Regular ArticleEvaluation of Triacetyloleandomycin, α-Nasymphthoflavone and Diethyldithiocarbamate as Selective Chemical Probes for Inhibition of Human Cytochromes P450
Abstract
A variety of chemicals, including triacetyloleandomycin (TAO), α-nasymphthoflavone (ANF), and diethyldithiocarbamate (DDC), are widely used as inhibitory probes for select individual human cytochrome P450 (CYP) enzymes, despite the fact that the selectivity of these inhibitors has not been rigorously evaluated. In the present study we take advantage of recent advances in cDNA-directed human P450 expression to evaluate directly the P450 form selectivity of TAO, ANF, and DDC, using a panel of 10 individual cDNA-expressed human P450s. Under experimental conditions known to yield maximal TAO complexation with P450 hemoproteins, TAO (20 μM) inhibited the catalytic activity of expressed CYPs 3A3, 3A4, and 3A5, whereas it did not affect CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, or 2E1 activity. ANF inhibited not only CYPs 1A1 and 1A2 (IC50 = 0.4-0.5 μM), but it was also similarly effective against CYPs 2C8 and 2C9. Increasing the concentration of ANF to 10 μM led to inhibition of CYP2A6 and CYP2B6. Although a previous study suggested that DDC is a selective inhibitor of CYP2E1, the present investigation shows that at concentrations required to inhibit CYP2E1 (IC50 ≍ 125 μM when preincubated with NADPH), DDC also inhibited CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 3A3, and 3A4. Decreasing the concentration of DDC to 10 μM, however, led to inhibition of CYP2A6 (65% inhibition) and CYP2B6 (50% inhibition), but none of the other p450s examined, including CYP2E1. Overall, these results establish that (a) TAO is a selective inhibitor of the human CYP3A subfamily; (b) ANF potently inhibits CYP2C8 and CYP2C9, in addition to CYPs 1A1 and 1A2; and (c) DDC cannot be employed as a diagnostic inhibitory probe for CYP2E1.
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Exploration of inhibition potential of isoniazid and its metabolites towards CYP2E1 in human liver microsomes through LC-MS/MS analysis
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Risk assessment of the chiral pesticide fenamiphos in a human model: Cytochrome P450 phenotyping and inhibition studies
2020, Food and Chemical ToxicologyFenamiphos (FS) is a chiral organophosphate pesticide that is used to control nematodes in several crops. Enantioselective differences may be observed in FS activity, bioaccumulation, metabolism, and toxicity. Humans may be exposed to FS through occupational and chronic (food, water, and environmental) exposure. FS may cause undesirable CYP450 pesticide-drug interactions, which may impact human health. Here, the CYP450 isoforms involved in enantioselective FS metabolism were identified, and CYP450 inhibition by rac-FS, (+)-FS, and (−)-FS was evaluated to obtain reliable information on enantioselective FS risk assessment in humans. CYP3A4 and CYP2E1 metabolized FS enantiomers, and CYP2B6 may participate in rac-FS metabolism. In addition, rac-FS, (+)-FS, and (−)-FS were reversible competitive CYP1A2, CYP2C19, and CYP3A4/5 inhibitors. High stereoselective inhibition potential was verified; rac-FS and (−)-FS strongly inhibited and (+)-FS moderately inhibited CYP1A2. Stereoselective differences were also detected for CYP2C19 and CYP3A4/5, which were strongly inhibited by rac-FS, (+)-FS, and (−)-FS. Our results indicated a high potential for CYP450 drug-pesticide interactions, which may affect human health. The lack of stereoselective research on the effect of chiral pesticides on the activity of CYP450 isoforms highlights the importance of assessing the risks of such pesticides in humans.
3-Methylcholanthrene impacts on the female germ cells of rats without causing systemic toxicity
2020, ToxicologyWe have previously shown that daily exposure to the environmental pollutant 3-methylcholanthrene (3MC) alters the ovarian function by affecting follicle growth and ovulation. To extend our findings, the aims of this work were to study the effects of daily and non-daily exposure to 3MC on oocyte morphology and integrity and the meiosis process. To this end, immature female rats were daily (0.1–1.0 mg/kg) and non-daily (0.1 mg/kg, three times a week) exposed to 3MC and/or α-naphthoflavone (αNF) (80 mg/kg) for 19 and 20 days, respectively. The latter was used to study its ability to prevent the 3MC action. Follicular growth was examined by histology, apoptosis by in situ cell death detection, oocyte integrity by morphological parameters and fluorescent dyes, and the meiotic spindle by immunostaining. Compared with controls (C), and in a dose-dependent manner, all 3MC-treated rats showed i) increased presence of apoptotic cells in antral follicles and decreased percentage of healthy oocytes, ii) increased oocyte area, perimeter and perivitelline space and decreased thickness of the zona pellucida, and ii) increased percentage of oocytes with abnormal meiotic spindle. In addition, the non-daily dose of 3MC caused DNA damage in oocytes, but not in blood or bone marrow cells. All 3MC-induced changes were prevented with the co-treatment with αNF. These results suggest that low doses of 3MC severely disrupt the ovarian function and that germ cells seem to be more sensitive to this environmental pollutant than other cells such as peripheral blood and bone marrow cells.
In vitro enantioselective study of the toxicokinetic effects of chiral fungicide tebuconazole in human liver microsomes
2019, Ecotoxicology and Environmental SafetyTebuconazole (TEB) is a chiral triazole fungicide that is globally marketed and used as a racemic mixture to control plant pathogens. Due to its use as a racemic mixture, TEB may exhibit enantioselective toxicokinetics toward nontarget organisms, including humans. Therefore, the in vitro enantioselective metabolism of TEB by cytochrome P450 enzymes (CYP450) was studied using human liver microsomes, and the in vivo toxicokinetic parameters were predicted. A new enantioselective, reversed-phase LC-MS/MS method was developed and validated to analyze the enantiomers of TEB and its main metabolite, 1-hydroxytebuconazole (TEBOH). In vitro metabolic parameters were obtained, and in vitro-in vivo extrapolations were performed. Michaelis-Menten and atypical biphasic kinetic profiles were observed with a total intrinsic clearance ranging from 53 to 19 mL min−1 mg−1. The in vitro-in vivo extrapolation results showed that TEB first passage effect by the liver seems to be negligible, with hepatic clearance and extraction ratios ranging from 0.53 to 5.0 mL min−1 kg−1 and 2.7–25%, respectively. Preferential metabolism of (+)-TEB to rac-TEB and (−)-TEB was observed, with preferential production of (+)-TEBOH. Furthermore, reaction phenotyping studies revealed that, despite the low hepatic clearance in the first pass metabolism of TEB, multiple human CYP450 isoforms were involved in TEB metabolism when TEBOH enantiomers were generated, mainly CYP3A4 and CYP2C9, which makes TEB accumulation in the human body more difficult due to multiple metabolic pathways.
Electrochemically-driven benzo[a]pyrene metabolism via human cytochrome P450 1A1 with reductase coated nitrogen-doped graphene nano-composites
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