to ESI in positive-ion mode. Following ip injection of this metabolite of m/z 217 (designated as m/z 217) to beta-naphthoflavone-treated mice, 57% of the total radioactivity was recovered in a 24-hr mouse urine sample. HPLC separation followed by MS analysis indicates that the urine sample contained m/z 217 (36 ± 3% of total recovered radioactivity) and two other peaks that gave rise to the [M + H]+ ions of m/z 393 (31 ± 4%, designated as m/z 393) and m/z 233 (14 ± 1%, designated as m/z 233). Beta-glucuronidase treatment of m/z 393 resulted in a radioactive peak corresponding to m/z 217. Electrospray ionization in combination with various mass spectrometry techniques, including multiple-stage mass spectrometry, exact mass measurements, and H-D exchange followed by tandem mass spectrometry was used for structural characterization. The urinary metabolites of m/z 217, 393, and 233 were identified as 1,2-dihydro-2-amino-5-hydroxy-3-methylimidazo[4,5-f]quinoline, 1,2-dihydro-2-amino-5-O-glucuronide-3-methylimidazo[4,5-f]quinoline, and 1,2-dihydro-2-amino-5,7-dihydroxy-3-methylimidazo[4,5-f]quinoline, respectively. Our results demonstrated that m/z 217 is biotransformed in vivo to m/z 393 by O-glucuronidation and to m/z 233 by oxidation. The observation of these more polar metabolites relative to IQ suggests that they may arise from a previously undescribed detoxicification pathway. PMID:19629964

Relationship between NH sub 4 sup + assimilation rate and in vivo phosphoenolpyruvate carboxylase activity

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

Vanlerberghe, G.C.; Schuller, K.A.; Smith, R.G.

The rate of NH{sub 4}{sup +} assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH{sub 4}{sup +} assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H{sup 14}CO{sub 3}{sup {minus}} into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinationsmore » of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, (1990) Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C{sub 3} organism.« less

Antifungal activity of secondary plant metabolites from potatoes (Solanum tuberosum L.): Glycoalkaloids and phenolic acids show synergistic effects.

PubMed

Sánchez-Maldonado, A F; Schieber, A; Gänzle, M G

2016-04-01

To study the antifungal effects of the potato secondary metabolites α-solanine, α-chaconine, solanidine and caffeic acid, alone or combined. Resistance to glycoalkaloids varied among the fungal species tested, as derived from minimum inhibitory concentrations assays. Synergistic antifungal activity between glycoalkaloids and phenolic compounds was found. Changes in the fluidity of fungal membranes caused by potato secondary plant metabolites were determined by calculation of the generalized polarization values. The results partially explained the synergistic effect between caffeic acid and α-chaconine and supported findings on membrane disruption mechanisms from previous studies on artificial membranes. LC/MS analysis was used to determine variability and relative amounts of sterols in the different fungal species. Results suggested that the sterol pattern of fungi is related to their resistance to potato glycoalkaloids and to their taxonomy. Fungal resistance to α-chaconine and possibly other glycoalkaloids is species dependent. α-Chaconine and caffeic acid show synergistic antifungal activity. The taxonomic classification and the sterol pattern play a role in fungal resistance to glycoalkaloids. Results improve the understanding of the antifungal mode of action of potato secondary metabolites, which is essential for their potential utilization as antifungal agents in nonfood systems. © 2016 The Society for Applied Microbiology.

Integration of a 'proton antenna' facilitates transport activity of the monocarboxylate transporter MCT4.

PubMed

Noor, Sina Ibne; Pouyssegur, Jacques; Deitmer, Joachim W; Becker, Holger M

2017-01-01

Monocarboxylate transporters (MCTs) mediate the proton-coupled transport of high-energy metabolites like lactate and pyruvate and are expressed in nearly every mammalian tissue. We have shown previously that transport activity of MCT4 is enhanced by carbonic anhydrase II (CAII), which has been suggested to function as a 'proton antenna' for the transporter. In the present study, we tested whether creation of an endogenous proton antenna by introduction of a cluster of histidine residues into the C-terminal tail of MCT4 (MCT4-6xHis) could facilitate MCT4 transport activity when heterologously expressed in Xenopus oocytes. Our results show that integration of six histidines into the C-terminal tail does indeed increase transport activity of MCT4 to the same extent as did coexpression of MCT4-WT with CAII. Transport activity of MCT4-6xHis could be further enhanced by coexpression with extracellular CAIV, but not with intracellular CAII. Injection of an antibody against the histidine cluster into MCT4-expressing oocytes decreased transport activity of MCT4-6xHis, while leaving activity of MCT4-WT unaltered. Taken together, these findings suggest that transport activity of the proton-coupled monocarboxylate transporter MCT4 can be facilitated by integration of an endogenous proton antenna into the transporter's C-terminal tail. © 2016 Federation of European Biochemical Societies.

[Synthetic biology toward microbial secondary metabolites and pharmaceuticals].

PubMed

Wu, Lin-Zhuan; Hong, Bin

2013-02-01

Microbial secondary metabolites are one of the major sources of anti-bacterial, anti-fungal, antitumor, anti-virus and immunosuppressive agents for clinical use. Present challenges in microbial pharmaceutical development are the discovery of novel secondary metabolites with significant biological activities, improving the fermentation titers of industrial microbial strains, and production of natural product drugs by re-establishing their biosynthetic pathways in suitable microbial hosts. Synthetic biology, which is developed from systematic biology and metabolic engineering, provides a significant driving force for microbial pharmaceutical development. The review describes the major applications of synthetic biology in novel microbial secondary metabolite discovery, improved production of known secondary metabolites and the production of some natural drugs in genetically modified or reconstructed model microorganisms.

Ruta graveolens Extracts and Metabolites against Spodoptera frugiperda.

PubMed

Ayil-Gutiérrez, Benjamin A; Villegas-Mendoza, Jesús M; Santes-Hernndez, Zuridai; Paz-González, Alma D; Mireles-Martínez, Maribel; Rosas-García, Ninfa M; Rivera, Gildardo

2015-11-01

The biological activity of Ruta graveolens leaf tissue extracts obtained with different solvents (ethyl acetate, ethanol, and water) and metabolites (psoralen, 2- undecanone and rutin) against Spodoptera frugiperda was evaluated. Metabolites levels in extracts were quantified by HPLC and GC. Ethyl acetate and ethanol extracts showed 94% and 78% mortality, respectively. Additionally, psoralen metabolite showed a high mortality as cypermethrin. Metabolite quantification in extracts shows the presence of 2-undecanone (87.9 µmoles mg(-1) DW), psoralen (3.6 µmoles mg(-1) DW) and rutin (0.001 pmoles mg(-1) DW). We suggest that these concentrations of 2-undecanone and psoralen in R. graveolens leaf tissue extracts could be responsible for S. frugiperda mortality.

Network Analysis of Enzyme Activities and Metabolite Levels and Their Relationship to Biomass in a Large Panel of Arabidopsis Accessions[C][W][OA

PubMed Central

Sulpice, Ronan; Trenkamp, Sandra; Steinfath, Matthias; Usadel, Bjorn; Gibon, Yves; Witucka-Wall, Hanna; Pyl, Eva-Theresa; Tschoep, Hendrik; Steinhauser, Marie Caroline; Guenther, Manuela; Hoehne, Melanie; Rohwer, Johann M.; Altmann, Thomas; Fernie, Alisdair R.; Stitt, Mark

2010-01-01

Natural genetic diversity provides a powerful resource to investigate how networks respond to multiple simultaneous changes. In this work, we profile maximum catalytic activities of 37 enzymes from central metabolism and generate a matrix to investigate species-wide connectivity between metabolites, enzymes, and biomass. Most enzyme activities change in a highly coordinated manner, especially those in the Calvin-Benson cycle. Metabolites show coordinated changes in defined sectors of metabolism. Little connectivity was observed between maximum enzyme activities and metabolites, even after applying multivariate analysis methods. Measurements of posttranscriptional regulation will be required to relate these two functional levels. Individual enzyme activities correlate only weakly with biomass. However, when they are used to estimate protein abundances, and the latter are summed and expressed as a fraction of total protein, a significant positive correlation to biomass is observed. The correlation is additive to that obtained between starch and biomass. Thus, biomass is predicted by two independent integrative metabolic biomarkers: preferential investment in photosynthetic machinery and optimization of carbon use. PMID:20699391

Metabolomics and Cheminformatics Analysis of Antifungal Function of Plant Metabolites

PubMed Central

Cuperlovic-Culf, Miroslava; Rajagopalan, NandhaKishore; Tulpan, Dan; Loewen, Michele C.

2016-01-01

Fusarium head blight (FHB), primarily caused by Fusarium graminearum, is a devastating disease of wheat. Partial resistance to FHB of several wheat cultivars includes specific metabolic responses to inoculation. Previously published studies have determined major metabolic changes induced by pathogens in resistant and susceptible plants. Functionality of the majority of these metabolites in resistance remains unknown. In this work we have made a compilation of all metabolites determined as selectively accumulated following FHB inoculation in resistant plants. Characteristics, as well as possible functions and targets of these metabolites, are investigated using cheminformatics approaches with focus on the likelihood of these metabolites acting as drug-like molecules against fungal pathogens. Results of computational analyses of binding properties of several representative metabolites to homology models of fungal proteins are presented. Theoretical analysis highlights the possibility for strong inhibitory activity of several metabolites against some major proteins in Fusarium graminearum, such as carbonic anhydrases and cytochrome P450s. Activity of several of these compounds has been experimentally confirmed in fungal growth inhibition assays. Analysis of anti-fungal properties of plant metabolites can lead to the development of more resistant wheat varieties while showing novel application of cheminformatics approaches in the analysis of plant/pathogen interactions. PMID:27706030

Determination of N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine and its metabolites in human plasma and urine by column-switching high-performance liquid chromatography with ultraviolet detection.