Hepatic CYP2E1 Research Articles

EFFECT OF CYP2E1 INDUCTION BY ETHANOL ON THE IMMUNOTOXICITY AND GENOTOXICITY OF EXTENDED LOW-LEVEL BENZENE EXPOSURE

Potential additive effects of ethanol consumption, a common life-style factor, and lowlevel benzene exposure, a ubiquitous environmental pollutant, were investigated. Ethanol is a potent inducer of the cytochrome P-450 2E1 (CYP2E1) enzyme, which bioactivates benzene to metabolites with known genotoxicity and immunotoxicity. A liquid diet containing 4.1% ethanol was used to induce hepatic CYP2E1 activity by 4-fold in female CD-1 mice. Groups of ethanol-treated or pair-fed control mice were exposed to benzene or filtered air in inhalation chambers for 7 h/d, 5 d/wk for 6 or 11 wk. The initial experiment focused on immunotoxicity endpoints based on literature reports that ethanol enhances high-dose benzene effects on spleen, thymus, and bone marrow cellularity and on peripheral red blood cell (RBC) and white blood cell (WBC) counts. No statistically significant alterations were found in spleen lymphocyte cellularity, subtype profile, or function (mitogen-induced proliferation, cytokine production, or natural killer cell lytic activity) after 6 wk of ethanol diet, 0.44 ppm benzene exposure, or both. This observed absence of immunomodulation by ethanol alone, a potential confounding factor, further validates our previously established murine model of sustained CYP2E1 induction by dietary ethanol. Subsequent experiments involved a 10-fold higher benzene level for a longer time of 11 wk and focused on genotoxic endpoints in known target tissues. Bone marrow and spleen cells were evaluated for DNA-protein cross-links, a sensitive transient index of genetic damage, and spleen lymphocytes were monitored for hprt -mutant frequency, a biomarker of cumulative genetic insult. No treatment-associated changes in either genotoxic endpoint were detected in animals exposed to 4.4 ppm benzene for 6 or 11 wk with or without coexposure to ethanol. Thus, our observations suggest an absence of genetic toxicity in CD-1 mice exposed to environmentally relevant levels of benzene with or without CYP2E1 induction.

Attenuation of Alcohol-Induced Apoptosis of Hepatocytes in Rat Livers by Polyenylphosphatidylcholine (PPC)

Background: Alcohol consumption increases apoptosis of hepatocytes. This effect appears to be mediated by the induction of hepatic cytochrome P-4502E1(CYP2E1) and its generation of free radicals, which results in an enhanced lipid peroxidation that initiates apoptosis. Because polyenylphosphatidylcholine (PPC), a soybean extract rich in polyunsaturated phosphatidylcholines, decreases the induction of ethanol-specific CYP2E1 and opposes oxidative stress, we hypothesized that PPC supplementation may attenuate hepatocyte apoptosis caused by ethanol ingestion. Methods: Twenty-eight male Sprague Dawley® rats were pair-fed Lieber-DeCarli liquid diets containing 36% of energy as alcohol or an isocaloric amount of carbohydrate for 28 days. Half of the rats were given PPC (3 g/liter), whereas the other half received the same amount of linoleate (as safflower oil) and of choline as the bitartrate. An additional dose of alcohol (3 g/kg) was given intragastrically 90 min before the livers were removed. We assessed apoptosis in formalin-fixed, paraffin-embedded liver sections by using the TUNEL (terminal transferase dUTP nick end labeling) assay. Apoptotic hepatocytes were identified by positive TUNEL staining in conjunction with condensation of nucleoplasm or margination of chromatin. In each rat, 20,000 to 60,000 hepatocytes were counted by light microscopy by using Image-Pro Plus computer software, and the incidence of apoptosis was expressed as the percentage of total hepatocytes. Results: Alcohol feeding resulted in a 4.5-fold increase in apoptosis of hepatocytes compared to pair-fed control rats; PPC supplementation decreased the alcohol-induced apoptosis to less than half. No difference in the incidence of apoptosis between the control and PPC-supplemented rats was found in the absence of alcohol. Apoptosis was distributed randomly in the liver lobules of the rats fed the control diet, whereas the alcohol-induced apoptosis was significantly increased in the perivenular area. PPC supplementation strikingly reduced this effect. Conclusions: PPC attenuates alcohol-induced apoptosis of hepatocytes; this effect may provide a mechanism for PPC's protection against liver injury, possibly in association with its antioxidative action via the down-regulation of ethanol-mediated CYP2E1 induction.

Attenuation of Alcohol‐Induced Apoptosis of Hepatocytes in Rat Livers by Polyenylphosphatidylcholine (PPC)

Alcohol consumption increases apoptosis of hepatocytes. This effect appears to be mediated by the induction of hepatic cytochrome P-4502E1(CYP2E1) and its generation of free radicals, which results in an enhanced lipid peroxidation that initiates apoptosis. Because polyenylphosphatidylcholine (PPC), a soybean extract rich in polyunsaturated phosphatidylcholines, decreases the induction of ethanol-specific CYP2E1 and opposes oxidative stress, we hypothesized that PPC supplementation may attenuate hepatocyte apoptosis caused by ethanol ingestion. Twenty-eight male Sprague Dawley rats were pair-fed Lieber-DeCarli liquid diets containing 36% of energy as alcohol or an isocaloric amount of carbohydrate for 28 days. Half of the rats were given PPC (3 g/liter), whereas the other half received the same amount of linoleate (as safflower oil) and of choline as the bitartrate. An additional dose of alcohol (3 g/kg) was given intragastrically 90 min before the livers were removed. We assessed apoptosis in formalin-fixed, paraffin-embedded liver sections by using the TUNEL (terminal transferase dUTP nick end labeling) assay. Apoptotic hepatocytes were identified by positive TUNEL staining in conjunction with condensation of nucleoplasm or margination of chromatin. In each rat, 20,000 to 60,000 hepatocytes were counted by light microscopy by using Image-Pro Plus computer software, and the incidence of apoptosis was expressed as the percentage of total hepatocytes. Alcohol feeding resulted in a 4.5-fold increase in apoptosis of hepatocytes compared to pair-fed control rats; PPC supplementation decreased the alcohol-induced apoptosis to less than half. No difference in the incidence of apoptosis between the control and PPC-supplemented rats was found in the absence of alcohol. Apoptosis was distributed randomly in the liver lobules of the rats fed the control diet, whereas the alcohol-induced apoptosis was significantly increased in the perivenular area. PPC supplementation strikingly reduced this effect. PPC attenuates alcohol-induced apoptosis of hepatocytes; this effect may provide a mechanism for PPC's protection against liver injury, possibly in association with its antioxidative action via the down-regulation of ethanol-mediated CYP2E1 induction.

Constitutive and Inducible Expression of Hepatic CYP2E1 in Leptin-deficient ob/ob Mice

In this study we have analyzed the inducible as well as constitutive hepatic expression of Cyp2e1 in a genetic model of obesity and non-insulin dependent (type II) diabetes, the leptin-deficient ob/ob mouse. In obese mice, Cyp2e1 levels were decreased compared to lean littermates. Treatment with leptin increased hepatic Cyp2e1 in obese mice to the levels observed in lean animals, but failed to alter Cyp2e1 expression in lean animals. As expected, leptin also reduced food intake in treated mice compared to saline-treated controls. In obese mice pair-fed the reduced amount of food, there was a significant increase in Cyp2e1 mRNA but no increase in Cyp2e1 protein or enzyme activity. Fasting and administration of acetone and 4-methylpyrazole increased Cyp2e1 mRNA as well as protein and activity in both obese and lean mice. The present data indicate that while Cyp2e1 is still inducible in obese mice by xenobiotics and fasting, full constitutive expression of Cyp2e1 requires leptin to be present. This effect of leptin appears to be at least partly independent of the hypothalamic control of food intake.

Effects of Tetrachloroethylene and 1, 1, 1-Trichloroethane on the Expression of P450 Isoforms in Rat Lung and Liver

Tetrachloroethylene (PCE) and 1, 1, 1-trichloroethane (1, 1, 1-TCA) were administered intraperitoneally at a moderately toxic dose of 1 g/kg/d for three consecutive days to male and female Wistar rats weighing about 200g (7 weeks old). The agents were given alone or simultaneously with phenobarbital (PB, 80mg/kg/d) and the animals were sacrificed 24h after the last treatment. Microsomal fractions prepared from both lungs and livers were tested for the monooxygenase activity and protein content of four cytochrome P450 (CYP) isoforms, i.e., CYP1A, 2B, 2E1 and 3A. As far as the monooxygenase activity was concerned, CYP1A, 2E1 and 3A but not 2B were constitutive in liver while only CYP2B was detected in lung. PCE suppressed significantly pulmonary CYP2B and hepatic CYP2E1 but enhanced hepatic CYP1A. In contrast, 1, 1, 1-TCA suppressed hepatic CYP2E1 under the conditions used. Although PB induced hepatic CYP2B and enhanced CYP1A several-fold, it had no effect on CYP2E1 and 3A, as well as pulmonary CYP2B. The effects of PCE and 1, 1, 1-TCE on CYP isoforms in microsomal fractions prepared from PB-cotreated animals were also studied comparatively in terms of sex and organ. The response of pulmonary enzymes to both chemicals was the same as that of PB-untreated animals. As far as PB-induced hepatic CYP2B was concerned, PCE was suppressive while 1, 1, 1-TCA showed a degree of potentiation. Furthermore, in the case of CYP1A a sex difference was noted in the response to these chemicals. As far as the protein levels of CYP isoforms were concerned, they were generally proportional to the enzyme activities.

The induction of the human hepatic CYP2E1 gene by interleukin 4 is transcriptional and regulated by protein kinase C.

Cytochrome P4502E1 (CYP2E1) plays a key role in the metabolism of numerous drug substrates, mostly in mammalian liver. Both the apoprotein and mRNA levels are increased in response to interleukin 4 (IL-4) in primary human hepatocyte cultures. We developed a human hepatoma cell model that faithfully reproduces the responsiveness of the CYP2E1 gene to IL-4 at least in part through transcriptional activation, upon treatment with 150 U/ml of IL-4. As expected, IL-4 induced tyrosine phosphorylation of the STAT6 transcription factor, an effect prevented by the tyrosine kinase inhibitor tyrphostin A25. However, this inhibitor as well as genistein (another inhibitor of tyrosine kinases) had no effect on the IL-4 induction of CYP2E1. Similarly, protein kinase A activators (forskolin and dibutyryl-cAMP) and inhibitor (H89) did not influence the response to IL-4. However, PKC inhibitors (H7 and calphostin C) strongly blocked any induction of the gene, as well as the IL-4-dependent translocation of PKCS. Taken together, our results show that IL-4 coordinately induces CYP2E1 transcription, mRNA and apoprotein levels in human hepatoma cells in a PKC-dependent manner, potentially through the activity of the PKCzeta isoform.

CYP2E1 Expression in Human Lymphocytes From Various Ethnic Populations

Background: Monitoring CYP2E1 levels in alcoholic individuals holds inherent appeal because such determinations might indicate individuals at increased risk for alcoholic liver disease. We previously demonstrated that lymphocyte CYP2E1 expression reflects in vivo activity of the hepatic enzyme. Methods: To further validate this approach, the current investigation compared lymphocyte CYP2E1 content and chlorzoxazone pharmacokinetics in 51 alcoholic and nonalcoholic White, Navajo, and Mexican American subjects. After an oral dose of chlorzoxazone, blood samples were collected and lymphocytes isolated. Results: Alcoholics exhibited a 2-fold clevation in lymphocyte CYP2E1 messenger ribonucleic acid (mRNA) and protein compared to nonalcoholics. Chlorzoxazone clearance rates were 1.9-fold higher and area under the concentration curve (AUC) values 1.8-fold lower in alcoholic individuals compared to nonalcoholics. Furthermore, chlorzoxazone clearance rates correlated (r= 0.55, p < 0.01, n= 38) with lymphocyte CYP2E1 mRNA content, and transcript levels further correlated (r= 0.52, p < 0.001, n= 38) with CYP2E1 protein content. To compare phenotype with genotype, restriction fragment length polymorphism analyses on deoxyribonuclcic acid samples were performed to identify polymorphisms in the CYP2E1 gene. No subjects were homozygous for rare alleles c2 or C. Nonetheless, 27% of the Navajos and 15% of the Mexican Americans were heterozygous for the c2 allele. Two White subjects appeared heterozygous (c1/c2) when RsaI was used to characterize CYP2E1 genotype but homozygous (c1/c1) at the PstI locus. Fifteen percent of Mexican American subjects, 20% of Navajo subjects, and 6% of White subjects were heterozygous for the C allele. Neither CD nor c1/c2 genotypes were associated with alcoholism. Conclusions: Human lymphocyte CYP2E1 mRNA levels may be useful predictors of alcohol-mediated alterations in hepatic CYP2E1 activity. Moreover, ethnicity does not appear to play a major role in the levels of expression of lymphocyte CYP2E1.

CYP2E1 Expression in Human Lymphocytes From Various Ethnic Populations

Monitoring CYP2E1 levels in alcoholic individuals holds inherent appeal because such determinations might indicate individuals at increased risk for alcoholic liver disease. We previously demonstrated that lymphocyte CYP2E1 expression reflects in vivo activity of the hepatic enzyme. To further validate this approach, the current investigation compared lymphocyte CYP2E1 content and chlorzoxazone pharmacokinetics in 51 alcoholic and nonalcoholic White, Navajo, and Mexican American subjects. After an oral dose of chlorzoxazone, blood samples were collected and lymphocytes isolated. Alcoholics exhibited a 2-fold elevation in lymphocyte CYP2E1 messenger ribonucleic acid (mRNA) and protein compared to nonalcoholics. Chlorzoxazone clearance rates were 1.9-fold higher and area under the concentration curve (AUC) values 1.8-fold lower in alcoholic individuals compared to nonalcoholics. Furthermore, chlorzoxazone clearance rates correlated (r = 0.55, p < 0.01, n = 38) with lymphocyte CYP2E1 mRNA content, and transcript levels further correlated (r = 0.52, p < 0.001, n = 38) with CYP2E1 protein content. To compare phenotype with genotype, restriction fragment length polymorphism analyses on deoxyribonucleic acid samples were performed to identify polymorphisms in the CYP2E1 gene. No subjects were homozygous for rare alleles c2 or C. Nonetheless, 27% of the Navajos and 15% of the Mexican Americans were heterozygous for the c2 allele. Two White subjects appeared heterozygous (c1/c2) when RsaI was used to characterize CYP2E1 genotype but homozygous (c1/c1) at the PstI locus. Fifteen percent of Mexican American subjects, 20% of Navajo subjects, and 6% of White subjects were heterozygous for the C allele. Neither CD nor cl/c2 genotypes were associated with alcoholism. Human lymphocyte CYP2E1 mRNA levels may be useful predictors of alcohol-mediated alterations in hepatic CYP2E1 activity. Moreover, ethnicity does not appear to play a major role in the levels of expression of lymphocyte CYP2E1.

A Comparison of the Effect of Five Phenothiazines on Hepatic CYP Isoenzymes in Rats

We examined the influence of five phenothiazine derivatives on the activity and the expression of hepatic cytochrome P450 (CYP) 1A, 2B, 2C6, 2C11, 2E1 and 3A2 in male Sprague-Dawley rats. Twenty mg/kg of phenothiazine, chlorpromazine, and thioridazine, or 1 mg/kg of trifluoperazine and perphenazine was administered intraperitoneally for 4 consecutive days. Phenothiazine increased the total CYP content and induced CYP1A, CYP2B, and CYP3A. Western blot analysis showed marked induction of CYP1A1 by phenothiazine. Chlorpromazine induced CYP2B, and CYP3A without a significant increase in the CYP content. Thioridazine decreased the total CYP content and reduced CYP2C11, CYP2E1, and CYP3A. Neither trifluoperazine nor perphenazine significantly altered the catalytic activity or the protein level of any enzyme examined. These results suggest that the antipsychotic phenothiazine derivatives examined here hardly affect CYP1A at the pharmacological doses, while phenothiazine does induce it. Although a relatively short period and small dose of administration might counteract the influence, trifluoperazine and perphenazine have less effect on CYP subfamilies than chlorpromazine or thioridazine.

Characterization of a Dietary Ethanol Protocol for Cyp2e1 Induction in the CD-1 Mouse without Evident Hepatic Toxicity or Genotoxicity

Although the CD-1 mouse strain has been used to investigate the toxicity of numerous substrates of Cyp2e1, limited information is available about responses of this strain to ethanol, a potent and clinically relevant inducer of this cytochrome P450 isozyme. Our goal was to characterize a dietary ethanol protocol for greater than threefold induction of hepatic Cyp2e1 in CD-1 mice without confounding alterations to other biotransformation enzymes or injury to known target tissues. Female CD-1 mice were fed the Lieber-DeCarli liquid diet containing 1.4 to 6.4% ethanol (v/v) for time periods of 1 to 12 weeks. A series of range-finding experiments indicated that the stock 6.4% ethanol diet caused rapid weight loss, whereas dietary ethanol concentrations less than or equal to 3.2% produced inadequate (i.e., less than threefold) induction of hepatic Cyp2e1. Suitable responses were observed in mice fed a 4.1% ethanol diet, namely, body weight gain equivalent to both pair-fed or rodent chow control groups plus consistent and stable induction of hepatic Cyp2e1 activities by greater than threefold without evidence of hepatic lipid peroxidation or histopathology. Evaluations of other representative biotransformation activities, including bone marrow quinone reductase and hepatic aldehyde dehydrogenase, showed no alterations with the 4.1% ethanol diet, except for a modest 20% decline in hepatic glutathione peroxidase. Unlike observations in other species, Cyp2e1 induction was not evident in bone marrow or spleen by Western blot. Mice given the 4.1% ethanol diet for 6 and/or 12 weeks showed no changes in cellularity of the spleen or bone marrow, frequency of hprt mutations in splenic lymphocytes, or percentage of DNA-protein crosslinks in bone marrow cells. These parameters were monitored because ethanol at high exposures is known to cause immunosuppression and mild genotoxicity. Female CD-1 mice fed a 4.1% ethanol liquid diet showed substantial (greater than threefold) induction of hepatic Cyp2e1 without confounding detrimental effects on the fiver, spleen, or bone marrow. Thus, this dietary ethanol protocol should be useful for future investigations of the role of Cyp2e1 induction on genotoxicity responses to Cyp2e1 substrates.

Autoantibodies against cytochromes P-4502E1 and P-4503A in alcoholics.

Autoantibodies against soluble liver enzymes have been reported among alcoholics, but the targets of self-reactivity toward membrane proteins of the liver have not been characterized. Previously, among alcoholics, we found antibodies against ethanol-derived radical protein adducts that are dependent on cytochrome P-4502E1 (CYP2E1) for their formation. To further investigate autoantibodies against cytochrome P-450s during alcohol abuse, sera of rats chronically treated with ethanol in the total enteral nutrition model and sera from alcoholics with or without alcohol liver disease and from control subjects were analyzed by enzyme-linked immunosorbent assay and Western blotting for the presence of IgG against rat and human CYP2E1, rat CYP3A1, and human CYP3A4. A time-dependent appearance of IgG against rat CYP3A1 and CYP2E1 was evident during chronic ethanol feeding of rats. Anti-CYP2E1 reactivity showed positive correlation with the levels of hepatic CYP2E1 and was inhibited by the CYP2E1 transcriptional inhibitor chlormethiazole. Screening of the human sera by enzyme-linked immunosorbent assay revealed reactivity against CYP3A4 and CYP2E1 in about 20 to 30% and 10 to 20% of the alcoholic sera, respectively. No difference were noted between sera from alcoholics with or without hepatitis C virus infection, and only very little reactivity was seen in sera from control subjects. Western blotting analysis revealed anti-human CYP2E1 reactivity in 8 of 85 alcoholic sera and 3 of 58 control sera, whereas anti-CYP3A4 reactivity was detected in 18 of 85 alcoholic sera and 4 of 58 control sera, which were different from the sera reactive with CYP2E1. Immunoblot reactivity of CYP3A4-positive alcoholic sera was found against glutathione-S-transferase fusion proteins containing truncated forms of CYP3A4, and such sera were also able to immunoprecipitate in vitro translated CYP3A4. Seven of eight sera showed reactivity toward domains C-terminal of position Ser281, and 1 of 8 sera recognized autoepitopes within the region Thr207-Ser281. These findings indicate that alcoholics develop autoantibodies against CYP2E1 and CYP3A4 that the CYP3A4 C-terminal domain is a target for the autoantibody reactions among a subset of alcoholics. The novel finding of CYP3A4 autoantibodies and their significant expression among alcoholics warrants further investigation. Attention should be given to immune toxicity associated with CYP3A4 autoantibodies and cases of alcohol abuse that are accompanied by exposure to drugs and substances that are CYP3A substrates.

Hormonal regulation of microsomal cytochrome P4502E1 and P450 reductase in rat liver and kidney

1. The relative roles of pituitary hormones (especially growth hormone) and testicular hormones (especially testosterone) in the regulation of renal and hepatic CYP2E1 and cytochrome P450 reductase have been studied in the male rat. 2. Depletion of pituitary hormones by hypophysectomy (Hx) resulted in 12-14-fold increases in renal CYP2E1 (p ⩽ 0.05) and a 40% drop in NADPH-dependent cytochrome c reductase activity (p ⩽ 0.05) compared with 6-fold increases in CYP2E1 (p ⩽ 0.05) and a 60% drop in P450 reductase apoprotein (p ⩽ 0.05) in the liver. 3. The increase in hepatic CYP2E1 was associated with increased gene transcription in nuclear run-on experiments. 4. Restoration of renal CYP2E1 to control levels by hormone treatment required both growth hormone and an intact testis, whereas partial restoration of CYP2E1 apoprotein levels in liver was accomplished by growth hormone, but not testosterone. 5. Renal NADPH-dependent cytochrome c reductase activity was restored by growth hormone and testosterone treatment, whereas the hepatic reductase appeared to be regulated by other pituitary hormones. 6. CYP2E1 and P450 reductase appear to be under complex endocrine regulation by pituitary and testicular hormones in a tissue specific manner.

Acarbose alone or in combination with ethanol potentiates the hepatotoxicity of carbon tetrachloride and acetaminophen in rats.

Acarbose reduces the absorption of monosaccharides derived from dietary carbohydrates, which play an important role in the metabolism and toxicity of some chemical compounds. We studied the effects of acarbose on the hepatotoxicity of carbon tetrachloride (CCl4) and acetaminophen (AP) in rats, both of which exert their toxic effects through bioactivation associated with cytochrome P450 2E1 (CYP2E1). Male Sprague-Dawley rats were kept on a daily ration (20 g) of powdered chow diet containing 0, 20, 40, or 80 mg/100 g of acarbose, with drinking water containing 0% or 10% of ethanol (vol/vol). Three weeks later, the rats were either killed for an in vitro metabolism study or challenged with 0.50 g/kg CCl4 orally or 0. 75 g/kg AP intraperitoneally. The ethanol increased the hepatic microsomal CYP2E1 level and the rate of dimethylnitrosamine (DMN) demethylation. The 40- or 80-mg/100 g acarbose diet, which alone increased the CYP2E1 level and the rate of DMN demethylation, augmented the enzyme induction by ethanol. The 40- or 80-mg/100 g acarbose diet alone potentiated CCl4 and AP hepatotoxicity, as evidenced by significantly increased levels of both alanine transaminase (ALT) and aspartate transaminase (AST) in the plasma of rats pretreated with acarbose. Ethanol alone also potentiated the toxicity of both chemicals. When the 40- or 80-mg/100 g acarbose diet was combined with ethanol, the ethanol-induced potentiation of CCl4 and AP hepatotoxicity was augmented. Our study demonstrated that high doses of acarbose, alone or in combination with ethanol, can potentiate CCl4 and AP hepatotoxicity in rats by inducing hepatic CYP2E1.

Comparative Study on the Effect of Trichloroethylene on the Expression of P450 Isoforms in Rat Lung and Liver(PROCEEDINGS OF 24TH SYMPOSIUM ON TOXICOLOGY AND ENVIRONMENTAL HEALTH)

Trichloroethylene (TCE) is a highly toxic compound which belongs to the class of volatile halogenated hydrocarbons, which are well-known pollutants of drinking water and the atmosphere. The effects of moderately toxic concentration of TCE on the levels of cytochrome P450 (CYP) isoforms and on their phenobarbital (PB) induction in rats were studied. Monooxygenase activities associated with individual CYP isoforms, i.e., CYP1A, 2B, 2E1 and 3A, were measured in microsomal fractions prepared from both lungs and livers of male and female Wistar rats (7-weeks old). Differences in constitutive levels of CYP isoforms were observed between liver and lung of mock-treated rat ; microsomal CYP2B activity was solely detected in lung while the activities of CYP1A, 2EI and 3A, but not 2B were detected in liver. Among these, the pulmonary CYP2B and hepatic CYP2E1 activities were reduced by TCE-treatment. PB-treatment resulted in the detection of increased levels of hepatic CYP1A and 2E1 and of pulmonary CYP2B activities, and also the appearance of hepatic CYP2B activity. Coadministration of TCE was suppressive against the activities of CYP isoforms except for hepatic CYP2E1 in PB-treated rats. The lowered CYP2B activity in the presence of TCE was accompanied by the reduction in the amount of CYP2B apoprotein.

Diet and risk of ethanol-induced hepatotoxicity: carbohydrate-fat relationships in rats.

Nutritional status is a primary factor in the effects of xenobiotics and may be an important consideration in development of safety standards and assessment of risk. One important xenobiotic consumed daily by millions of people worldwide is alcohol. Some adverse effects of ethanol, such as alcohol liver disease, have been linked to diet. For example, ethanol-induced hepatotoxicity in animal models requires diets that have a high percentage of the total calories as unsaturated fat. However, little attention has been given to the role of carbohydrates (or carbohydrate to fat ratio) in the effects of this important xenobiotic on liver injury. In the present study, adult male Sprague-Dawley rats (8-10/group) were infused (intragastrically) diets high in unsaturated fat (25 or 45% total calories), sufficient protein (16%) and ethanol (38%) in the presence or absence of adequate carbohydrate (21 or 2.5%) for 42-55 days (d). Animals infused ethanol-containing diets adequate in carbohydrate developed steatosis, but had no other signs of hepatic pathology. However, rats infused with the carbohydrate-deficient diet had a 4-fold increase in serum ALT levels (p < 0.05), an unexpectedly high (34-fold) induction of hepatic microsomal CYP2E1 apoprotein (p < 0.001), and focal necrosis. The strong positive association between low dietary carbohydrate, enhanced CYP2E1 induction and hepatic necrosis suggests that in the presence of low carbohydrate intake, ethanol induction of CYP2E1 is enhanced to levels sufficient to cause necrosis, possibly through reactive oxygen species and other free radicals generated by CYP2E1 metabolism of ethanol and unsaturated fatty acids.

Dietary carbohydrate intake plays an important role in preventing alcoholic fatty liver in the rat

Background/Aims: Dietary carbohydrate intake during ethanol ingestion augments the induction of hepatic cytochrome P450 2E1 (CYP2E1) by ethanol. This study addresses the role of carbohydrate intake in the development of alcoholic fatty liver in the rat. Methods: Male Sprague-Dawley rats were pair-fed on liquid diets containing ethanol (3.5 g/day, 36% of total calories) with different amounts of carbohydrate and fat for 4 weeks, and the development of fatty liver was observed biochemically and morphologically. Results: An ethanol-containing low-carbohydrate diet (protein 17%; fat 36%; carbohydrate 11%; ethanol 36%) had more markedly adverse effects on the liver of rats than did an isocaloric ethanol-containing high-carbohydrate diet (protein 17%; fat 5%; carbohydrate 42%; ethanol 36%). The hepatic triglyceride level in the rats that consumed the low-carbohydrate diet was higher than that in the rats kept on the high-carbohydrate diet, a finding that was confirmed histologically. The ethanol-containing low-carbohydrate diet caused a marked increase in the activity of hepatic CYP2E1. The CYP2E1 protein level, as measured by Western blot analysis, matched the activity of CYP2E1, as measured by the rates of dimethylnitrosamine, p-nitrophenol and ethanol metabolism. The severity of the fatty liver was well correlated with the increased CYP2E1 activity. Conclusions: Dietary carbohydrate intake plays an important role in the development of alcoholic fatty liver by affecting CYP2E1 activity in the liver. A liquid diet containing ethanol in which the ethanol is included at the expense of fat is more acceptable to rats than a diet in which the ethanol replaces carbohydrate.

Chlorzoxazone pharmacokinetics as a marker of hepatic cytochrome P4502E1 in humans.

Previous in vitro studies have demonstrated that hepatic P4502E1 metabolizes chlorzoxazone (CZX, a commonly used muscle relaxant) to 6-hydroxychlorzoxazone (6-OH-CZX). We thus assessed whether measurement of the plasma 6-OH-CZX/CZX ratio after a CZX challenge could serve as a marker of hepatic P4502E1 content. Three subject groups were included: recently drinking alcoholics (N = 6), abstinent alcoholics (N = 5), and nonalcoholic subjects with liver disease (N = 5) undergoing liver biopsy. Excess tissue was procured for immunochemical determination of hepatic P4502E1 content. Within an hour of the biopsy, 750 mg CZX was administered orally and serial plasma samples were collected for 6 h. Recently drinking alcoholic subjects had a higher area under the curve for plasma 6-OH-CZX (1.354 +/- 0.258 microg x min x ml(-1)) then abstinent alcoholic subjects (0.296 +/- 0.080 microg x min x ml(-1), p < 0.005) and subjects with nonalcoholic liver disease (0.428 +/- 0.061 microg x min x ml(-1), p < 0.005). The use of the plasma 6-OH-CZX/CZX ratio at 90, 120, and 180 min discriminated between recently drinking alcoholic and nondrinking subjects. Hepatic P4502E1 content significantly correlated with the maximal 6-OH-CZX concentration (r = 0.76, p = 0.001) and other pharmacokinetic parameters. In the recently drinking group, the area under the curve for plasma 6-OH-CZX significantly decreased after 8 days of abstinence. Measurement of plasma 6-OH-CZX after administration of a CZX challenge can serve as a marker of hepatic P4502E1 activity and thus help avoid adverse drug reactions secondary to P4502E1 induction, particularly in heavy drinkers.

Chlorzoxazone pharmacokinetics as a marker of hepatic cytochrome P4502E1 in humans

Objective: Previous in vitro studies have demonstrated that hepatic P4502E1 metabolizes chlorzoxazone (CZX, a commonly used muscle relaxant) to 6-hydroxychlorzoxazone (6-OH-CZX). We thus assessed whether measurement of the plasma 6-OH-CZX/CZX ratio after a CZX challenge could serve as a marker of hepatic P4502E1 content. Methods: Three subject groups were included: recently drinking alcoholics (N = 6), abstinent alcoholics (N = 5), and nonalcoholic subjects with liver disease (N = 5) undergoing liver biopsy. Excess tissue was procured for immunochemical determination of hepatic P4502E1 content. Within an hour of the biopsy, 750 mg CZX was administered orally and serial plasma samples were collected for 6 h. Results: Recently drinking alcoholic subjects had a higher area under the curve for plasma 6-OH-CZX (1.354 ± 0.258 μg · min · ml −1) then abstinent alcoholic subjects (0.296 ± 0.080 μg · min · ml −1, p < 0.005) and subjects with nonalcoholic liver disease (0.428 ± 0.061 μg · min · ml −1, p < 0.005). The use of the plasma 6-OH-CZX/CZX ratio at 90, 120, and 180 min discriminated between recently drinking alcoholic and nondrinking subjects. Hepatic P4502E1 content significantly correlated with the maximal 6-OH-CZX concentration (r = 0.76, p = 0.001) and other pharmacokinetic parameters. In the recently drinking group, the area under the curve for plasma 6-OH-CZX significantly decreased after 8 days of abstinence. Conclusions: Measurement of plasma 6-OH-CZX after administration of a CZX challenge can serve as a marker of hepatic P4502E1 activity and thus help avoid adverse drug reactions secondary to P4502E1 induction, particularly in heavy drinkers.

Inactivation of cytochrome P450 2E1 by tert-butylisothiocyanate.

Several naturally occurring and synthethic isothiocyanates were evaluated for their ability to inactivate the major ethanol-inducible hepatic cytochrome P450 2E1. Of the compounds tested, tert-butylisothiocyanate (tBITC) was found to be the most selective inactivator of the 2E1 p-nitrophenol hydroxylation activity. tBITC was more specific for inactivating P450 2E1 activity than for rat P450 1A1, 1A2, 3A2, and 2B1, or the human cytochromes P450 3A4 and 2B6. The kinetics of inactivation of P450 2E1 by tBITC were characterized. P450 2E1, either in rat liver microsomes or in a purified reconstituted system containing the bacterially expressed rabbit cytochrome, was inactivated by tBITC in a mechanism-based manner. The loss of activity followed pseudo-first-order kinetics and was NADPH- and tBITC-dependent. The maximal rates for inactivation of P450 2E1 in microsomes or for the purified P450 2E1 at 30 degrees C were 0.72 and 0.27 min-1 and the apparent KI values were 11 and 7.6 microM, respectively. When cytochrome b5 was co-reconstituted with P450 2E1, the apparent KI for P450 2E1 inactivation by tBITC was similar to that seen in microsomes (14 microM). P450 2E1 T303A was also inactivated by tBITC with kinetic constants similar to that of the wild type enzyme. Co-incubations with an alternate substrate protected P450 2E1 from inactivation by tBITC. The extent of P450 2E1 inactivation by tBITC resulted in a comparable loss of the ability of the enzyme to form a reduced CO complex.

Chemopreventive effects of 2-(allylthio)pyrazine on hepatic lesion, mutagenesis and tumorigenesis induced by vinyl carbamate or vinyl carbamate epoxide.

2-(Allylthio)pyrazine (2-AP), synthesized for its possible use as a hepatoprotective agent, has been found to selectively inhibit rat hepatic cytochrome P450 2E1 (Kim et al., Biochem. Pharmacol., 53, 261-269, 1997), while it enhances the activities of phase II detoxification enzymes such as glutathione S-transferase and epoxide hydrolase. As part of a program in evaluating the chemopreventive potential of 2-AP, we have determined its effects on hepatotoxicity, mutagenicity and tumorigenicity of vinyl carbamate (VC), a prototypic hepatocarcinogen preferentially activated by P450 2E1 to the ultimate carcinogenic metabolite vinyl carbamate epoxide (VCO), which undergoes detoxification by glutathione conjugation and oxirane hydrolysis. Administration of 2-AP (100 mg/kg body wt) to male Sprague-Dawley rats by gavage, 2 days, 1 day and 4 h prior to VC or VCO, markedly ameliorated the hepatotoxicity of these compounds as determined by decreased serum aspartate aminotransferase and alanine aminotransferase activities. Furthermore, 2-AP pre-treatment significantly suppressed the VC-induced hepatocarcinogenesis in infant male B6C3F1 mice. In a separate experiment, the multiplicities of skin tumors formed in female ICR mice treated with 5.8 micromol of VC or VCO were inhibited 58 and 70%, respectively, by pre-treatment with 2-AP by oral administration. The mutational spectrum of ras-oncogene in papillomas was not altered by 2-AP pre-treatment. 2-AP also inhibited the mutagenicity of VC in the Salmonella-microsome assay. Taken together, these findings suggest that 2-AP is a potential chemopreventive agent.