Chlorzoxazone 6-hydroxylation Research Articles

Isoniazid is a mechanism-based inhibitor of cytochrome P 450 1A2, 2A6, 2C19 and 3A4 isoforms in human liver microsomes

In order to evaluate the inhibitory effects of isoniazid on cytochrome P450 (CYP) mediated drug metabolism, the in vitro inhibitory potency and specificity as well as the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-, time- and concentration dependency of isoniazid as an inhibitor of the activity of the major human CYP isoforms were studied. Using pooled human liver microsomes, the in vitro inhibitory effects of isoniazid on CYP1A2 (phenacetin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide hydroxylation), CYP2CI9 (S-mephenytoin 4'-hydroxylation), CYP2D6 (dextromethorphan O-demethylation), CYP2E1 (chlorzoxazone 6-hydroxylation) and CYP3A4 (midazolam 1'-hydroxylation) activities were examined. After a 15-min preincubation without NADPH, isoniazid reversibly inhibited microsomal CYP2C19- and CYP3A4-mediated reactions with apparent Ki values of 36 microM and 73 microM, respectively. However, isoniazid had only weak inhibitory effects on the five other CYP-mediated reactions (Ki > 110 microM). After a 15-min preincubation with NADPH, isoniazid showed an increased inhibitory potency toward CYP1A2, CYP2A6, CYP2C19 and CYP3A4 activities (Ki = 56, 60, 10 and 36 microM, respectively). In addition, the inactivation of CYP1A2, CYP2A6, CYP2C19 and CYP3A4 by isoniazid was NADPH-, time- and concentration dependent, and was characterised by Kinact values of 0.11, 0.13, 0.09 and 0.08 min(-1), and K1 values of 285, 173, 112 and 228 microM, respectively. As the peak plasma concentrations of isoniazid are around 30-50 microM, isoniazid at clinically relevant concentrations reversibly inhibits CYP2C19 and CYP3A4 activities, and mechanistically inactivates CYP1A2, CYP2A6, CYP2C19 and CYP3A4 in human liver microsomes. Co-administration of isoniazid and drugs that are primarily metabolised by these CYP isoforms, particularly by CYP2C19 and CYP3A4, may result in significant drug interactions.

Cytochrome CYP2E1 phenotyping and genotyping in the evaluation of health risks from exposure to polluted environments

Humans are exposed to over 70 000 man-made chemicals including drugs, food additives, herbicides, pesticides, and industrial agents. It is well established that environmental chemicals are the cause of numerous human diseases including cancer. In most cases, chemical carcinogens require metabolic activation, which is mainly achieved by P450s enzymes. CYP2E1 is of clinical relevance because it is inducible by ethanol, and it metabolizes many common organic solvents such as benzene, alcohols and halogenated solvents. Therefore, alteration in the level of CYP2E1 might influence the health effects of the environmental pollutants. This hypothesis needs to be validated by epidemiological studies and the objective of the “Biomed-2” project was to develop new tests to assess the individual metabolic capacity of workers exposed to volatile organic compounds in order to predict their occupational risk. In vivo chlorzoxazone 6-hydroxylation was validated as a non-invasive and selective test for the determination of liver CYP2E1 activity. Preliminary data in workers exposed to organic solvents indicated that chlorzoxazone metabolism may be a biomarker of occupational exposure to organic solvents. Other approaches, such as use of salicylate as catalytic probe or measurement of catalytic activity in lymphocytes, were not conclusive. Attempts to use CYP2E1 genotyping for estimating human risks from chemical exposure did not bring convincing data as genetic polymorphism of CYP2E1 could not be clearly related to its catalytic activity.

Inactivation of cytochrome P450 by the food-derived complex phenol oleuropein

Complex phenols, such as the glycoside oleuropein and hydroxytyrosol, are found in high concentrations in the typical components of the Mediterranean diet. We have previously reported that oleuropein inhibits androstenedione 6β-hydroxylase activity, a CYP3A marker in human liver microsomes (Stupans, I., Stretch, G., Hayball, P., 2000. Olive oil phenols inhibit human hepatic microsomal activity. Journal of Nutrition. 130 2367–2370). Oleuropein, but not the structurally similar compounds hydroxytyrosol and secologanin, was found to be a mechanism-based inhibitor of androstenedione 6β-hydroxylase activity. Preincubation with 100 μ m oleuropein and NADPH resulted in a significantly lower androstenedione 6β-hydroxylase activity when compared to preincubation carried out with oleuropein without NADPH, 0.11±0.01 nmol/mg microsomal protein/min compared with 0.29±0.03 nmol/mg microsomal protein/min ( P<0.05). The inactivation exhibited pseudo-first-order kinetics. The maximal rate constant for inactivation (k inactivation) was calculated to be 0.09 min −1 and the concentration of inactivator required for half maximal inactivation (Ki) was calculated to be 22.2 μ m. Oleuropein was found to be a relatively weak inhibitor of CYP1A2-mediated 7-methoxyresorufin- O-deethylation (24% inhibition at 100 μ m oleuropein), but not CYP2E1-mediated chlorzoxazone 6-hydroxylation. CYP1A2 did not undergo mechanism-based inactivation by oleuropein.

Chlorzoxazone: a probe drug the metabolism of which can be used to monitor one-point blood sampling in the carbon tetrachloride-intoxicated rat.

In this study, we have carried out an investigation to determine if chlorzoxazone (CZX) is a suitable probe drug for predicting hepatic injury in carbon tetrachloride (CCl4)-intoxicated rats. The animals received oral doses of CCl4 (0.25, 0.5 and 1 ml/kg) 24 h prior to intraperitoneal administration of CZX. The total CYP and CYP2E1 content, as well as the aniline and CZX hydroxylase activity (Vmax and CLint), was reduced depending on the dose of CCl4 administered. At the highest concentration (128 mM) of diethyldithiocarbamate, a specific inhibitor of CYP2E1, the production of 6-hydroxychlorzoxazone (HCZX) in microsomes from CCl4-treated rats was reduced by about 85%. The IC50 value in microsomes from CCl4-treated rats was between 3 and 5 microM. The production of HCZX and the activity of aniline hydroxylase in CCl4-treated rats correlated with the amount of rat CYP2E1 protein (r=0.881, P<0.001 and r=0.822, P<0.001, respectively). The elimination of CZX by CCl4-treated rats was reduced and the HCXZ production in the CCl4-treated group was less than that in the olive oil-treated control group. The correlations between the intrinsic clearance [CLint: Vmax/Km) in vitro and the total body clearance (CLtot) of CZX hydroxylation and the elimination half-life (t1/2) of CZX in vivo in CCl4-treated rats were high (r=0.839, P<0.001 and r= -0.828, P<0.001, respectively). In addition, the metabolic plasma HCZX/CZX ratio did not require multiple blood sampling and, 2 h after CZX administration in vivo, there was also a high correlation with the CLint (Vmax/Km) in vitro (r= -0.909, P<0.01). In conclusion, these results from this study demonstrate that CZX is a good probe for monitoring the inhibition of metabolism in rats due to CCl4 treatment.

Real-Time Quantification of Cytochrome P4502E1 mRNA in Human Peripheral Blood Lymphocytes by Reverse Transcription-PCR: Method and Practical Application

Assessing the activity of a particular drug-metabolizing enzyme (i.e., its phenotypic status) has practical implications in the fields of therapeutic drug monitoring and clinical toxicology as well as for the biological monitoring of environmental and/or occupational exposure to chemical agents. This report describes a novel, convenient, and rapid analytical method for phenotyping cytochrome P4502E1 (CYP2E1), an important drug-metabolizing enzyme in industrial toxicology (1). The measurement of CYP2E1 expression also has applications for monitoring alcoholic patients (2). Although the most accurate assessment of CYP2E1 activity is based on direct hepatic measurement, which requires a liver biopsy, alternative noninvasive approaches have been developed. Until now, the “gold standard” method for CYP2E1 phenotyping has used a probe drug, chlorzoxazone (CZX), administered orally (500 or 250 mg) to fasting individuals and metabolized to 6-hydroxychlorzoxazone (HCZX) by CYP2E1. Various CZX pharmacokinetic measures have been proposed to reflect CYP2E1 activity, as reviewed recently (3): plasma CZX t 1/2β, HCZX renal excretion, CZX oral clearance, fractional clearance to HCZX, and HCZX area under the concentration curve (AUC). However, these procedures are time-consuming, laborious (e.g., urine collection), and require subject compliance and catheterization. Furthermore, recent in vitro studies suggest that CYP1A1, CYP1A2, and possibly CYP3A are involved in CZX 6-hydroxylation, limiting the specificity of CZX for CYP2E1 phenotyping (3). CYP2E1 phenotyping for screening or follow-up of large numbers of individuals, in epidemiological studies, or even as an adjunct to improve biological monitoring programs requires the implementation of more convenient methods. Circulating lymphocytes have been used as surrogates because CYP2E1 expression in these cells appears to parallel the concentration of the hepatic enzyme, suggesting that both cell types are influenced by the same factors, including xenobiotics and physiological state (4). In a recent report involving alcoholics and controls (2), pharmacokinetic parameters for CZX hydroxylation (CZX clearance …

Characterization of xenobiotic-metabolizing cytochrome P450 (CYP) forms in ringed and grey seals from the Baltic Sea and reference sites

Earlier studies have shown that members of the cytochrome P4501 (CYP1) enzyme family are constitutively expressed, and are elevated in the livers of ringed seals ( Phoca hispida) and grey seals ( Halichoerus grypus) living in the heavily polluted Baltic Sea. In this study, we compared the expression profiles of several additional CYP enzymes in the liver and extrahepatic tissues of Baltic ringed and grey seals with the corresponding CYP expression in seals from relatively unpolluted waters. We used marker enzyme activity levels, diagnostic inhibitors and immunoblot analysis to assess members of the CYP2A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A sub-families. Coumarin 7-hydroxylation (COH), a marker of CYP2A activity, was high in the liver and the lungs of all the studied seal populations. The presence of a putative CYP2A form in these seals was further supported by the strong inhibition of COH activity by a chemical inhibitor and by an anti-CYP2A5 antibody. However, antibodies to human and rodent CYP2B, CYP2C and CYP2E forms did not recognize any proteins in these seal species. Dextromethorphan O-demethylation (marker for CYP2D activity) and chlorzoxazone 6-hydroxylation (marker for CYP2E activity) were measurable in the livers of all the seals we studied. Both activities were elevated in the Baltic seal populations, showed a strong positive correlation with CYP1A activity and were at least partly inhibited by a typical CYP1A inhibitor, α-naphthoflavone. Further studies are needed to determine the presence and characteristics of CYP2D and CYP2E enzymes in ringed and grey seals. Testosterone 6β-hydroxylation, a CYP3A marker, showed a relatively high level of activity in the livers of both seal species and was potently inhibited by ketoconazole, a CYP3A-selective inhibitor. The putative CYP3A activity showed an opposing geographical trend to that of CYP2D and CYP2E, since it was elevated in the control area. CYP3A protein levels, revealed by immunoblotting, showed a positive correlation with testosterone 6β-hydroxylation. We conclude tentatively that CYP2A- and CYP3A-like enzymes are expressed in ringed and grey seals, but that CYP2B- and CYP2C-like ones are not. Further information on the individual contaminant profile is needed before any conclusions can be drawn on a possible connection between the varying CYP expressions and the contaminant load.

Inhibition of ethanol-induced liver disease in the intragastric feeding rat model by chlormethiazole.

The purpose of this investigation was to assess the effect of chlormethiazole treatment on liver damage in the experimental rat intragastric ethanol-feeding model of alcoholic liver disease. Chlormethiazole has been used in the treatment of alcoholic withdrawal and has been shown to inhibit cytochrome P4502E1. Since treatment of experimental alcoholic liver disease with CYP2E1 inhibitors had an ameliorating effect on liver injury in the rat, chlormethiazole was used to see if it had a similar effect. Rats fed ethanol for 2 months had significantly less liver injury when chlormethiazole was added to the diet, fed intragastrically. The CYP2E1 apoprotein levels, which were increased by ethanol feeding, were also increased when chlormethiazole was fed with ethanol. Chlormethiazole inhibited the increase in the ethanol-induced CYP2E1 activity in vivo, as measured by chlorzoxazone 6-hydroxylation, but did not affect the level of CYP2E1 apoprotein. Likewise, the reduction in proteasome proteolytic enzyme activity produced by ethanol feeding was blunted in chlormethiazole-fed rats. These results support the conclusion that chlormethiazole treatment partially protects the liver from injury by inhibiting CYP2E1 activity in vivo.

Chlorzoxazone: a probe drug whose metabolism can be used to monitor toluene exposure in rats.

In this study we investigated cytochrome P450 (CYP) 2E1 expression using a probe drug, chlorzoxazone (CZX), whose metabolism can be used to monitor toluene exposure in rats. The animals received an i.p. injection of toluene (0.25, 0.5 and 1 ml/kg) once a day for 3 days. The total CYP and CYP2E1 content and the aniline and CZX hydroxylase activity (Vmax and CL(int)) increased depending on the dose of toluene administered. At the highest concentration (128 mM) of diethyldithiocarbamate, a specific inhibitor of CYP2E1, the production of 6-hydroxychlorzoxazone (HCZX) in microsomes from toluene-treated rats was reduced by about 80%. The IC50 values in microsomes from toluene-treated rats were between 3 and 5 microM. The production of HCZX and the activity of aniline hydroxylase in toluene-treated rats were correlated with the amount of rat CYP2E1 protein (r = 0.88 and r = 0.88, respectively). The elimination of CZX by toluene-treated rats was increased and the HCXZ production in the toluene-treated group was greater than that in the olive oil control group. The correlations between intrinsic clearance (CL(int): Vmax/Km) in vitro and total body clearance (CL(tot)) of CZX hydroxylation and the elimination half-life (t1/2) of CZX in vivo in toluene-treated rats were high (r = 0.784, P < 0.001; r = -0.678, P < 0.001, respectively). In addition, the metabolic plasma HCZX/CZX ratio did not require multiple blood sampling and 2 h after CZX administration in vivo there was also a high correlation with CL(int) (Vmax/Km) in vitro (r = -0.729, P < 0.001). In conclusion, these results demonstrate that CZX is a very good probe for monitoring induction in toluene-treated rats.

Changes in microsomal activity in alcoholism and obesity.

Liver damage is more prevalent among obese alcoholics, and cytochrome P-4502E1(CYP2E1) induction is involved in its pathogenesis. The study was undertaken to assess microsomal function, in alcoholic and nonalcoholic male subjects with different body compositions, through pharmacokinetics of chlorzoxazone (CLZ). We also intended to study the relationship between CLZ hydroxylation and urinary levels of 8-hydroxydiguanosine, and between CLZ levels and liver histology. Serial measurements of CLZ serum concentration, after a 750 mg dose, were performed in 17 alcoholics (9 normal weight and 8 obese) and 21 nonalcoholic subjects (10 normal weight and 11 obese). Concentration of 6-hydroxy-chlorzoxazone (6-OH-CLZ) was determined at the second hour. Anthropometry, clinical laboratory tests, and urinary 8-hydroxydiguanosine concentrations were measured. Liver biopsies were performed in alcoholics. Five biopsies revealed severe lesions, one in normal-weight and four in obese patients (p = NS). Area under the curve (AUC) of CLZ was higher in normal-weight controls compared with the rest of the groups (ANOVA p = 0.001). This parameter correlated negatively with adiposity in nonalcoholic subjects and did not correlate with liver histology. 6-OH-CLZ/CLZ ratio was lower in normal-weight controls, compared with obese subjects and normal-weight alcoholics (p = 0.02). Both alcoholism and obesity were included as predictors of CLZ AUC in a multiple regression analysis. The two-factor ANOVA showed an additive effect of centripetal body fat distribution and alcoholism. Urinary 8-hydroxydiguanosine levels were extremely variable. Centripetal adiposity and alcoholism are associated with induction of CYP2E1. This may explain the higher prevalence of liver damage among obese alcoholics and also nonalcoholic steatohepatitis.

Changes in Microsomal Activity in Alcoholism and Obesity

Background: Liver damage is more prevalent among obese alcoholics, and cytochrome P‐450–2E1(CYP2E1) induction is involved in its pathogenesis.Objectives The study was undertaken to assess microsomal function, in alcoholic and nonalcoholic male subjects with different body compositions, through pharmacokinetics of chlorzoxazone (CLZ). We also intended to study the relationship between CLZ hydroxylation and urinary levels of 8‐hydroxydiguanosine, and between CLZ levels and liver histology.Methods: Serial measurements of CLZ serum concentration, after a 750 mg dose, were performed in 17 alcoholics (9 normal weight and 8 obese) and 21 nonalcoholic subjects (10 normal weight and 11 obese). Concentration of 6‐hydroxy‐chlorzoxazone (6‐OH‐CLZ) was determined at the second hour. Anthropometry, clinical laboratory tests, and urinary 8‐hydroxydiguanosine concentrations were measured. Liver biopsies were performed in alcoholics.Results: Five biopsies revealed severe lesions, one in normal‐weight and four in obese patients (p= NS). Area under the curve (AUC) of CLZ was higher in normal‐weight controls compared with the rest of the groups (ANOVA p= 0.001). This parameter correlated negatively with adiposity in nonalcoholic subjects and did not correlate with liver histology. 6‐OH‐CLZ/CLZ ratio was lower in normal‐weight controls, compared with obese subjects and normal‐weight alcoholics (p= 0.02). Both alcoholism and obesity were included as predictors of CLZ AUC in a multiple regression analysis. The two‐factor ANOVA showed an additive effect of centripetal body fat distribution and alcoholism. Urinary 8‐hydroxydiguanosine levels were extremely variable.Conclusions: Centripetal adiposity and alcoholism are associated with induction of CYP2E1. This may explain the higher prevalence of liver damage among obese alcoholics and also nonalcoholic steatohepatitis.

Inhibition of human cytochrome P450 enzymes by 1,4-dihydropyridine calcium antagonists: prediction of in vivo drug-drug interactions.

1,4-Dihydropyridine calcium antagonists such as nifedipine are potent vasodilators. It is now commonly agreed that the oxidation of 1,4-dihydropyridine into pyridine, which is one of the main metabolic pathways, is catalysed by the cytochrome P450 (CYP) 3A4 isoform. In the present study, the inhibitory effects of 13 kinds of 1,4-dihydropyridine calcium antagonists clinically used in Japan on human CYP-isoform-dependent reactions were investigated to predict the drug interactions using microsomes from human B-lymphoblast cells expressing CYP. The specific activities for human CYP isoforms included 7-ethoxyresorfin O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), 7-benzyloxyresorufin O-dealkylation (CYP2B6), S-warfarin 7-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylaion (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and testosterone 6beta-hydroxylation (CYP3A4). Benidipine and amlodipine competitively inhibited the CYP1A1 activity. Nifedipine, nisoldipine and aranidipine competitively inhibited the CYP1A2 activity. No 1,4-dihydropyridie calcium antagonists used in this study inhibited the CYP2A6 activity. Barnidipine and amlodipine inhibited the CYP2B6 activity. Nicardipine, benidipine, manidipine and barnidipine competitively inhibited the CYP2C9 and CYP2D6 activities. Inhibition extent of the CYP2E1 activity by nifedipine and aranidipine were weak. Nicardipine, benidipine and barnidipine inhibited the CYP2C19 and CYP3A4 activities. Among the human CYP isoforms investigated, the inhibitory effects of 1,4-dihydropyridine calcium antagonists were potent on human CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2D6 as well as CYP3A4. Furthermore, the isoform selectivity of inhibition by 1,4-dihydropyridine calcium antagonists was clarified. In consideration of the Ki values obtained in the in vitro inhibition study and the concentration of 1,4-dihydropyridine calcium antagonists in human liver, the possibility of in vivo drug interactions of nicardipine and other drugs which are mainly metabolised by CYP2C9 and/or CYP3A4 was suggested. The inhibition of human CYP isoforms by 1,4-dihydropyridine calcium antagonists except nicardipine might be clinically insignificant.

CYP2E1 expression in bone marrow and its intra- and interspecies variability: approaches for a more reliable extrapolation from one species to another in the risk assessment of chemicals.

When characterizing the health risks for man by exposure to chemicals, species-specific differences have to be taken into consideration, otherwise extrapolation from animal data to the human situation would be inadequate. The site-specific toxicity of chemicals may be explained by the following alternatives: (1) reactive metabolites are generated in the liver and subsequently transported to the target tissue(s); (2) metabolism of the parent compound occurs in the target tissue, a pathway by which the enzymes necessary for activation must be expressed in the target tissue. Cytochrome P450 2E1 (CYP2E1) is an important phase-I enzyme activating several chemicals. In the study described in this paper, myeloid intra- and interspecies variability in the expression of CYP2E1 has been investigated in rats, rabbits and man, because the bone marrow represents an important target organ for toxic effects of several chemicals, e.g. benzene. CYP2E1 at the protein level was detected by Western blotting and enzyme activities were determined by CYP2E1-dependent hydroxylation of chlorzoxazone (CLX). In the bone marrow of Wistar rats, the CLX hydroxylase activities were within the same order of magnitude (range: 0.1-0.4 pmol/mg protein per min) as previously described for mice (range 0.2-0.8 pmol/mg protein per min), whereas the CYP2E1 activities in two strains of rabbits were significantly higher (range: 1.7-4.7 pmol/mg protein per min) than in the rodents (P < 0.05). In human CD34+ bone marrow stem cells, CYP2E1 could also be detected on the protein level by Western blotting. The data demonstrate a presence of CYP2E1 in the bone marrow of all species investigated, thus supporting the hypothesis of CYP2E1-dependent local metabolism of several chemicals as a factor possibly contributing to their myelotoxicity and haematotoxicity. The data show that intraspecies/intrastrain variability of CYP2E1 activity in rodents is small. However, CYP2E1 activity between rodents and a non-rodent species was quite different indicating considerable interspecies variability.

Characterization of cytochrome P450 enzymes involved in drug oxidations in mouse intestinal microsomes

1. Cytochrome P450 (P450, CYP) enzymes involved in drug oxidations in mouse intestines were characterized for their role in the first-pass metabolism of xenobiotics. 2. Preparation of mouse intestinal microsomes using a buffer containing glycerol and protease inhibitors including (p-amidinophenyl) methanesulphonyl fluoride, EDTA, soybean trypsin inhibitor, aprotinin, bestatin and leupeptine gave the highest testosterone 6β-hydroxylase activity among several preparation buffers tested in this study. Testosterone 6β-hydroxylase activity catalysed by mouse intestinal microsomes subjected to freezing and thawing was lower than that catalysed by unfrozen intestinal microsomes. 3. Low but significant catalytic activities of nifedipine oxidation, midazolam 1′ - and 4-hydroxylation, chlorzoxazone 6-hydroxylation, bufuralol 1′ - and 6-hydroxylations and tolbutamide methylhydroxylation were observed in mouse intestinal microsomes. Testosterone 6β-hydroxylation, chlorzoxazone 6-hydroxylation, and bufuralol 1′ - and 6-hydroxylations were inhibited by ketoconazole, diethyldithiocarbamate and quinine respectively. 4. Immunoblot analysis using anti-rat CYP3A antibodies demonstrated two immunoreactive bands showing similar migration in mouse intestinal and hepatic microsomes, although studies using anti-CYP1A, anti-CYP2C, anti-CYP2D and anti-CYP2E1 antibodies did not detect any band in mouse intestinal microsomes. 5. The results suggest that mouse intestinal microsomes should be prepared with glycerol and several protease inhibitors and that Cyp3a enzymes probably play an important role in drug oxidations catalysed by mouse intestine.

Cytochrome P4502E (CYP2E) in Brain: Constitutive Expression, Induction by Ethanol and Localization by Fluorescence in Situ Hybridization

Cytochrome P4502E (P4502E), the major ethanol-inducible P450 metabolizes ethanol to acetaldehyde and bioactivates procarcinogens to ultimate carcinogens. Metabolism of ethanol to acetaldehyde in the brain could be deleterious since it can react with cytoskeletal proteins, forming adducts. In the present study, rats were administered ethanol chronically to evaluate its effect on chlorzoxazone hydroxylation in rat brain regions. Chlorzoxazone hydroxylation in brains from the treated rats was induced in hippocampus and cortex, downregulated in brainstem, and unchanged in cerebellum, striatum, and thalamus. The presence of functionally active P4502E was also seen in human brain regions obtained at autopsy from traffic accident victims. Northern blot analysis of rat and human brain poly(A)+ RNA hybridized with cDNA to rat CYP2E1 revealed the constitutive presence of a corresponding transcript in rat and human brain. Localization of CYP2E by fluorescence in situ hybridization demonstrated the constitutive expression of CYP2E preferentially in the neuronal cells in rat and human brain. CYP2E expression was seen in neurons within the cerebral cortex, Purkinje and granule cell layers of cerebellum, granule cell layer of dentate gyrus, and pyramidal neurons of CA1, CA2, and CA3 subfields of hippocampus in both rat and human brain. The present studies demonstrate constitutive expression of P4502E1 in brain, its differential induction in rat brain regions by chronic ethanol treatment, and its topographic distribution in rat and human brain.

Metabolic activation of o-phenylphenol to a major cytotoxic metabolite, phenylhydroquinone: role of human CYP1A2 and rat CYP2C11/CYP2E1

1. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested. 2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the presence of 1 μm CuCl2, were the most cytotoxic chemicals examined to cultured mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2′-, 4,4′-, 2,3- and phenylhydroquinone. 3. Rat and human liver microsomes catalysed the formation of phenylhydroquinone, but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of metabolic activation of o-phenylphenol was observed with livers of themale than the female rats by 5.6- and 2.6-fold respectively. 4. Inhibitory antibodies against the male-specific CYP2C11 inhibited hepatic o-phenylphenol p-hydroxylation in the male F344 and Sprague-Dawley rat by > 70%. Liver microsomes from the isoniazid-treated rats produced 1.8- and 3-fold induction of o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-dependent activity) respectively. 5. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems, exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concentrationsof 5 (> 5-fold), 50 (> 2-fold) and 500 μm (> 2-fold) than CYP2A, CYP2B, CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450. 6. Among various CYP inhibitors tested here, 7,8-benzoflavone and furafylline, typical human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol in human livers most potently by 70 and 50% respectively. 7. The results thus indicate the involvement of rat CYP2C11/CYP2E1 and human CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.

RELATIONSHIPS BETWEEN GENETIC POLYMORPHISMS IN &lt;I&gt;CYP2D6&lt;/I&gt; AND &lt;I&gt;CYP2E1&lt;/I&gt; GENES AND LIVER MICROSOMAL DRUG OXIDATION ACTIVITIES

Twenty one types of genetic polymorphisms in CYP2D6 gene were determined in liver DNA of Japanese and Caucasians and compared these CYP2D6 genotypes with CYP2D6 protein levels and bufuralol 1'- and 6-hydroxylation activities in liver microsomes of these human samples. We detected 11 types of CYP2D6 genetic polymorphisms and classified these humans into 17 genotypes; 7 types were found in the Japanese and 13 types in the Caucasian. CYP2D6* 10B, but not CYP2D6*10A, was the most frequent in mutation at 34.6% in the Japanese, whereas in Caucasians, CYP2D6 polymorphisms including CYP2D6*4A, *4D, *4E, *4L *3, *9, and *M12 (frequencies at 6.8, 3.4, 4.5, 9.1, 2.3, 2.3, 4.5%) respectively, were detected. A Caucasian having homozygous CYP2D6*3/*3 had a protein with slower gel mobility (immunoblotting with anti-CYP2D6 antibody) and a very low activity for bufuralol 1'-hydroxylation. Five Caucasian samples with CYP2D6*4A/*4A, *4A/*4L, or *4D/*4L had no measurable CYP2D6 protein and very low bufuralol 1'-hydroxylation activities. Seven Japanese subjects with CYP2D6*10B/*IOB had CYP2D6 protein at levels of -20% of those present in humans with CYP2D6*1 and *2 and catalyzed bufuralol 1'-hydroxylation at low rates. These results support the view that CYP2D6*3, *4A, *4D, and *4L are major genotypes in producing poor metabolizer phenotypes in CYP2D6 in Caucasians, whereas CYP2D6*10B is a major causes in decreasing CYP2D6 protein expression and catalytic activities in Japanese. We also determine three types of CYP2E1 genetic polymorphisms, namely RsaI/PstI-, DraI-, and MspI-types, and compared these genotypes with levels of CYP2E1 and activities of 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation in liver microsomes from these human samples. The results obtained collectively indicated that RsaI/PstI-, DraI-, and MspI-types of CYP2E1 polymorphisms may not cause significant alterations in protein expression and enzyme catalytic activities of CYP2E1 enzyme in human livers.

Relationship between mRNA Levels Quantified by Reverse Transcription-Competitive PCR and Metabolic Activity of CYP3A4 and CYP2E1 in Human Liver

Reverse transcription-competitive polymerase chain reaction is a powerful and sensitive tool for quantifying the absolute amount of mRNA. Using this method with β-actin as the standard, we measured the mRNA level of CYP3A and CYP2E1 isoforms in human livers. We also determined the metabolic activities for both CYP isoforms. The absolute amounts of CYP3A4 and CYP2E1 mRNA in 15 liver tissues ranged from 15 to 5127 and 15163 to 69289 copies/104 copies of β-actin (341- & 3.6-fold), respectively. The testosterone 6β-hydroxylation for CYP3A4 and chlorzoxazone 6-hydroxylation activity for CYP2E1 ranged from 30 to 505 pmol/mg/min (16-fold) and from 0.59 to 2.73 nmol/mg/ml (3.6-fold), respectively. The correlation between the mRNA level and activity of CYP3A4 was significant (r = 0.94), while there was no significant correlation for CYP2E1 (r = 0.04). In conclusion, we observed a significant correlation between enzyme activity and mRNA expression for CYP3A4 but not for CYP2E1. This fact indicates that CYP2E1, in addition to being less variable between individuals than CYP3A4, differs in its regulation mechanism.

Chlorzoxazone, a selective probe for phenotyping CYP2E1 in humans.

The ability of human cytochromes P450 other than CYP2E1 to catalyse the 6-hydroxylation of chlorzoxazone (6-OH-CHZ) was examined in vitro using human liver microsomal preparations and in vivo using chlorzoxazone as a metabolic probe. Chlorzoxazone 6-hydroxylation activity was significantly correlated with 4-nitrophenol 2-hydroxylase activity and immunodetected CYP2E1 in 14 human liver samples (r = 0.92 and 0.81, P < 0.001, respectively). Conversely, this catalytic activity was not correlated with CYP 3A or CYP1A activities. Diethyldithiocarbamate (DEDTC), a specific CYP2E1 inhibitor, reduced chlorzoxazone 6-hydroxylase activity by 92.3 +/- 7.6% (n = 14 samples) while ketoconazole, a specific CYP3A inhibitor, reduced this activity by 8.6 +/- 6.3% (n = 14). The residual activity following preincubation with DEDTC was significantly correlated with nifedipine oxidation and tamoxifen N-demethylations, both specific to CYP3A (r = 0.76 and 0.68, respectively). Genetically produced pure human CYP2E1 and 3A4 hydroxylated chlorzoxazone with turnover numbers of 19.7 and 0.14 min(-1), respectively. Furthermore, cytochrome b5 stimulated chlorzoxazone 6-hydroxylation. From examination of the relative liver content of CYP2E1 and 3A, it can be asserted that CYP2E1 is the major enzyme involved in chlorzoxazone 6-hydroxylation and that the contribution of CYP3A is very minor. CYP2E1 activity was evaluated by the plasmatic metabolic ratio 6-OH-CHZ/CHZ (CHZ-MR) measured 2 h after ingestion of 500 mg CHZ. Smoker status did not influence the rate of CHZ hydroxylation. The CHZ-MR was 0.30 +/- 0.13 (mean +/- SD) n = 39 non-smokers versus 0.32 +/- 0.15, n = 75 smokers. This result suggests that CYP1A, inducible by cigarette smoking, is not significantly involved in chlorzoxazone hydroxylation. Women exhibited a slightly lower CHZ-MR than men (0.29 +/- 0.15, n = 44 versus 0.34 +/- 0.15 n = 49, respectively). Obesity increased CHZ-MR, especially in non-insulin-dependent diabetic individuals (0.45 +/- 0.21, n = 13 versus 0.30 +/- 0.15, n = 42 control individuals, P = 0.007). Furthermore, exposure of workers to volatile organics in a shoe factory decreased CHZ-MR (0.19 +/- 0.09, n = 10 Mexican workers versus 0.34 +/- 0.12, n = 16 Mexican control individuals, P = 0.001). Concomitant administration of grapefruit juice (known to be an inhibitor of CYP3A4) with chlorzoxazone did not significantly modify the CHZ metabolic ratio: 0.29 +/- 0.1 versus 0.31 +/- 0.1, for nine control individuals without and with grapefruit juice, respectively. In conclusion, all these results demonstrate that chlorzoxazone is a very selective probe for phenotyping CYP2E1 in humans.

Effect of protein-calorie malnutrition on cytochromes P450 and glutathione S-transferase.

Protein-calorie malnutrition (PCM) can develop both from inadequate food intake and as a consequence of diseases such as cancer and AIDS. Several studies have shown that PCM can alter drug clearance but little information is available on the effect of PCM on individual cytochrome P450 isoforms and phase II conjugation enzymes. The aim of the present study was to begin a systematic evaluation of the effect of PCM on the activity of individual drug metabolizing enzymes in a rat model of PCM. Control and PCM rats received isocaloric diets which contained either 21% or 5% (deficient) protein. After 3 weeks, the animals were sacrificed and microsomal and cytosolic fractions prepared. Ethoxyresorufin O-deethylation (EROD), chlorzoxazone 6-hydroxylation, dextromethorphan N- and O-demethylation and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation were used as measures of CYP1A, CYP2E1, CYP3A2, CYP2D1 and glutathione S-transferase (GST) activity, respectively. Additionally, NADPH-cytochrome P450 reductase activity was measured in the liver microsomes. PCM significantly reduced the maximum velocity (Vmax) of all model reactions studied. However, differential effects were observed with respect to K(m) values of the reactions. The K(m) values for EROD and dextromethorphan N-demethylation were significantly increased in PCM animals, whereas the K(m) values for chlorzoxazone 6-hydroxylation and dextromethorphan O-demethylation were decreased. In contrast, the K(m) value for CDNB conjugation was unchanged. When NADPH-cytochrome P450 reductase activity was compared, a 29% reduction in reductase activity was noted in PCM animals as compared to controls. Thus, it appears that PCM decreases the overall activity of certain phase I and phase II metabolism enzymes in rat liver while exhibiting differential effects on K(m). Furthermore, this reduction in activity may be due in part to diminished activity of cytochrome P450 reductase.

Inhibition of human prenatal biosynthesis of all- trans-retinoic acid by ethanol, ethanol metabolites, and products of lipid peroxidation reactions : A possible role for CYP2E1

Biotransformation of all- trans-retinol (t-ROH) and all- trans-retinal (t-RAL) to all- trans-retinoic acid (t-RA) in human prenatal hepatic tissues (53–84 gestational days) was investigated with HPLC using human adult hepatic tissues as positive controls. Catalysis of the biotransformation of t-ROH by prenatal human cytosolic fractions resulted in accumulation of t-RAL with minimal t-RA. Oxidations of t-ROH catalyzed by prenatal cytosol were supported by both NAD + and NADP +, although NAD + was a much better cofactor. In contrast, catalysis of the oxidation of t-RAL to t-RA appeared to be solely NAD + dependent. Substrate K m values for conversions of t-ROH to t-RAL and of t-RAL to t-RA were 82.4 and 65.8 μM, respectively. At concentrations of 10 and 90 mM, ethanol inhibited the conversion of t-ROH to t-RAL by 25 and 43%, respectively, but did not inhibit the conversion of t-RAL to t-RA significantly. In contrast, acetaldehyde reduced the conversion of t-RAL to t-RA by 25 and 87% at 0.1 and 10 mM respective concentrations. Several alcohols and aldehydes known to be generated from lipid peroxides also exhibited significant inhibition of t-RA biosynthesis in human prenatal hepatic tissues. Among the compounds tested, 4-hydroxy-2-nonenal (4-HNE) was highly effective in inhibiting the conversion of t-RAL to t-RA. A 20% inhibition was observed at a concentration of only 0.001 mM, and nearly complete inhibition was produced at 0.1 mM. Human fetal and embryonic hepatic tissues each exhibited significant CYP2E1 expression as assessed with chlorzoxazone 6-hydroxylation, a highly sensitive western blotting technique, and reverse transcriptase-polymerase chain reaction (PCR) (RT-PCR), suggesting that lipid peroxidation can be initiated via CYP2E1-catalyzed ethanol oxidation in human embryonic hepatic tissues. In summary, these studies suggest that ethanol may affect the biosynthesis of t-RA in human prenatal hepatic tissues directly and indirectly. Ethanol and its major oxidative metabolite, acetaldehyde, both inhibit the generation of t-RA. Concurrently, the CYP2E1-catalyzed oxidation of ethanol can initiate lipid peroxidation via generation of a variety of free radicals. The lipid peroxides thereby generated could then be further converted via CYP2E1-catalyzed reactions to alcohols and aldehydes, including 4-HNE, that act as potent inhibitors of t-RA synthesis.