Cytochrome P450 2E1 and its roles in disease.

Cytochrome P450 (P450) 2E1 is the major P450 enzyme involved in ethanol metabolism. That role is shared with two other enzymes that oxidize ethanol, alcohol dehydrogenase and catalase. P450 2E1 is also involved in the bioactivation of a number of low molecular weight cancer suspects, as validated in vivo in mouse models where cancers could be attenuated by deletion of Cyp2e1. P450 2E1 does not have a role in global production of reactive oxygen species but localized roles are possible, e.g. in mitochondria. The structures, conformations, and catalytic mechanisms of P450 2E1 have some unusual features among P450s. The concentration of hepatic P450 varies ≥10-fold among humans, possibly in part due to single nucleotide variants. The level of P450 2E1 may have relevance in the rates of oxidation of drugs, particularly acetaminophen and anesthetics.

Metabolism and Toxicity of Trichloroethylene and Tetrachloroethylene in Cytochrome P450 2E1 Knockout and Humanized Transgenic Mice.

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are structurally similar olefins that can cause liver and kidney toxicity. Adverse effects of these chemicals are associated with metabolism to oxidative and glutathione conjugation moieties. It is thought that CYP2E1 is crucial to the oxidative metabolism of TCE and PCE, and may also play a role in formation of nephrotoxic metabolites; however, inter-species and inter-individual differences in contribution of CYP2E1 to metabolism and toxicity are not well understood. Therefore, the role of CYP2E1 in metabolism and toxic effects of TCE and PCE was investigated using male and female wild-type [129S1/SvlmJ], Cyp2e1(-/-), and humanized Cyp2e1 [hCYP2E1] mice. To fill in existing gaps in our knowledge, we conducted a toxicokinetic study of TCE (600 mg/kg, single dose, i.g.) and a subacute study of PCE (500 mg/kg/day, 5 days, i.g.) in 3 strains. Liver and kidney tissues were subject to profiling of oxidative and glutathione conjugation metabolites of TCE and PCE, as well as toxicity endpoints. The amounts of trichloroacetic acid formed in the liver was hCYP2E1≈ 129S1/SvlmJ > Cyp2e1(-/-) for both TCE and PCE; levels in males were about 2-fold higher than in females. Interestingly, 2- to 3-fold higher levels of conjugation metabolites were observed in TCE-treated Cyp2e1(-/-) mice. PCE induced lipid accumulation only in liver of 129S1/SvlmJ mice. In the kidney, PCE exposure resulted in acute proximal tubule injury in both sexes in all strains (hCYP2E1 ≈ 129S1/SvlmJ > Cyp2e1(-/-)). In conclusion, our results demonstrate that CYP2E1 is an important, but not exclusive actor in the oxidative metabolism and toxicity of TCE and PCE.

Characterization of novel cytochrome P450 2E1 knockout rat model generated by CRISPR/Cas9.

A bacterial CRISPR-associated protein-9 nuclease (CRISPR/Cas9) from Streptococcus pyogenes has generated considerable excitement as a new tool to edit the targeted genome. Cytochrome P450 (CYP) 2E1 not only plays an important role in the xenobiotic metabolism and chemical toxicity, but also is involved in many kinds of diseases, such as alcoholic liver diseases and diabetes. Despite its importance, few animal models are used to predict CYP2E1 properties in physiology, pathology, as well as carcinogen activation. To establish a novel model for investigating the functions of CYP2E1 in vivo, this study has successfully generated the Cyp2e1 knockout (KO) rat model without detectable off-target effects using CRISPR/Cas9 system. The Cyp2e1 KO rats were viable and fertile and did not display any obvious physiological abnormities. The absent expression of CYP2E1 in KO rats also resulted in inactive behaviors in the metabolism of CYP2E1 substrates. The Cyp2e1 KO rats as a novel and available rodent animal model provide a powerful tool for the study of CYP2E1 in the chemical metabolism, toxicity, carcinogenicity, and its core factor in drug-drug interactions.

Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury.

UNLABELLED: Hepatocyte cell death via apoptosis and necrosis are major hallmarks of ethanol-induced liver injury. However, inhibition of apoptosis is not sufficient to prevent ethanol-induced hepatocyte injury or inflammation. Because receptor-interacting protein kinase (RIP) 3-mediated necroptosis, a nonapoptotic cell death pathway, is implicated in a variety of pathological conditions, we tested the hypothesis that ethanol-induced liver injury is RIP3-dependent and RIP1-independent. Increased expression of RIP3 was detected in livers of mice after chronic ethanol feeding, as well as in liver biopsies from patients with alcoholic liver disease. Chronic ethanol feeding failed to induce RIP3 in the livers of cytochrome P450 2E1 (CYP2E1)-deficient mice, indicating CYP2E1-mediated ethanol metabolism is critical for RIP3 expression in response to ethanol feeding. Mice lacking RIP3 were protected from ethanol-induced steatosis, hepatocyte injury, and expression of proinflammatory cytokines. In contrast, RIP1 expression in mouse liver remained unchanged following ethanol feeding, and inhibition of RIP1 kinase by necrostatin-1 did not attenuate ethanol-induced hepatocyte injury. Ethanol-induced apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive nuclei and accumulation of cytokeratin-18 fragments in the liver, was independent of RIP3. CONCLUSION: CYP2E1-dependent RIP3 expression induces hepatocyte necroptosis during ethanol feeding. Ethanol-induced hepatocyte injury is RIP3-dependent, but independent of RIP1 kinase activity; intervention of this pathway could be targeted as a potential therapeutic strategy.

Increased oxidative stress and cytotoxicity by hydrogen sulfide in HepG2 cells overexpressing cytochrome P450 2E1.

The main objectives of this work were to evaluate the effects of hydrogen sulfide on oxidative stress and cytotoxicity parameters in HepG2 cells and to assess the extent to which cytochrome P450 2E1 (CYP2E1) activity modulates the effects of hydrogen sulfide on oxidative stress and cytotoxicity. Sodium hydrosulfide (NaHS) caused time- and concentration-dependent cytotoxicity in both non-P450-expressing HepG2 cells (C34 cells) and CYP2E1-overexpressing HepG2 cells (E47 cells); however, NaHS-dependent cytotoxicity was higher in E47 than C34 cells. Cytotoxicity by NaHS in C34 and E47 cells was mainly necrotic in nature and associated with an early decrease in mitochondrial membrane potential. NaHS caused increased oxidation of lipophilic (C11-BODIPY(581/591)) and hydrophilic (DCFH-DA) probes only in E47 cells, at a time point prior to overt cytotoxicity. Trolox, an amphipathic antioxidant, partially inhibited both the cytotoxicity and the increased oxidative stress detected in E47 cells exposed to NaHS. Cell-permeable iron chelators and CYP2E1 inhibitors significantly inhibited the oxidation of C11-BODIPY(581/591) in E47 cells in the presence of NaHS. NaHS produced lipid peroxidation and cytotoxicity in E47 cells supplemented with a representative polyunsaturated fatty acid (docosahexaenoic acid) but not in C34 cells; these effects were inhibited by α-tocopherol, a lipophilic antioxidant. These data suggest that CYP2E1 enhances H(2)S-dependent cytotoxicity in HepG2 cells through the generation of iron-dependent oxidative stress and lipid peroxidation.

Serum metabolomics reveals irreversible inhibition of fatty acid beta-oxidation through the suppression of PPARalpha activation as a contributing mechanism of acetaminophen-induced hepatotoxicity.

Metabolic bioactivation, glutathione depletion, and covalent binding are the early hallmark events after acetaminophen (APAP) overdose. However, the subsequent metabolic consequences contributing to APAP-induced hepatic necrosis and apoptosis have not been fully elucidated. In this study, serum metabolomes of control and APAP-treated wild-type and Cyp2e1-null mice were examined by liquid chromatography-mass spectrometry (LC-MS) and multivariate data analysis. A dose-response study showed that the accumulation of long-chain acylcarnitines in serum contributes to the separation of wild-type mice undergoing APAP-induced hepatotoxicity from other mouse groups in a multivariate model. This observation, in conjunction with the increase of triglycerides and free fatty acids in the serum of APAP-treated wild-type mice, suggested that APAP treatment can disrupt fatty acid beta-oxidation. A time-course study further indicated that both wild-type and Cyp2e1-null mice had their serum acylcarnitine levels markedly elevated within the early hours of APAP treatment. While remaining high in wild-type mice, serum acylcarnitine levels gradually returned to normal in Cyp2e1-null mice at the end of the 24 h treatment. Distinct from serum aminotransferase activity and hepatic glutathione levels, the pattern of serum acylcarnitine accumulation suggested that acylcarnitines can function as complementary biomarkers for monitoring the APAP-induced hepatotoxicity. An essential role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the regulation of serum acylcarnitine levels was established by comparing the metabolomic responses of wild-type and Ppara-null mice to a fasting challenge. The upregulation of PPARalpha activity following APAP treatment was transient in wild-type mice but was much more prolonged in Cyp2e1-null mice. Overall, serum metabolomics of APAP-induced hepatotoxicity revealed that the CYP2E1-mediated metabolic activation and oxidative stress following APAP treatment can cause irreversible inhibition of fatty acid oxidation, potentially through suppression of PPARalpha-regulated pathways.

Cytochrome P450 2E1 contributes to ethanol-induced fatty liver in mice.

Cytochrome P450 2E1 (CYP2E1) is suggested to play a role in alcoholic liver disease, which includes alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. In this study, we investigated whether CYP2E1 plays a role in experimental alcoholic fatty liver in an oral ethanol-feeding model. After 4 weeks of ethanol feeding, macrovesicular fat accumulation and accumulation of triglyceride in liver were observed in wild-type mice but not in CYP2E1-knockout mice. In contrast, free fatty acids (FFAs) were increased in CYP2E1-knockout mice but not in wild-type mice. CYP2E1 was induced by ethanol in wild-type mice, and oxidative stress induced by ethanol was higher in wild-type mice than in CYP2E1-knockout mice. Peroxisome proliferator-activated receptor alpha (PPARalpha), a regulator of fatty acid oxidation, was up-regulated in CYP2E1-knockout mice fed ethanol but not in wild-type mice. A PPARalpha target gene, acyl CoA oxidase, was decreased by ethanol in wild-type but not in CYP2E1-knockout mice. Chlormethiazole, an inhibitor of CYP2E1, lowered macrovesicular fat accumulation, inhibited oxidative stress, and up-regulated PPARalpha protein level in wild-type mice fed ethanol. The introduction of CYP2E1 to CYP2E1-knockout mice via an adenovirus restored macrovesicular fat accumulation. These results indicate that CYP2E1 contributes to experimental alcoholic fatty liver in this model and suggest that CYP2E1-derived oxidative stress may inhibit oxidation of fatty acids by preventing up-regulation of PPARalpha by ethanol, resulting in fatty liver.

Role of CYP3A and CYP2E1 in alcohol-mediated increases in acetaminophen hepatotoxicity: comparison of wild-type and Cyp2e1(-/-) mice.

CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.

Comparative metabolism and disposition of trichloroethylene in Cyp2e1-/-and wild-type mice.

Trichloroethylene (TCE)1 is an important environmental contaminant, a well established rodent carcinogen, and a "probable human carcinogen". Metabolism of TCE occurs primarily via cytochrome P450 (P450)-dependent oxidation. In vitro studies suggested that CYP2E1 is the principal high-affinity enzyme responsible for TCE metabolism. The objective of the present work is to more directly assess the role of CYP2E1 in the metabolism and disposition of 1,2-14C-TCE administered at 250 or 1000 mg/kg (gavage) using Cyp2e1-/-[knockout (KO)] versus wild-type (WT) mice. After dosing, animals were individually placed in glass metabolism cages that allowed the collection of expired air, urine, and feces. Exhalation of TCE-derived 14CO2 increased in a dose-dependent manner in mice of both genotypes and was significantly higher in WT versus KO mice. A significantly greater percentage of the dose was exhaled in KO versus WT mice as organic volatiles (mainly as TCE). Urinary excretion was the major route of TCE metabolism in WT mice, and the percentage of dose eliminated in urine was significantly higher at the 250 versus 1000 mg/kg dose. Furthermore, urinary excretion and CO2 exhalation significantly decreased in KO versus WT mice. Pretreatment with 1-aminobenzotriazole clearly inhibited TCE metabolism as evident from increased exhalation of parent TCE, and decreased urinary excretion and CO2 exhalation in mice of both genotypes. In conclusion, these data showed that whereas CYP2E1 plays an important role in TCE metabolism and disposition, other P450s also play a significant role and may explain earlier results showing that TCE causes lung damage in KO and WT mice.

Role of CYP2E1 in the epoxidation of acrylamide to glycidamide and formation of DNA and hemoglobin adducts.

Acrylamide (AA) is an animal carcinogen, neurotoxin, and reproductive toxin. AA is formed in baked and fried carbohydrate-rich foods. Metabolism of AA occurs via epoxidation to glycidamide (GA) or direct conjugation with glutathione. Using CYP2E1-null mice, recent studies in this laboratory demonstrated that induction of somatic and germ cell mutagenicity in AA-treated mice is dependent on CYP2E1. We hypothesized that AA metabolism to GA is a prerequisite for the induction of AA-induced mutagenicity. Current studies were undertaken to assess the role of CYP2E1 in the epoxidation of AA to GA and the formation of DNA and hemoglobin (HGB) adducts. AA was administered to CYP2E1-null or wild-type mice at 50 mg/kg ip. Mice were euthanized 6 h later and blood and tissues were collected. Using LC-ES/MS/MS, AA, GA, and DNA- and HGB-adducts were measured. While the plasma levels of AA and GA were 115 +/- 14.0 and 1.7 +/- 0.31 microM in CYP2E1-null mice, they were 0.84 +/- 0.80 and 33.0 +/- 6.3 microM in the plasma of AA-treated wild-type mice. Administration of AA to wild-type mice caused a large increase in N7-GA-Gua and N3-GA-Ade adducts in the liver, lung, and testes. While traces of N7-GA-Gua adducts were measured in the tissues of AA-treated CYP2E1-null mice, these levels were 52- to 66-fold lower than in wild-type mice. Significant elevation of both AA- and GA-HGB adducts was detected in AA-treated wild-type mice. In AA-treated CYP2E1-null mice, levels of AA-HGB adducts were roughly twice as high as those in wild-type mice. In conclusion, current work demonstrated that CYP2E1 is the primary enzyme responsible for the epoxidation of AA to GA, which leads to the formation of GA-DNA and HGB adducts.

Increased bioaccumulation of urethane in CYP2E1-/- versus CYP2E1+/+ mice.

Urethane is a fermentation by-product and a potent animal carcinogen. Human exposure to urethane occurs through consumption of alcoholic beverages and fermented foods. Recently, CYP2E1 was identified as the primary enzyme responsible for the metabolism of [(14)C]carbonyl-labeled urethane. Subsequently, attenuation of urethane-induced cell proliferation and genotoxicity in CYP2E1-/- mice was reported. The present work compares the metabolism of single versus multiple exposures of CYP2E1-/- and CYP2E1+/+ mice to (14)C-ethyl-labeled urethane. Urethane was administered as a single 10 or 100 mg/kg gavage dose or at 100 mg/kg/day for 5 consecutive days. CYP2E1+/+ mice administered single or multiple doses exhaled 78 to 88% of dose as (14)CO(2)/day. CYP2E1-/- mice eliminated 30 to 38% of a single dose as (14)CO(2) in 24 h and plateaued after day 3 at approximately 52% of dose/day. The concentrations of urethane-derived radioactivity in plasma and tissues were dose-dependent, increased as a function of the number of doses administered, and were significantly higher in CYP2E1-/- versus CYP2E1+/+ mice. Whereas urethane was the main chemical found in the plasma and tissues of CYP2E1-/- mice, it was not detectable in CYP2E1+/+ mice. In conclusion, multiple dosing led to considerable bioaccumulation of urethane in mice of both genotypes; however, greater retention occurred in CYP2E1-/- versus CYP2E1+/+ mice. Furthermore, greater bioaccumulation of (14)C-ethyl-labeled than [(14)C]carbonyl-labeled urethane was observed in mice. Comparison of the metabolism of ethyl-versus carbonyl-labeled urethane was necessary for tracing the source of CO(2) and led us to propose for the first time that C-hydroxylation is a likely pathway of urethane metabolism.

Differential metabolism of acrylonitrile to cyanide is responsible for the greater sensitivity of male vs female mice: role of CYP2E1 and epoxide hydrolases.

Acrylonitrile (AN) is a potent toxicant and a known rodent carcinogen. AN epoxidation to cyanoethylene oxide (CEO) via CYP2E1 and its subsequent metabolism via epoxide hydrolases (EH) to yield cyanide is thought to be responsible for the acute toxicity and mortality of AN. Recent reports showed that male mice are more sensitive than females to the acute toxicity/mortality of AN. The present work was undertaken to assess the metabolic and enzymatic basis for the greater sensitivity of male vs female mice to AN toxicity. Male and female wild-type and CYP2E1-null mice received AN at 0, 2.5, 10, 20, or 40 mg/kg by gavage. Cyanide concentrations were measured at 1 or 3 h after dosing. Current data demonstrated that cyanide levels in blood and tissues of AN-treated wild-type mice of both sexes were significantly greater than in vehicle-treated controls and increased in a dose-dependent manner. In contrast, cyanide levels in AN-treated CYP2E1-null mice were not statistically different from those measured in vehicle-treated controls. Furthermore, higher levels of cyanide were detected in male wild-type mice vs females in association with greater sensitivity of males to the acute toxicity/mortality of this chemical. Using Western blot analysis, negligible difference in CYP2E1 expression with higher levels of soluble and microsomal EH (sEH and mEH) was detected in the liver of male vs female mice. In kidneys, male mice exhibited higher expression of both renal CYP2E1 and sEH than did female mice. In conclusion, higher blood and tissue cyanide levels are responsible for the greater sensitivity of male vs female mice to AN. Further, higher expression of CYP2E1 and EH in male mice may contribute to greater formation of CEO and its subsequent metabolism to yield cyanide, respectively.

Hemizygous mice for the angiotensin II type 2 receptor gene have attenuated susceptibility to azoxymethane-induced colon tumorigenesis.

Evidence suggests that the use of angiotensin-converting enzyme inhibitors potentially reduces the risk of cancer, though the mechanism is unclear. To clarify a potential involvement of angiotensin II (Ang II) signaling in cancer risk, we have examined the effect of Ang II receptor deficiency on azoxymethane (AOM)-induced colon tumorigenesis. Male Ang II type 2 receptor gene-disrupted (AT(2)-null) mice with a 129/Ola and C57BL/6J genetic background, AT(2)-null mice with an SWR/J genetic background, and their corresponding control wild type mice were treated once a week with AOM (10 mg/kg, i.p., 4 consecutive weeks) or saline vehicle. All mice were killed 23-26 weeks after the initial injection of AOM, and tumor burdens were examined. AOM treatment caused the development of colon tumors in all wild type control mice regardless of genetic background (100% tumor prevalence), but only one tumor was present in AT(2)-null mice with a 129/Ola and C57BL/6J genetic background (11.1% tumor prevalence). Although the introduction of the AOMsusceptible SWR/J genetic background induced AOM susceptibility in AT(2) null mice, the tumor multiplicity (6.3) and tumor size (19.8 +/- 3.0 mm(3)) were significantly smaller than those in wild type mice (multiplicity, 12.0 and size, 36.8 +/- 3.2 mm(3)). AOM efficiently downregulated cytochrome P450 2E1 (CYP2E1) in the liver of wild type mice significantly more than in AT(2)-null mice. The levels of DNA methyl adducts formed in wild type mouse colon epithelium by AOM treatment were also significantly higher than in AT(2)-null mice. These results imply that the AT(2) receptor functions to augment AOM-induced downregulation of CYP2E1 expression in the liver, and thus increases AOM-induced tumorigenesis in the colon. The AT(2) receptor function in the liver may be a potential determinant of tumor susceptibility in chemical carcinogen-induced colon tumorigenesis.

Differential effects of CYP2E1 status on the metabolic activation of the colon carcinogens azoxymethane and methylazoxymethanol.

Methylazoxymethanol (MAM) and its chemical and metabolic precursor, azoxymethane (AOM), both strong colon carcinogens in rodents, can be metabolically activated by CYP2E1 in vitro. Using CYP2E1-null mice, we found that CYP2E1 deficiency differentially affects the activation of AOM and MAM, as reflected in DNA guanine alkylation in the colon and in the formation of colonic aberrant crypt foci (ACF). Male and female inbred 129/SV wild-type (WT) and CYP2E1-null (null) mice were treated with 189 micromol/kg of either AOM or methylazoxymethyl acetate (MAMAc), and 7-methylguanine (7-MeG) and O(6)-methylguanine (O(6)-MeG) were measured in the DNAs of various organs. The levels of O(6)-MeG (as pmol/nmol guanine) in the liver, colon, kidney, and lung of male null mice treated with AOM were 87, 48, 70, and 43% lower, respectively, than in AOM-treated WT mice. In null mice treated with MAMAc, the DNA O(6)-MeG levels were lower by 38% in the liver but were higher by 368, 146, and 194% in the colon, kidney, and lung, respectively, compared with the same organs of WT mice treated in the same way. Determination of ACF revealed that although AOM-induced ACF formation was significantly lower in the null group than in the WT group, MAMAc-induced ACF formation was significantly higher in the null group than in the WT group. These results demonstrate an important role for CYP2E1 in the in vivo activation of AOM and MAM and suggest that agents that modify CYP2E1 activity at the tumor initiation stage might either enhance or inhibit colon carcinogenesis, depending on whether AOM or MAMAc is used as the carcinogen. The mechanism of this effect is discussed.

Hepatic and pulmonary microsomal benzene metabolism in CYP2E1 knockout mice.

Benzene is an occupational and environmental toxicant. The major health concern for humans is acute myelogenous leukemia. To exert its toxic effects, benzene must be metabolized via cytochrome P450. CYP2E1 has been identified as the most important cytochrome, P450 isozyme in hepatic benzene metabolism in mice, rats, and humans. In pulmonary microsomes CYP2E1 and members of the CYP2F subfamily are both significantly involved. In the current study CYP2E1 knockout mice and wild-type controls were used to further examine the cytochrome P450 isozymes involved in metabolism of 24 microM benzene. The results show that CYP2E1 is the most important isozyme in the liver, accounting for 96% of the total hydroxylated metabolite formation. However, in the lung CYP2E1 was responsible for only 45% of the formation of total hydroxylated metabolite. Chemical inhibitors of CYP2E1 and CYP2F2 were used to further examine the contributions of these isozymes to benzene metabolism. The results confirmed the finding that while CYP2E1 is the most important isozyme in the liver, CYP2F2 and CYP2E1 are both significantly involved in the lung.

Metabolism of methyl tert-butyl ether and other gasoline ethers in mouse liver microsomes lacking cytochrome P450 2E1.

To reduce the production of pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE) and other ethers such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Metabolism of these gasoline ethers is catalyzed by cytochrome P450 (P450) enzymes. P450 2E1, which metabolizes diethyl ether, was suggested to be an enzyme involved. The present study used 2E1 knock-out mice (2E1-/-) to assess the contribution of 2E1 to the metabolism of MTBE, ETBE and TAME. Liver microsomes prepared from the 2E1 knock-out mice lacked 2E1 activity (assayed as N-nitrosodimethylamine demethylation), but were still active in metabolizing all three gasoline ethers. The levels of ether-metabolizing activity (nmol/min per mg) in the liver microsomes from 7 week old female 2E1 knock-out mice were 0.54+/-0.17 for MTBE, 0.51+/-0.24 for ETBE and 1.14+/-0.25 for TAME at a 1 mM substrate concentration. These activity levels were not significantly different from those of the sex- and age-matched C57BL/6N and 129/Sv mice, which are the parental lineage strains of the 2E1 knock-out mice and are both 2E1+/+. Our results clearly demonstrate that 2E1 plays a negligible role in the metabolism of MTBE, ETBE and TAME in mouse livers.

Ethanol interactions with other cytochrome P450 substrates including drugs, xenobiotics, and carcinogens.

Chronic ethanol abuse is associated with increased activity of the microsomal ethanol-oxidizing system. This effect is due primarily to induction by ethanol of a specific cytochrome P450 (CYP2E1) responsible for enhanced oxidation of ethanol and other P450 substrates and, consequently, for metabolic tolerance to these substances. Furthermore, cytochrome 450 induction increases the activation of numerous xenobiotics to toxic metabolites and of chemical carcinogens to reactive metabolites, thereby accelerating their adverse effects. Microsomal enzyme induction has been associated with increased reactive oxygen species production and enhanced lipid peroxidation, as well as with decreased enzymatic and nonenzymatic scavenger activity, providing another possible explanation for ethanol-mediated toxicity. Yet another effect of chronic alcohol abuse is chronic immune system activation, which is the mechanism underlying alcohol-related liver disease. The metabolism of steroids and vitamins is catalyzed by P450 and is altered in chronic alcoholics. This article reviews recent advances in the understanding of ethanol interactions with drugs, toxic agents, and carcinogens, as well as with steroids and vitamins.

Reduction of benzene metabolism and toxicity in mice that lack CYP2E1 expression.

Transgenic CYP2E1 knockout mice (cyp2e1-/-) were used to investigate the involvement of CYP2E1 in the in vivo metabolism of benzene and in the development of benzene-induced toxicity. After benzene exposure, absence of CYP2E1 protein was confirmed by Western blot analysis of mouse liver samples. For the metabolism studies, male cyp2e1-/- and wild-type control mice were exposed to 200 ppm benzene, along with a radiolabeled tracer dose of [14C]benzene (1.0 Ci/mol) by nose-only inhalation for 6 hr. Total urinary radioactivity and all radiolabeled individual metabolites were reduced in urine of cyp2e1-/- mice compared to wild-type controls during the 48-hr period after benzene exposure. In addition, a significantly greater percentage of total urinary radioactivity could be accounted for as phenylsulfate conjugates in cyp2e1-/- mice compared to wild-type mice, indicating the importance of CYP2E1 in oxidation of phenol following benzene exposure in normal mice. For the toxicity studies, male cyp2e1-/-, wild-type, and B6C3F1 mice were exposed by whole-body inhalation to 0 ppm (control) or 200 ppm benzene, 6 hr/day for 5 days. On Day 5, blood, bone marrow, thymus, and spleen were removed for evaluation of micronuclei frequencies and tissue cellularities. No benzene-induced cytotoxicity or genotoxicity was observed in cyp2e1-/- mice. In contrast, benzene exposure resulted in severe genotoxicity and cytotoxicity in both wild-type and B6C3F1 mice. These studies conclusively demonstrate that CYP2E1 is the major determinant of in vivo benzene metabolism and benzene-induced myelotoxicity in mice.