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S9 Fraction (s9 + fraction)
Kinds of S9 Fraction Selected AbstractsAnalysis of oxycodol and noroxycodol stereoisomers in biological samples by capillary electrophoresisELECTROPHORESIS, Issue 10 2005Andrea Baldacci Abstract A capillary electrophoresis (CE) method for the separation of the diastereoisomers of 6-oxycodol (6OCOL) and nor-6-oxycodol (N6OCOL), the 6-keto-reduced metabolites of oxycodone (OCOD) and noroxycodone (NOCOD), respectively, is reported and employed to assess the stereoselectivity of these metabolic steps in vivo, in vitro, and in chemical synthesis. CE in an untreated fused-silica capillary with acidic buffers containing 2-hydroxypropyl-,-cyclodextrin, randomly sulfated ,-cyclodextrin, or single isomer heptakis(2,3-diacetyl-6-sulfato)-,-cyclodextrin (HDAS-,-CD) is shown to permit the simultaneous separation of the stereoisomers of 6OCOL and N6OCOL. A 100 mM phosphate buffer of pH 2.0 containing 2.05% w/v HDAS-,-CD provides a medium for rapid analysis and unambiguous identification of these stereoisomers in solid-phase extracts of (i) urines stemming from patients under pharmacotherapy with OCOD, (ii) incubations of OCOD and NOCOD with human liver cytosol and the human liver S9 fraction, and (iii) after chemical synthesis from OCOD and NOCOD using NaBH4. In all cases, ,-N6OCOL is shown to be the predominant stereoisomer of N6OCOL. For 6OCOL, the same is true for in vitro formation and for chemical synthesis. In urine, however, ,-6OCOL is observed to be excreted in a higher amount than ,-6OCOL. For the urinary ,-/,-isomer ratio of 6OCOL and N6OCOL, there are no differences between the data obtained for nonhydrolyzed and enzymatically hydrolyzed urines. The data document the stereoselectivity of the 6-keto-reduction of OCOD and NOCOD in man. [source] Metabolism of chlorpyrifos and chlorpyrifos oxon by human hepatocytes,JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2006Kyoungju Choi Abstract The metabolism of chlorpyrifos (CPS) and chlorpyrifos oxon (CPO) by human hepatocytes and human liver S9 fractions was investigated using LC-MS/MS. Cytochrome P450 (CYP)-dependent and phase II-related products were determined following incubation with CPS and CPO. CYP-related products, 3,5,6-trichloro-2-pyridinol (TCP), diethyl thiophosphate, and dealkylated CPS, were found following CPS treatment and dealkylated CPO following CPO treatment. Diethyl phosphate was not identified because of its high polarity and lack of retention with the chromatographic conditions employed. Phase II-related conjugates, including O- and S-glucuronides as well as 11 GSH-derived metabolites, were identified in CPS-treated human hepatocytes, although the O -sulfate of TCP conjugate was found only when human liver S9 fractions were used as the enzyme source. O -Glucuronide of TCP was also identified in CPO-treated hepatocytes. CPS and CPO were identified using HPLC,UV after CPS metabolism by the human liver S9 fraction. However, CPO was not found following treatment of human hepatocytes with either CPS or CPO. These results suggest that human liver plays an important role in detoxification, rather than activation, of CPS. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:279,291, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20145 [source] Isolation and characterization of metabolites of centpropazine in rat liver, intestine, and red blood cell homogenatesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2002Bhattaram V. Atul Abstract The potential sites for metabolism of centpropazine (CPZ) (an antidepressant) were evaluated in male Sprague-Dawley rats. The isolation and identification of the major metabolites formed in the presence of rat liver S9 fraction, intestine, and red blood cells under aerobic conditions were performed using high-performance liquid chromatography and electrospray ionization mass spectrometry. CPZ was found to be extensively metabolized to seven possible metabolites by liver S9 fraction in the presence of a nicotinamide adenine dinucleotide phosphate generating system at 37°C. Both intestinal wall and red blood cells were also found to metabolize the compound. This metabolite structure was confirmed by comparison with that of its synthetic standard. The drug was stable in intestinal contents. On the basis of our finding, we propose the in vitro metabolic pathways for CPZ. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2067,2075, 2002 [source] Mutagenic and antimutagenic potential of the medicinal plants M. laevigata and C. xanthocarpaPHYTOTHERAPY RESEARCH, Issue 3 2003J. B. F. Fernandes Abstract Aqueous extracts of medicinal plants (Mikania laevigata and Campomanesia xanthocarpa) were screened for the presence of mutagenic activity in the Salmonella/microsome assay. The extracts of Campomanesia xanthocarpa showed frameshift (TA97a strain) signs of mutagenic activity without exogenous metabolism (S9 fraction). The infusions of Mikania laevigata, negative for mutagenic activity, showed high percentages of inhibition of mutagenesis induced by mutagens 2AF (2-amino,uorene), in the presence of exogenous metabolism (S9 fraction), for frameshift (TA98) and base pair substitution (TA100) lesions. In addition, these inhibitions were observed against mutagen SAZ (sodium azide) in assays with the TA100 strain, without exogenous metabolism (S9 fraction). A synergistic effect was also observed in frameshift mutagenic events, with direct action in the presence of 4NQO (4-oxide-1-nitroquinoline) and a tendency to a low percentage of action enhancement, in the presence of the 2AF mutagen. The variable responses observed in the extract assays show the potentials for interaction of the different active principles in genetic material. Copyright © 2003 John Wiley & Sons, Ltd. [source] Cytotoxicity of settling particulate matter and sediments of the Neckar River (Germany) during a winter floodENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2000Henner Hollert Abstract To investigate the cytotoxic and genotoxic potentials of settling particulate matter (SPM) carried by the Neckar River, a well-studied model for a lock-regulated river in central Europe, during a flood, acute cytotoxicity was investigated using the fibroblast-like fish cell line RTG-2 with the neutral red retention, the succinic acid dehydrogenase (MTT), and the lactatedehydro-genase (LDH) release assays as well as microscopic inspection as endpoints. Genotoxicity of water, pore water, sediments, and SPM were assessed using the Ames test. Different extraction methods (Soxhlet extraction with solvents of variable polarity as well as a fluid/fluid extraction according to pH) in addition to a supplementation of biotests with S9 fractions from the liver of ,-naphthoflavone/phenobarbital-induced rats allowed a further characterization of the biological damage. Both sediments and SPM extracts caused cytotoxic effects in RTG-2 cells. Cytotoxicity was found to increase significantly with polarity of extracting solvents (NR50 = effective concentration for 50% cell death in the neutral red test: 80 [65], 100 [70], 180 [220], and 225 [270] mg/ml for ethanol, acetone, dichloromethane, and n -hexane extracts, respectively, if measured with [without] S9 supplementation). Following extraction according to pH, cytotoxicity could be attributed mainly to neutral substances (NR50: 80 and 218 mg dry SPM/ml test medium for the neutral and the acid fractions, respectively), whereas the slightly acid and basic fractions already showed little or no cytotoxicity. Samples taken during the period of flood rise showed the highest cytotoxic activities. Cytotoxicity was significantly enhanced by the addition of S9 preparations. In contrast, no genotoxic activity was found in native surface waters, pore waters, and SPM. [source] Metabolism of chlorpyrifos and chlorpyrifos oxon by human hepatocytes,JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2006Kyoungju Choi Abstract The metabolism of chlorpyrifos (CPS) and chlorpyrifos oxon (CPO) by human hepatocytes and human liver S9 fractions was investigated using LC-MS/MS. Cytochrome P450 (CYP)-dependent and phase II-related products were determined following incubation with CPS and CPO. CYP-related products, 3,5,6-trichloro-2-pyridinol (TCP), diethyl thiophosphate, and dealkylated CPS, were found following CPS treatment and dealkylated CPO following CPO treatment. Diethyl phosphate was not identified because of its high polarity and lack of retention with the chromatographic conditions employed. Phase II-related conjugates, including O- and S-glucuronides as well as 11 GSH-derived metabolites, were identified in CPS-treated human hepatocytes, although the O -sulfate of TCP conjugate was found only when human liver S9 fractions were used as the enzyme source. O -Glucuronide of TCP was also identified in CPO-treated hepatocytes. CPS and CPO were identified using HPLC,UV after CPS metabolism by the human liver S9 fraction. However, CPO was not found following treatment of human hepatocytes with either CPS or CPO. These results suggest that human liver plays an important role in detoxification, rather than activation, of CPS. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:279,291, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20145 [source] Automated software-guided identification of new buspirone metabolites using capillary LC coupled to ion trap and TOF mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2006Anabel S. Fandiño Abstract The identification and structure elucidation of drug metabolites is one of the main objectives in in vitro ADME studies. Typical modern methodologies involve incubation of the drug with subcellular fractions to simulate metabolism followed by LC-MS/MS or LC-MSn analysis and chemometric approaches for the extraction of the metabolites. The objective of this work was the software-guided identification and structure elucidation of major and minor buspirone metabolites using capillary LC as a separation technique and ion trap MSn as well as electrospray ionization orthogonal acceleration time-of-flight (ESI oaTOF) mass spectrometry as detection techniques. Buspirone mainly underwent hydroxylation, dihydroxylation and N -oxidation in S9 fractions in the presence of phase I co-factors and the corresponding glucuronides were detected in the presence of phase II co-factors. The use of automated ion trap MS/MS data-dependent acquisition combined with a chemometric tool allowed the detection of five small chromatographic peaks of unexpected metabolites that co-eluted with the larger chromatographic peaks of expected metabolites. Using automatic assignment of ion trap MS/MS fragments as well as accurate mass measurements from an ESI oaTOF mass spectrometer, possible structures were postulated for these metabolites that were previously not reported in the literature. Copyright © 2006 John Wiley & Sons, Ltd. [source] | |||||||||||||