Role Of Cytochrome P450 2E1 Research Articles

Role of cytochrome P450 2E1 in protein nitration and ubiquitin-mediated degradation during acetaminophen toxicity

It is well established that following a toxic dose of acetaminophen (APAP), nitrotyrosine protein adducts (3-NT), a hallmark of peroxynitrite production, were colocalized with necrotic hepatic centrilobular regions where cytochrome P450 2E1 (CYP2E1) is highly expressed, suggesting that 3-NT formation may be essential in APAP-mediated toxicity. This study was aimed at investigating the relationship between CYP2E1 and nitration (3-NT formation) followed by ubiquitin-mediated degradation of proteins in wild-type and Cyp2e1-null mice exposed to APAP (200 and 400mg/kg) for 4 and 24h. Markedly increased centrilobular liver necrosis and 3-NT formation were only observed in APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for CYP2E1 in APAP biotransformation and toxicity. However, the pattern of 3-NT protein adducts, not accompanied by concurrent activation of nitric oxide synthase (NOS), was similar to that of protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed wild-type mice, confirming the fact that nitrated proteins are more susceptible than the native proteins for ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in APAP-exposed wild-type mice. These data suggest that CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many proteins in the wild-type mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity.

In vivo Role of Cytochrome P450 2E1 and Glutathione-S-Transferase Activity for Acrylamide Toxicokinetics in Humans

Acrylamide, a potential food carcinogen in humans, is biotransformed to the epoxide glycidamide in vivo. Both acrylamide and glycidamide are conjugated with glutathione, possibly via glutathione-S-transferases (GST), and bind covalently to proteins and nucleic acids. We investigated acrylamide toxicokinetics in 16 healthy volunteers in a four-period change-over trial and evaluated the respective role of cytochrome P450 2E1 (CYP2E1) and GSTs. Participants ingested self-prepared potato chips containing acrylamide (1 mg) without comedication, after CYP2E1 inhibition (500 mg disulfiram, single dose) or induction (48 g/d ethanol for 1 week), and were phenotyped for CYP2E1 with chlorzoxazone (250 mg, single dose). Unchanged acrylamide and the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-cysteine (GAMA) accounted for urinary excretion [geometric mean (percent coefficient of variation)] of 2.9% (42), 65% (23), and 1.7% (65) of the acrylamide dose in the reference period. Hemoglobin adducts increased clearly following the acrylamide test-meal. The cumulative amounts of acrylamide, AAMA, and GAMA excreted and increases in AA adducts changed significantly during CYP2E1 blockade [point estimate (90% confidence interval)] to the 1.34-fold (1.14-1.58), 1.18-fold (1.02-1.36), 0.44-fold (0.31-0.61), and 1.08-fold (1.02-1.15) of the reference period, respectively, but were not changed significantly during moderate CYP2E1 induction. Individual baseline CYP2E1 activity, CYP2E1*6, GSTP1 313A>G and 341T>C single nucleotide polymorphisms, and GSTM1-and GSTT1-null genotypes had no major effect on acrylamide disposition. The changes in acrylamide toxicokinetics upon CYP2E1 blockade provide evidence that CYP2E1 is a major but not the only enzyme mediating acrylamide epoxidation in vivo to glycidamide in humans. No obvious genetic risks or protective factors in xenobiotic-metabolizing enzymes could be determined for exposed subjects.

The role of oxidative stress in alcoholic liver injury

Oxidative stress plays an important role in pathogenesis of alcoholic liver injury. The main source of free oxygen species is cytochrome P450-dependent monooxygenase, which can be induced by ethanol. ROLE OF CYTOCHROME P4502E1 IN ETHANOL-INDUCED OXIDATIVE STRESS: Reactive oxygen species produced by this enzyme are more important in intracellular oxidative damage compared to species derived from activated phagocytes. Free radicals lead to lipid peroxidation, enzymatic inactivation and protein oxidation. ROLE OF MITOCHONDRIA IN ALCOHOL-INDUCED OXIDATIVE STRESS. Production of mitochondrial reactive oxygen species is increased, and glutathione content is decreased in chronically ethanol-fed animals. Oxidative stress in mitochondria leads to mitochondrial DNA damage and has a dual effect on apoptosis. ROLE OF KUPFFER CELLS IN ALCOHOL-INDUCED LIVER INJURY: Chronic ethanol consumption is associated with increased release of endotoxin from gut lumen into portal circulation. Endotoxin activates Kupffer cells, which then release proinflammatory cytokines and oxidants. ROLE OF NEUTROPHILS IN ALCOHOL-INDUCED LIVER INJURY: Alcoholic liver injury leads to the accumulation of neutrophils, which release reactive oxygen species and lysosomal enzymes and contribute to hepatocyte damage and necrosis. ROLE OF NITRIC OXIDE IN ALCOHOL-INDUCED OXIDATIVE STRESS: High amounts of nitric oxide contribute to the oxidative damage, mainly by generating peroxynitrites. ROLE OF ANTIOXIDANTS IN ETHANOL-INDUCED OXIDATIVE STRESS: Chronic ethanol consumption is associated with reduced liver glutathione and alpha-tocopherol level and with reduced superoxide dismutase, catalase and glutathione peroxidase activity. Oxidative stress in alcoholic liver disease is a consequence of increased production of oxidants and decreased antioxidant defense in the liver.

103 The Role of Cytochrome P-4502e1 in DNA Damage and Ethanol-Mediated Hepatic Carcinogenesis

103 The Role of Cytochrome P-4502e1 in DNA Damage and Ethanol-Mediated Hepatic Carcinogenesis

Enhancement by pyrazole of lipopolysaccharide-induced liver injury in mice: Role of cytochrome P450 2E1 and 2A5

The mechanisms by which alcohol causes liver injury are still not certain. Either LPS or CYP2E1 are considered independent risk factors involved in alcoholic liver disease, but mutual relationships or interactions between them are unknown. In the present study, the possible synergistic action of CYP2E1 and LPS in liver injury was investigated by evaluating the effects of pyrazole (inducer of CYP2E1), Chlormethiazole (CMZ), an inhibitor of CYP2E1, and CYP2E1-knockout mice. Mice were injected with pyrazole (150 mg/kg, ip) daily for 2 days, followed by LPS injection (4 mg/kg, ip). CMZ (50mg/kg, ip) was administered 15 h before and 30 min after LPS treatment, respectively. LPS-induced liver injury was enhanced by pyrazole, as indicated by pathological changes and increases in ALT and AST, and positive TUNEL staining. LPS-induced oxidative stress was also enhanced by pyrazole as indicated by increases in 4-hydroxy-2-nonenal and 3-nitrotyrosine adduct formation. CMZ protected against the pyrazole enhanced LPS liver injury and oxidative stress. CYP2E1 but also CYP2A5 were increased by the pyrazole/LPS treatment. CMZ decreased the elevated CYP2E1 activity by 90%, but CYP2A5 activity was also lowered (30%-50%). CYP2E1-knockout mice exhibited only minor liver injury after treatment with pyrazole/LPS, but wild-type mice exhibited severe liver injury. While no CYP2E1 was present in the CYP2E1 knockout mice, CYP2A5 activity was also lower. In conclusion, induction of CYP2E1 plays an important role in the enhancement of LPS liver injury by pyrazole, but some contribution by CYP2A5 cannot be excluded.

Zinc Supplementation Prevents Alcoholic Liver Injury in Mice through Attenuation of Oxidative Stress

Alcoholic liver disease is associated with zinc decrease in the liver. Therefore, we examined whether dietary zinc supplementation could provide protection from alcoholic liver injury. Metallothionein-knockout and wild-type 129/Sv mice were pair-fed an ethanol-containing liquid diet for 12 weeks, and the effects of zinc supplementation on ethanol-induced liver injury were analyzed. Zinc supplementation attenuated ethanol-induced hepatic zinc depletion and liver injury as measured by histopathological and ultrastructural changes, serum alanine transferase activity, and hepatic tumor necrosis factor-alpha in both metallothionein-knockout and wild-type mice, indicating a metallothionein-independent zinc protection. Zinc supplementation inhibited accumulation of reactive oxygen species, as indicated by dihydroethidium fluorescence, and the consequent oxidative damage, as assessed by immunohistochemical detection of 4-hydroxynonenal and nitrotyrosine and quantitative analysis of malondialdehyde and protein carbonyl in the liver. Zinc supplementation suppressed ethanol-elevated cytochrome P450 2E1 activity but increased the activity of alcohol dehydrogenase in the liver, without affecting the rate of blood ethanol elimination. Zinc supplementation also prevented ethanol-induced decreases in glutathione concentration and glutathione peroxidase activity and increased glutathione reductase activity in the liver. In conclusion, zinc supplementation prevents alcoholic liver injury in an metallothionein-independent manner by inhibiting the generation of reactive oxygen species (P450 2E1) and enhancing the activity of antioxidant pathways.

Role of Cytochrome P4502E1 Activation in Proximal Tubular Cell Injury Induced by Hydrogen Peroxide

Background. There is now good evidence to suggest that cytochrome P450 (CYP450) may act as an iron‐donating catalyst for the production of hydroxyl ion (OH·), which contributes to proximal tubular cell injury. However, it remains unclear which isoform of CYP450 is involved in this process. Cytochrome P4502E1 (CYP2E1) is a highly labile isoform which is not only involved in free radical generation, but has also been shown to be a source of iron in cisplatin‐induced renal injury. This study investigates the role of CYP2E1 in the proximal tubular cell injury induced by hydrogen peroxide (H2O2). Methods. Porcine proximal tubular cells (LLC‐PK1) were incubated with H2O2 (1 mM) for 4 h in the presence or absence of 0.1 mM of two CYP2E1 inhibitors; diallyl sulfide (DAS), or disulfiram (DSF), desferrioxamine (DFO) (0.1–0.4 mM), or catalase (CT) (78, 150, 300 U/mL). Cell death was determined by measuring LDH release. CYP2E1 activity was determined by p‐nitrophenol hydroxylation after 2 h incubation with H2O2. Results. Exposure of LLC‐PK1 to H2O2 significantly increased cell death. CT, DFO, DAS and DSF significantly reduced H2O2‐mediated cell death. Incubation with H2O2 increased CYP2E1 activation in time‐ and dose‐dependent manner, which was significantly reduced by CT, DFO, DAS and DSF. Conclusion. We propose that CYP2E1 activation occurs possibly due to OH· and contributes to H2O2‐mediated LLC‐PK1 cell necrosis by acting as a source of iron and perpetuating the generation of OH· via the Fenton reaction. Inhibition of CYP2E1 may be a novel approach for the prevention of tubular injury caused by oxidative stress.

Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug-induced hepatitis

Most cases with antituberculosis drug-induced hepatitis have been attributed to isoniazid. Isoniazid is metabolized by hepatic N-acetyltransferase (NAT) and cytochrome P450 2E1 (CYP2E1) to form hepatotoxins. However, the role of CYP2E1 in this hepatotoxicity has not yet been reported. The aim of this study was to evaluate whether the polymorphism of the CYP2E1 gene is associated with antituberculosis drug-induced hepatitis. A total of 318 tuberculosis patients who received antituberculosis treatment were followed prospectively. Their CYP2E1 and NAT2 genotypes were determined using a polymerase chain reaction with restriction fragment length polymorphism method. Twenty-one healthy volunteers were recruited for CYP2E1 phenotype study using a chlorzoxazone test. Forty-nine (15.4%) patients were diagnosed to have drug-induced hepatotoxicity. Patients with homozygous wild genotype CYP2E1 c1/c1 had a higher risk of hepatotoxicity (20.0%; odds ratio [OR], 2.52) than those with mutant allele c2 ( CYP2E1 c1/c2 or c2/c2, 9.0%, P = .009). If CYP2E1 c1/c2 or c2/c2 genotype combined with rapid acetylator status was regarded as the reference group, the risk of hepatotoxicity increased from 3.94 for CYP2E1 c1/c1 with rapid acetylator status to 7.43 for CYP2E1 c1/c1 with slow acetylator status. After adjustment for acetylator status and age, the CYP2E1 c1/c1 genotype remained an independent risk factor for hepatotoxicity (OR, 2.38; P = .017). Furthermore, under the administration of isoniazid, the volunteers with CYP2E1 c1/c1 genotype had higher CYP2E1 activity than those with other genotypes had and, hence, might produce more hepatotoxins. In conclusion, CYP 2E1 genetic polymorphism may be associated with susceptibility to antituberculosis drug-induced hepatitis. (H epatology 2003;37:924-930.)

Role of cytochrome P450 2E1 in the metabolism of acrylamide and acrylonitrile in mice.

Acrylonitrile (AN) and acrylamide (AM) are commonly used in the synthesis of plastics and polymers. In rodents, AM and AN are metabolized to the epoxides glycidamide and cyanoethylene oxide, respectively. The aim of this study was to determine the role of cytochrome P450 in the metabolism of AM and AN in vivo. Wild-type (WT) mice, WT mice pretreated with aminobenzotriazole (ABT, 50 mg/kg ip, 2 h pre-exposure), and mice devoid of cytochrome P450 2E1 (P450 2E1-null) were treated with 50 mg/kg [(13)C]AM po. WT mice and P450 2E1-null mice were treated with 2.5 or 10 mg/kg [(13)C]AN po. Urine was collected for 24 h, and metabolites were characterized using (13)C NMR. WT mice excreted metabolites derived from the epoxides and from direct GSH conjugation with AM or AN. Only metabolites derived from direct GSH conjugation with AM or AN were observed in the urine from ABT-pretreated WT mice and P450 2E1-null mice. On the basis of evaluation of urinary metabolites at these doses, these data suggest that P450 2E1 is possibly the only cytochrome P450 enzyme involved in the metabolism of AM and AN in mice, that inhibiting total P450 activity does not result in new pathways of non-P450 metabolism of AM, and that mice devoid of P450 2E1 do not excrete metabolites of AM or AN that would be produced by oxidation by other cytochrome P450s. P450 2E1-null mice may be an appropriate model for the investigation of the role of oxidative metabolism in the toxicity or carcinogenicity of these compounds.

Evaluation of rat hepatic 2E1 activity in function of age, sex and inducers: choice of an experimental model capable of testing the hepatotoxicity of low molecular weight compounds

The aim of this work on rat hepatic P450 2E1 activity was to seek the most suitable experimental model to study the role of cytochrome P450 2E1 in the metabolism of industrial chemicals. Two sets of experiments were devoted to selecting the age and sex of animals and to estimating the response of male and female rats to different inducers. In the first set, the effect of three inducers (fasting; ethanol; acetone) was studied in male rats aged 5, 7 and 9 weeks. In the second set, the effect of different inducers, namely β-naphthoflavone (BNF), phenobarbital (PB), ethanol, acetone and pyridine, on PNP and chlorzoxazone (CLZO) hydroxylase activities was studied in 7 week old male and female rats. The results demonstrate firstly that microsomal p-nitrophenol (PNP) hydroxylase activity significantly decreases in control male rats in inverse function of age, and secondly that induction by ethanol decreases with age. The PNP hydroxylase activity level of controls and the significant increases in PNP hydroxylase activity observed in 7 week old male rats show that this is the most suitable age for the second set of experiments. In this second set, it was shown that P450 1A (induced by BNF) is involved in CLZO hydroxylase activity only. PB increased the hydroxylase activities in male and female rats by about 1.5 and 1.7 times those of the controls, respectively. The effects of P450 2E1 inducers in function of sex show that male rats exhibited more significant increases in PNP and CLZO hydroxylase activities than female. The specificity of these two substrates is discussed. Neither of these two reactions was specifically catalysed by P450 2E1, but PNP may be considered as the most specific and the least sensitive substrate. In addition, the linear relationship observed between the two substrates (PNP and CLZO) showed a good correlation between their activities ( r=0.90, P<0.001). In conclusion, these results suggest the use of the 7 week old male rat as the experimental model to study the role of cytochrome P450 2E1 in the hepatotoxicity of low molecular weight industrial chemicals.

Cytochrome P4502E1 inducibility and hydroxyethyl radical formation among alcoholics

Background/Aims: Animal studies have shown that the induction of cytochrome P4502E1 (CYP2E1) modulates oxidative damage induced by ethanol. Since CYP2E1 activity varies substantially in humans, we have investigated whether differences in CYP2E1 activity might influence the formation of hydroxyethyl free radicals and the stimulation of lipid peroxidation among alcohol abusers. Methods: Chlorzoxazone oxidation, an index of CYP2E1 activity, and the levels of antibodies reacting with hydroxyethyl radical and malonyldialdehyde protein adducts were investigated in 51 alcoholic patients. Results: We observed that in 40 out of 51 (78%) alcoholics, chlorzoxazone oxidation was increased over the control levels, consistently with CYP2E1 induction by ethanol. However, in the remaining 22% of the patients, in spite of a similar alcohol intake, chlorzoxazone oxidation was within the control range, indicating a lack of CYP2E1 inducibility. IgG reacting with hydroxyethyl free radical-protein adducts were absent in subjects without CYP2E1 induction, while they were significantly increased in alcoholics with induced CYP2E1 activity. IgG against malonyldialdehyde protein-adducts were increased in all patients, irrespective of CYP2E1 inducibility. Moreover, chlorzoxazone oxidation was significantly lower in alcoholics without clinical and biochemical signs of liver disease as compared to patients with alcoholic liver disease. Conclusions: These results indicate that CYP2E1 activity greatly influences the formation of hydroxyethyl radicals in humans, and suggest a possible role of CYP2E1 in the development of alcoholic liver disease.

Ferritin Stimulation of Lipid Peroxidation by Microsomes after Chronic Ethanol Treatment: Role of Cytochrome P4502E1

Ferritin is the major storage form of iron within cells, and iron released from ferritin has been shown to stimulate lipid peroxidation. Microsomes from rats chronically fed ethanol are more active in generating reactive oxygen intermediates than control microsomes. Since superoxide is one of the reductants capable of releasing iron from ferritin, and superoxide generation by microsomes is increased after chronic ethanol treatment, the ability of ferritin to stimulate lipid peroxidation of microsomes isolated from control rats and rats treated chronically with ethanol was evaluated. Ferritin was much more effective in stimulating lipid peroxidation of microsomes after ethanol treatment; net increases in thiobarbituric acid-reactive components by ferritin were 4-fold greater in the presence of NADPH with microsomes from the ethanol-treated rats compared to pair-fed controls and 10-fold greater with NADH as the microsomal reductant. Net increases in chemiluminescence by ferritin were about 10-fold greater with microsomes from the ethanol-treated rats. The NADPH- and NADH-dependent increases in lipid peroxidation produced by ferritin were prevented by superoxide dismutase, which lowered the rates found in the presence of ferritin to values found in the absence of ferritin. Catalase and hydroxyl radical scavengers had no effect on the stimulation by ferritin. Nonheme iron chelators prevented the ferritin stimulation as did glutathione, propylgallate, and trolox. Basal rates of lipid peroxidation were inhibited by anti-CYP2E1 IgG; the stimulation by ferritin was decreased by anti-CYP2E1 IgG. These results show that microsomes from ethanol-fed rats are more reactive than control microsomes in interacting with ferritin to produce oxidants capable of catalyzing lipid peroxidation. The inhibition of the ferritin-catalyzed lipid peroxidation by superoxide dismutase and anti-CYP2E1 IgG is consistent with a role for CYP2E1-generated superoxide radical in mobilizing iron from ferritin and in the subsequent catalysis of lipid peroxidation. Since ferritin is the major cellular storage form of iron, increased mobilization of iron from ferritin by CYP2E1-derived superoxide radical may play a role in the development of oxidative stress after ethanol treatment.

Identification of the cytochrome P450 isozymes involved in the metabolism of N-nitrosodipropyl-,N-nitrosodibutyl- and N-nitroso-n-butyl-n-propylamine.

The metabolism of N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitroso-n-butyl-n-propylamine (NBPA) was investigated in vitro using liver microsomes and purified isoforms of cytochrome P450 in a reconstituted system. Liver microsomes were prepared from rats pretreated with phenobarbital (PB), pyridine (PYR), beta-naphthoflavone (BNF), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), clofibrate (CLO) or from untreated rats. The purified cytochrome P450s used in the reconstituted system were rat 1A1 and 2B1 and rabbit 2E1. The rates of metabolism and the product profiles for NDPA, NDBA and NBPA changed significantly depending on the pretreatment of the rats or the identity of the purified cytochrome P450 isoforms. Induction by PB dramatically increased cleavage of NDPA, NDBA and NBPA at C-N bonds, leading to substantial increases in formation of the respective aldehydes and the overall metabolic rates. Microsomes from PYR-pretreated rats exhibited increased activities for formation of formaldehyde and propionaldehyde from NDPA and NBPA. Microsomes from BHT-pretreated rats showed a slight increase in activity for N-dealkylation of NDBA and BNPA. Treatment with BHA decreased the overall metabolism of NDBA, but slightly increased N-dealkylation of NBPA. Microsomal metabolism of NDPA, NDBA and NBPA was decreased by pretreatment with BNF and CLO. Results from studies using the reconstituted system with purified cytochrome P450 isoforms demonstrated that cytochrome P450 2B1 specifically catalyzed alpha-hydroxylation of these three long chain nitrosamines with high activity. Cytochrome P450 2E1 catalyzed formation of formaldehyde and propionaldehyde from NDPA and NBPA, but did not catalyze formation of acetaldehyde or butyraldehyde. Cytochrome P450 1A1 exhibited no activity for metabolism of NDPA, NDBA and NBPA. The contributions of cytochrome P450 2B1 and 2E1 to N-dealkylation reactions were determined using inhibitory monoclonal antibodies (mAb). With microsomes from PB-pretreated rats, inhibition by mAb-2B1 indicated a 62% contribution by cytochrome P450 2B1 to debutylation of NDBA and 65% to depropylation of NDPA. In microsomes from PYR-pretreated rats inhibition by mAbs also showed a role for cytochrome P450 2E1 in depropylation of NDPA. These studies provide a better understanding of the role of various forms of cytochrome P450 in metabolic activation of these long chain N-nitrosodialkylamines to potentially toxic, mutagenic and carcinogenic intermediates.

DNA strand cleavage as a sensitive assay for the production of hydroxyl radicals by microsomes: role of cytochrome P4502E1 in the increased activity after ethanol treatment.

There is increasing interest in the role of reactive oxygen radicals in the hepatotoxicity associated with ethanol consumption. Reactive oxygen intermediates interact with DNA and can cause single-strand breaks of supercoiled DNA. Experiments were carried out to evaluate the utility of this system as a sensitive assay for the detection of potent oxidants generated by rat liver microsomes isolated from pair-fed control rats and rats treated chronically with ethanol. DNA strand cleavage was assayed by monitoring the migration of the supercoiled and open circular forms in agarose. Microsomes catalysed DNA strand breakage with either NADPH or NADH as cofactors; iron was required to catalyse the reaction and various ferric complexes were effective in promoting the reaction. DNA strand cleavage was prevented by catalase, superoxide dismutase, GSH and hydroxyl-radical-scavenging agents, suggesting that a hydroxyl-radical-like species was the oxidant responsible for the breakage. This assay system proved to be much more sensitive in detecting hydroxyl radicals than are other methods, such as e.s.r. spectroscopy or oxidation of chemical scavenging agents with respect to the amount of microsomal protein and the nature and concentration of the iron catalyst required. Microsomes from ethanol-treated rats were more reactive than control microsomes in catalysing the DNA strand cleavage with either NADPH or NADH; increased catalytic activity was observed with various ferric complexes and was sensitive to the above antioxidants. Compared with preimmune IgG, anti-(cytochrome P4502E1) IgG had no effect on DNA strand cleavage by the control microsomes, but completely prevented the NADPH- and the NADH-dependent increased activity found with microsomes from the ethanol-treated rats. Inhibitors of cytochrome P4502E1, such as diethyl dithiocarbamate and tryptamine, also lowered the extent of increase of DNA strand cleavage produced by microsomes from the ethanol-treated rats. These results indicate that DNA strand cleavage is a very sensitive assay for detecting the production of hydroxyl radicals by microsomes and to demonstrate increased activity by microsomes after chronic ethanol treatment. This increased activity with NADPH and NADH is due, at least in part, to induction of cytochrome P4502E1.

Kinetic Analysis of Furan Biotransformation by F-344 Rats in Vivo and in Vitro

Furan is both hepatotoxic and hepatocarcinogenic in rats. The kinetics of furan biotransformation by male F-344 rats were studied in vivo and in vitro in order to understand target tissue dosimetry. A physiologically based pharmacokinetic (PBPK) model for furan in rats was developed from gas uptake studies using initial furan concentrations of 100, 500, 1050, and 3850 ppm. Tissue partition coefficients for furan were determined in vitro using vial equilibration techniques. Furan gas uptake kinetics in vivo were described by a single saturable process with a Vmax of 27.0 μmol/hr/250 g rat and a KM of 2.0 μM. Furan metabolism in vivo was inhibited by pyrazole. The furan PBPK model adequately simulated blood and liver furan concentrations following 4-hr inhalation exposures to 52, 107, and 208 ppm furan. The biotransformation of furan was studied in freshly isolated rat hepatocytes in vitro and compared to biotransformation in vivo. Furan biotransformation by isolated rat hepatocytes exhibited a KM of 0.4 μM and a Vmax of 0.018 μmol/hr/106 cells. Inhibition and induction studies indicated that cytochrome P450 was the catalyst of furan oxidation. Acetone pretreatment of the rats produced a five-fold increase in the rate of the hepatocyte oxidation of furan, suggesting an important role for cytochrome P450 2E1. The Vmax determined in hepatocytes in vitro extrapolated to 23.0 μmol/hr/250 g rat, assuming 128 × 106 hepatocytes/g liver. Incorporation of the in vitro hepatocyte kinetic parameters into the PBPK model for furan accurately simulated in vivo pharmacokinetics. These results suggest that freshly isolated hepatocytes are a valuable in vitro system for predicting chemical pharmacokinetics in vivo.

Sex-Related Differences in Mouse Renal Metabolism and Toxicity of Acetaminophen

The objective of this study is to elucidate the role of cytochrome P450 2E1 in the metabolic activation of acetaminophen (APAP) in mouse kidneys. With the kidney microsomes from C3H/HeJ mice, a significant sex-related difference was observed in the NADPH-dependent formation of a reactive APAP metabolite which was trapped as a glutathione conjugate. The enzyme activity in male mouse kidney was about 35- and 50-fold higher than that in the females at substrate concentrations 50 and 500 μM, respectively, and treatment of female mice with testosterone significantly induced the enzyme activity in the mouse kidney. No sex-related difference in this enzyme activity, however, was observed in the livers. The oxidative metabolism of APAP in control male mouse kidney microsomes displayed an apparent low Km of 43-45 μM and an apparent high Km of 603-702 μM. Studies using monoclonal antibodies against P450 2E1 showed that P450 2E1 accounted for about half of the activity in the metabolic activation of APAP in both mouse liver and kidney. Furthermore, there was a strong correlation between the renal P450 2E1 content (measured by Western blot analysis) and the rate of APAP oxidation by renal microsomes. To test the hypothesis that the susceptibility of renal tissue to APAP toxicity is determined by the distribution and level of P450 2E1, toxicity studies were conducted. A time-dependent depletion of hepatic and renal cortical glutathione was observed in both male and female mice following a dose of 1200 mg/kg APAP. At 4 hr after APAP dosing, the level of renal cortical glutathione depletion in male mice was significantly greater than that in the females. The level of blood creatinine elevation in male mice was higher than that in the females 8 hr after APAP dosing. Histopathology studies by light and electron microscopic assessments demonstrated that renal damage by APAP was restricted mostly to the epithelial cells of the proximal convoluted tubules where P450 2E1 was localized. The renal proximal tubular necrosis induced by APAP was more severe in males than that in the females. Results from this study suggest that P450 2E1 plays an important role in the metabolic activation of APAP and is a key factor in determining the sex-related difference of APAP-mediated toxicity in the mouse kidney.

Role of cytochrome P-450 2E1 in ethanol-, carbon tetrachloride- and iron-dependent microsomal lipid peroxidation.

This study investigated the role of cytochrome P-450 2E1 in enhanced microsomal lipid peroxidation in experimental alcoholic liver disease. We also examined the contribution of this isoform to the increased microsomal injury in alcoholic liver disease caused by carbon tetrachloride-induced or iron-induced oxidant stress. Adult male Wistar rats were intragastrically infused with a high-fat diet and ethanol or glucose for 16 wk; this resulted in hepatic lipid peroxidation and fibrogenesis in the ethanol-fed animals. Microsomes were isolated by differential centrifugation in the presence of 100 mumol/L deferoxamine, washed twice in buffer without deferoxamine and incubated in the absence or presence of ethanol (50 mmol/L), carbon tetrachloride (150 mumol/L), ferric citrate (50 mumol/L) or ferric citrate plus ethanol at 37 degrees C for 30 min in an NADPH-generating system. The basal rate of lipid peroxidation in microsomes isolated from ethanol-fed rats was increased by 52% compared with that in microsomes from controls. Carbon tetrachloride-induced and ferric citrate-induced lipid peroxidation were also accentuated in microsomes from ethanol-fed rats, by 76% and 108%, respectively. Ethanol added in vitro significantly reduced basal (-58%) and ferric citrate-induced (-48%) lipid peroxidation in microsomes from ethanol-fed rats, whereas it had an insignificant effect on that in control microsomes. In fact, this protective effect of ethanol on microsomes from ethanol-fed rats resulted in attenuation of the difference in the level of microsomal lipid peroxidation between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of cytochrome P4502E1 in chemically induced cytotoxicity

The role of cytochrome P4502E1 in chemically induced cytotoxicity