The effect of isoniazid on CYP2E1- and CYP4A-mediated hydroxylation of arachidonic acid in the rat liver and kidney.
(25/78)
Cytochrome P450 (P450) bioactivation of arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) has been reported to be isoform- and tissue-specific. To determine whether altered P450 expression affects the production of these metabolites, the formation of HETEs after isoniazid-mediated CYP2E1 induction was evaluated in the rat liver and kidney. Male Sprague-Dawley rats received isoniazid (200 mg/kg) or saline intraperitoneally once daily for 5 days. Chlorzoxazone, lauric acid, and arachidonic acid hydroxylation was measured in liver and kidney microsomes with and without preincubation with the specific CYP2E1 inhibitor, trans-1,2-dichloroethylene (DCE). P450 isoform content and tissue HETE metabolite concentrations were also determined. Isoniazid increased CYP2E1 protein, and the 6-hydroxychlorzoxazone formation rate was increased by 2.7 +/- 0.3- and 2.2 +/- 0.5-fold in liver and kidney, respectively. Formation of 19-HETE and 11-hydroxylauric acid was induced 2.3 +/- 0.6-fold and 2.2 +/- 0.4-fold in the liver, respectively, with no difference in the kidney. All of the induced activities were attenuated by DCE. An unanticipated decrease in liver CYP4A expression and in vitro 20-HETE formation rate was observed after isoniazid administration. Isoniazid decreased liver and kidney 20-HETE content to 34 +/- 10% and 15.6 +/- 5.3% of control, respectively, without significantly altering tissue 19-HETE concentration. Based on these findings, we conclude that under induced conditions, CYP2E1 is a primary enzyme involved in liver, but not kidney, formation of 19-HETE. In addition, formation of both CYP4A and 20-HETE is reduced in the liver by isoniazid. It was also demonstrated that tissue concentrations parallel in vitro inhibited formation rates for 20-HETE, but not the induced 19-HETE formation in the liver. (+info)
Robustness of chlorzoxazone as an in vivo measure of cytochrome P450 2E1 activity.
(26/78)
AIMS: Chlorzoxazone is metabolized by cytochrome P450 2E1 (CYP2E1) to a single oxidized metabolite, 6-hydroxychlorzoxazone. The aim of the study was to test the robustness of chlorzoxazone as an in vivo probe of CYP2E1 activity in humans, with emphasis on investigating short-term and long-term intra-individual variabilities and effects of different doses of the drug. In addition, the influences of body build, drug metabolizing enzyme genotype, blood sampling time, and moderate recent ethanol intake were investigated. METHODS: The 6-hydroxychlorzoxazone:chlorzoxazone (metabolic) ratio in plasma was measured at 2 h in 28 male and nine female volunteers following a single oral dose of 500 mg chlorzoxazone. Similarly, the metabolic ratios at 4 h and 6 h were measured in 20 of the males. The metabolic ratio at 2 h was also determined 1.5 and 2.5 years later in 13 and seven males, respectively, and weekly for 3 weeks in seven males, after a dose of 500 mg, once at higher (750 mg) and lower (250 mg) doses, and once (500 mg) following moderate ethanol intake (0.5 g kg(-1) body weight) the preceding evening. Genotypes were determined for CYP2E1 as well as for N-acetyltransferase 2 and glutathione transferase M1. RESULTS: Excluding an outlier (ratio = 1.6) the metabolic ratio at 2 h ranged from 0.12 to 0.61 (n = 36). A positive correlation with body weight (r = 0.61, P < 0.001) suggested dose-dependent metabolism of chlorzoxazone. The metabolic ratio decreased with increasing chlorzoxazone dose (P = 0.01), again suggesting dose-dependent metabolism. Long-term (yearly intervals) and short-term (weekly intervals) intra- and interindividual variabilities in metabolic ratio were similar (30% and 63%vs 28% and 54%, respectively). Both inter- and intra-individual variabilities tended to decrease with increasing dose of chlorzoxazone. There was no significant influence of moderate ethanol intake the preceding evening, or of CYP2E1 genotype on the metabolic ratio. CONCLUSIONS: The relatively low intra-individual variability in the metabolism of chlorzoxazone suggests that a single-sample procedure may suffice to assess CYP2E1 activity in vivo. However, chlorzoxazone metabolism is dose-dependent at commonly used doses and it is therefore advisable to adjust the dose for body weight. Moderate intake of ethanol the preceding evening did not significantly affect the chlorzoxazone metabolic ratio. (+info)
A convenient five-drug cocktail for the assessment of major drug metabolizing enzymes: a pilot study.
(27/78)
AIMS: To assess the feasibility of administering at the same time low doses of five probe drugs, metoprolol (25 mg), chlorzoxazone (250 mg), tolbutamide (250 mg), dapsone (100 mg) and caffeine (100 mg) to determine simultaneously the activities of CYP2D6, CYP2E1, CYP2C9, CYP3A4, CYP1A2, N-acetyltransferase-2 and xanthine oxidase. METHODS: Ten healthy young non-smoking males received the following drugs or combinations of drugs over a 5-week period: week 1) metoprolol; 2) tolbutamide; 3) caffeine, chlorzoxazone and dapsone; 4) caffeine, chlorzoxazone, dapsone and metoprolol; 5) caffeine, chlorzoxazone, dapsone, metoprolol and tolbutamide. The drugs were self-administered at bedtime and urine was collected for the following 8 h. RESULTS: Mean molar phenotypic ratios obtained after administering metoprolol (mean change of -11%) or tolbutamide (mean change of -0.3%) alone, were not significantly different from those obtained when other drugs were co-administered (P > 0.05). The mean within-subject coefficients of variation were 33%, 18%, 22%, 13%, 16%, 13% and 5% for CYP3A4, CYP2D6, CYP2C9, CYP2E1, CYP1A2, N-acetyltransferase 2 and xanthine oxidase metabolic ratios, respectively. No significant interactions (P > 0.5) were observed during the simultaneous administration of various combinations of the five probe drugs. CONCLUSIONS: We propose that this cocktail, composed of five widely available drugs, constitutes a promising means of simultaneously determining the activities of the major CYP enzymes in large populations. (+info)
Clinical assessment of effects of botanical supplementation on cytochrome P450 phenotypes in the elderly: St John's wort, garlic oil, Panax ginseng and Ginkgo biloba.
(28/78)
OBJECTIVES: Elderly patients are more likely to ingest prescription medications concurrently with botanical supplements, and may therefore be vulnerable to herb-drug interactions. Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Some evidence suggests that CYP activity may decrease in the elderly. If so, herb-mediated changes in CYP activity may take on greater clinical relevance in this population. In this study, single timepoint, phenotypic metabolic ratios were used to determine whether long-term supplementation of St John's wort, garlic oil, Panax ginseng, and Ginkgo biloba affected CYP1A2, CYP2D6, CYP2E1 or CYP3A4 activity in elderly subjects. METHODS: Twelve healthy volunteers between the ages of 60 and 76 years (mean age 67 years) were randomly assigned to receive each botanical supplement for 28 days followed by a 30-day washout period. Probe drug cocktails of midazolam, caffeine, chlorzoxazone and debrisoquine were administered before and at the end of supplementation. Pre- and post-supplementation phenotypic ratios were determined for CYP3A4, CYP1A2, CYP2E1 and CYP2D6 using 1-hydroxymidazolam/midazolam serum ratios (1-hour), paraxanthine/caffeine serum ratios (6-hour), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour) and debrisoquine urinary recovery ratios (8-hour), respectively. The content of purported 'active' phytochemicals was determined for each supplement. RESULTS: Comparisons of pre- and post-St John's wort phenotypic ratios revealed significant induction of CYP3A4 (approximately 140%) and CYP2E1 activity (approximately 28%). Garlic oil inhibited CYP2E1 activity by approximately 22%. P. ginseng inhibition of CYP2D6 was statistically significant, but the magnitude of the effect (approximately 7%) did not appear to be clinically relevant. None of the supplements tested in this study appeared to affect CYP1A2 activity. CONCLUSIONS: Elderly subjects, like their younger counterparts, are susceptible to herb-mediated changes in CYP activity, especially those involving St John's wort. Pharmacokinetic herb-drug interactions stemming from alterations in CYP activity may adversely affect drug efficacy and/or toxicity. When compared with earlier studies that employed young subjects, the data suggest that some age-related changes in CYP responsivity to botanical supplementation may exist. Concomitant ingestion of botanical supplements with prescription medications, therefore, should be strongly discouraged in the elderly. (+info)
Cytochrome P450 2E polymorphism in feline liver.
(29/78)
Only one isoform of cytochrome P450 (CYP) 2E subfamily was known in human and various animals. Three cDNAs corresponding to CYP 2E subfamily members (CYP2E-a, CYP2E-b and CYP2E-c) were obtained from feline liver. These cDNAs each had a 1488-bp nucleotide coding region encoding a predicted amino acid sequence of 495 residues. Eleven amino acid substitutions were observed between CYP2E-a and CYP2E-b, but only one substitution between CYP2E-b and CYP2E-c. The CO difference spectrums about 450 nm wave length and similar values of Vmax and Km of 6-hydoxygenase activity toward chlorzoxazone were observed in all three isoforms expressed in AH22 yeast cells. By PCR-RFLP, mRNA of the CYP2E-a was found to be expressed in liver, mononuclear cells, kidney, lung, stomach, intestine and pancreas, whereas CYP2E-b and CYP2E-c were expressed mainly in the liver and mononuclear cells. Expression of CYP2E-a was observed in the livers of all felines tested, but CYP2E-b and CYP2E-c were not expressed in all cats. The sequences of two different introns between exons I and II and between exons VII and VIII were obtained in genomic DNA from the feline liver. Based on these results, we conclude that cats have two highly similar CYP2E genes. (+info)
CYP2A5-mediated activation and early ultrastructural changes in the olfactory mucosa: studies on 2,6-dichlorophenyl methylsulfone.
(30/78)
2,6-Dichlorophenyl methylsulfone (2,6-diClPh-MeSO2) is a potent olfactory toxicant reported to induce endoplasmic reticulum (ER) stress, caspase activation, and extensive cell death in mice. The aim of the present study was to examine cytochrome P450 (P450)-dependent bioactivation, nonprotein sulfhydryl (NP-SH) levels, and early ultrastructural changes in mouse olfactory mucosa following an i.p. injection of 2,6-diClPh-MeSO2 (32 mg/kg). A high covalent binding of 2,6-diClPh-14C-MeSO2 in olfactory mucosa S9 fraction was observed, and the CYP2A5/CYP2G1 substrates coumarin and dichlobenil significantly decreased the binding, whereas the CYP2E1 substrate chlorzoxazone had no effects. An increased bioactivation was detected in liver microsomes of mice pretreated with pyrazole, known to induce CYP2A4, 2A5, 2E1, and 2J, and addition of chlorzoxazone reduced this binding. 2,6-DiClPh-14C-MeSO2 showed a marked covalent binding to microsomes of recombinant yeast cells expressing mouse CYP2A5 or human CYP2A6 compared with wild type. One and 4 h after a single injection of 2,6-diClPh-MeSO2, the NP-SH levels in the olfactory mucosa were significantly reduced compared with control, whereas there was no change in the liver. Ultrastructural studies revealed that ER, mitochondria, and secretory granules in nonneuronal cells were early targets 1 h after injection. We propose that lesions induced by 2,6-diClPh-MeSO2 in the mouse olfactory mucosa were initiated by a P450-mediated bioactivation in the Bowman's glands and depletion of NP-SH levels, leading to disruption of ion homeostasis, organelle swelling, and cell death. The high expression of CYP2A5 in the olfactory mucosa is suggested to play a key role for the tissue-specific toxicity induced by 2,6-diClPh-MeSO2. (+info)
Chlorzoxazone metabolism is increased in fasted Sprague-Dawley rats.
(31/78)
Earlier data showed that men fasted for 38 h had a reduced rate of chlorzoxazone metabolism, suggesting a decreased level of cytochrome P450 2E1 (CYP2E1). In contrast, the level of CYP2E1 in fasted rats had been shown to be elevated. In this study, we have investigated whether chlorzoxazone metabolism in fasted rats was changed by determining the pharmacokinetics of chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone (6-OHCZ), as a CYP2E1 probe, and by measuring liver CYP2E1 using immunoblot techniques. Chlorzoxazone was administered by gavage (50 mg kg(-1)) or intravenously (25 mg kg(-1)) to control (nine for oral and three for intravenous) and 24 h-fasted (nine for oral and four for intravenous) male Sprague-Dawley rats. Following sampling of blood through a jugular vein cannula, chlorzoxazone and 6-OHCZ plasma concentrations were measured by HPLC with UV detection. Pharmacokinetic parameters for chlorzoxazone and 6-OHCZ in each treatment group were determined by model fitting and non-compartmental analysis. In parallel with the increased liver CYP2E1 level, the elimination of chlorzoxazone and 6-OHCZ was significantly increased in fasted rats in the oral and the intravenous study. A multiple analysis of variance covariance analysis and a multiple regression analysis revealed a significant correlation between 1/t(1/2) and CYP2E1 level and aniline hydroxylase activity. However, the correlation between 1/t(1/2) and pentoxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase and erythromycin N-demethylase was not significant. Therefore the contribution of other P450s to chlorzoxazone metabolism seemed to be minor in the concentration range that we tested. In conclusion, fasting rats for 24 h caused a measurable induction of CYP2E1, which produced a significant increase in the rate of chlorzoxazone metabolism and elimination. (+info)
Selective effects of nitric oxide on the disposition of chlorzoxazone and dextromethorphan in isolated perfused rat livers.
(32/78)
The rapid and direct effects of nitric oxide (NO) donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) on the hepatic and biliary disposition of chlorzoxazone (CZX), a marker of CYP2E1, and dextromethorphan (DEM), a marker of CYP2D1, were studied in a single-pass isolated perfused rat liver model. Livers (n = 30) were perfused with constant concentrations of NO donors (0-120 min) in addition to infusion of CZX or DEM (60-120 min), and periodical outlet and bile samples were collected. Both ISDN and SNP significantly reduced (30 and 60%, respectively) the hepatic extraction ratio of CZX and decreased (50 and 70%, respectively) the recovery of the CYP2E1-mediated metabolite, 6-hydroxychlorzoxazone, in the outlet perfusate and bile. As for DEM, both NO donors increased (up to 3.5-fold) the recovery of the CYP2D1-mediated metabolite dextrorphan (DOR) in the outlet perfusate. However, this was associated with a simultaneous decrease (50-75%) in the excretion of the metabolite into the bile, thus resulting in no change in the overall recovery of DOR as a result of NO donor treatment. The decrease in the biliary excretion of DOR was caused by NO-induced simultaneous reductions in both the conjugation of DOR and biliary clearance of DOR conjugate. Additionally, both SNP and ISDN significantly reduced the metabolism of DEM to 3-hydroxymorphinan, which is mostly regulated by CYP3A2. These studies in an intact liver model confirm the selectivity of the inhibitory effects of NO donors on cytochrome P450 enzymes, which was recently reported in microsomal studies, and expand these inhibitory effects to conjugation pathways. (+info)