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Biomedical subjects

M Ingelman-Sundberg

Publications and source records attributed to M Ingelman-Sundberg.

At least 109 records · Page 6Linked to original sources
PubMed1996

High rates of substrate hydroxylation by human cytochrome P450 3A4 in reconstituted membranous vesicles: influence of membrane charge.

CYP3A4 represents the most important form of human cytochrome P450 active in drug metabolism. Reconstitution of this enzyme has in the past been a major problem. Using purified cDNA-expressed CYP3A4 incorporated into membranous vesicles made from microsomal phospholipids, rates of nifedipine and testosterone oxidation of about 60 nmol/nmol P450/min were achieved, whereas similar reconstitution into dilauroyl-phosphatidylcholine micelles was unsuccessful. A higher Vmax for nifedipine oxidation was obtained in negatively charged vesicles as compared to neutral membranes, whereas the membrane charge did not influence the Km. It is concluded that the native function of CYP3A4 requires a negatively charged microsomal membrane.

Animals↗
PubMed1996

Effects of diet and ethanol on the expression and localization of cytochromes P450 2E1 and P450 2C7 in the colon of male rats.

Local activation of procarcinogens in target tissues such as the colon by cytochrome P450-dependent microsomal monooxygenases is considered to be an important factor in the etiology of cancer. Diet and alcohol consumption are considered risk factors in colon cancer, and the cytochrome P450 isozymes CYP2E1 and CYP2C7 have been implicated in the biochemical mechanisms underlying colon cancer. The current study was conducted to determine the effects of diet and ethanol consumption on colonic and hepatic expression of these two enzymes. Adult male rat Sprague-Dawley rats were fed rat chow ad lib. or were infused intragastrically with control or ethanol-containing diets. Our results indicate that CYP2E1 is present in colonic epithelial cells, and expression of colonic and hepatic microsomal CYP2E1 and CYP2C7 was increased by chronic ethanol intake. As compared with rats having ad lib. access to standard rat food, rats receiving total enteral nutrition had significant (P < 0.01) reductions of CYP2C7 and slight, but not statistically significant, reductions in the expression of CYP2E1 in colon. Diet and ethanol differentially regulated CYP2E1 and CYP2C7 in a tissue-specific manner such that the ethanol induced CYP2E1 and CYP2C7 in the colon and liver, and the intragastric diet alone had a tendency to induce these isozymes in the liver and reduce them in the colon. These results may provide a partial explanation for the mechanism underlying effects of diet and ethanol on colon cancer.

Animals↗
PubMed1996

Role of cytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol.

We have previously shown that the treatment with diallyl sulfide (DAS) and phenylethyl isothiocyanate (PIC) of rats receiving ethanol in the alcohol tube-feeding model effectively suppressed the induction of cytochrome P4502E1 (CYP2E1) by ethanol. Here we report that rat treatment with DAS and PIC significantly decreased the trapping of hydroxyethyl free radicals in liver microsomes incubated in vitro with ethanol. Furthermore, these inhibitors also greatly reduced the production of hydroxyethyl radical-derived epitopes detectable in vivo in the liver of ethanol-fed rats. The action of DAS and PIC on the formation of hydroxyethyl radicals paralleled their inhibitory effect on lipid peroxidation as monitored using, respectively, liver malonildialdehyde (MDA) and plasma lipid hydroperoxide levels as well as by the titers of antibodies versus MDA adducts to proteins. Thus, these results indicated a link between the induction of CYP2E1 by ethanol, the formation of hydroxyethyl radicals and the stimulation of lipid peroxidation. The pathological scores in the livers of rats fed with ethanol plus or minus DAS and PIC also correlated with levels of hydroxyethyl radical-derived epitopes. Rats fed intragastrically with ethanol developed antibodies and the formation of these antibodies was greatly reduced by DAS and PIC. Taken together these results suggest that CYP2E1 plays an important role in the generation of hydroxyethyl radicals during chronic alcohol feeding and that ethanol-derived free radicals might play a role in the onset of liver injury in this model of alcohol administration.

Allyl Compounds↗
PubMed1996

The role of CYP2E1 and 2B1 in metabolic activation of benzene derivatives.

CYP2B1 and 2E1 oxidized toluene, aniline and monochlorobenzene (MCB) to water-soluble metabolites and to products covalently binding to microsomal proteins from male Wistar rats at high efficiency. Oxidation of benzene to covalently binding metabolites was catalysed by CYP2B1 and 2E1 more effectively than the formation of water-soluble metabolites, especially at low benzene levels. Thus, the formation of covalently binding products was inversely related but formation of soluble metabolites was proportional to benzene concentration. 1,4-Benzoquinone was responsible for the majority of covalent binding to microsomal proteins, being suppressed by ascorbate; 1,4-semiquinone was not important, since alpha-tocopherol did not inhibit the covalent binding and ESR showed its rapid decay, if NADPH was available. Specific antibodies and inhibitors confirmed the role of CYP2B1 and 2E1 induction. Covalent binding of benzene to DNA was largely due to benzene oxide; approximately 50% was due to N-7 guanine adduct. CYP2E1 oxidizing benzene via phenol to 1,4-hydroquinone appeared to mediate its further oxidation to 1,4-benzoquinone, which also occurred spontaneously, but was reversed in a reducing environment of microsomes with NADPH. Production of OH radicals in microsomes with NADPH was greatly stimulated by HQ and less by BQ, especially in CYP2E1 induced microsomes, although the quinones themselves failed to produce OH radicals. The quinones could act by simulation of the CYP futile cycle. Therefore, CYP2B1 and 2E1 in rats appeared essential for metabolic activation of benzene derivatives to potentially genotoxic products; BQ dominated the covalent binding of benzene to proteins, whereas DNA adducts were largely due to benzene oxide.

Animals↗
PubMed1996

Phenotype and genotype analysis of debrisoquine hydroxylase (CYP2D6) in a black Zimbabwean population. Reduced enzyme activity and evaluation of metabolic correlation of CYP2D6 probe drugs.

OBJECTIVE: Debrisoquine hydroxylase (CYP2D6) is responsble for the oxidative metabolism of many clinically used drugs. Since this enzyme has been poorly studied in the southern part of Africa, we examined the CYP2D6 phenotypes and genotypes in 103 unrelated black Zimbabweans. METHODS: Phenotyping for CYP2D6 activity was done using debrisoquine and metoprolol as probe drugs by measuring the urinary metabolic ratio (MR) of parent drug to metabolite concentration ratios. Genotyping was done using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP) and sequencing analyses with respect to CYP2D6 variants of interest. RESULTS AND CONCLUSION: Phenotyping with debrisoquine revealed two poor metabolisers (PMs), whereas 5 subjects out of 94 were PMs using metoprolol as probe drug. Genotypes predictive of the poor metaboliser status were observed for the two subjects who were PMs with both probe drugs, whereas no mutations could explain the PM phenotype for metoprolol among the three remaining subjects, a fact possibly explained by lack of compliance in metoprolol intake. There was a moderate correlation of 0.67 between the debrisoquine and metoprolol metabolic ratios in the 89 subjects who were extensive metabolisers for both probe drugs. The median values for the metabolic ratios for debrisoquine and metoprolol as probe drugs were 1.00 and 1.35, respectively, which are higher than those observed in Caucasian populations. This is indicative of a decreased capacity for metabolism of CYP2D6 substrates by Zimbabweans compared to Caucasians. Evaluation of the DNA samples for the known allelic variants CYP2D6A, CYP2D6B, CYP2D6C, CYP2D6D or CYP2D6Ch1 yielded no explanation for these results.

Cytochrome P-450 CYP2D6↗
PubMed1996

Hepatotoxicity induced by iron overload and alcohol. Studies on the role of chelatable iron, cytochrome P450 2E1 and lipid peroxidation.

BACKGROUND/AIMS: Clinical experience and studies with experimental animal models indicate a synergistic hepatotoxic effect of dietary iron overload and chronic alcohol ingestion. In order to elucidate the mechanism underlying this synergism, we examined the hepatic levels of ethanol-inducible cytochrome P450 2E1, glutathione and malondialdehyde, and the effect of iron chelation with desferrioxamine, in livers from rats treated with iron and/or ethanol. METHODS: Animals received diets with or without 2.5-3% carbonyl iron for 6-9 weeks, followed by an ethanol-containing diet or a liquid control diet for 5-9 weeks. Desferrioxamine was administered subcutaneously with mini-osmotic pumps. Alanine aminotransferase activity in serum and hepatic contents of glutathione and malondialdehyde were determined. The hepatic level of cytochrome P450 2E1 was determined with Western Blotting using a specific polyclonal antibody. RESULTS: The combination of iron and alcohol led to a marked increase in serum alanine aminotransferase activity as compared with all other treatment groups, and iron chelation with desferrioxamine reversed these increases. Treatment with alcohol alone led to slightly increased aminotransferases compared with controls. The level of cytochrome P450 2E1 was significantly elevated in microsomes isolated from ethanol-treated rats, but neither additional iron supplementation nor desferrioxamine influenced this level significantly. Glutathione contents were increased in the livers of animals treated with iron and/or ethanol. Malondialdehyde values were increased in iron-treated animals, whereas neither ethanol nor desferrioxamine altered malondialdehyde levels significantly. CONCLUSIONS: The toxic effects exerted by the combination of iron overload and chronic ethanol feeding on rat liver are dependent on a pool of chelatable iron. The hepatic level of cytochrome P450 2E1 is markedly induced by ethanol but not further altered by iron overload. Neither increased lipid peroxidation nor depletion of hepatic glutathione levels can explain the synergistic hepatotoxic effects of iron and ethanol in this model.

Alanine Transaminase↗
PubMed1996

A novel mutant variant of the CYP2D6 gene (CYP2D6*17) common in a black African population: association with diminished debrisoquine hydroxylase activity.

1. The debrisoquine hydroxylase (CYP2D6) is polymorphically distributed. Not only are there differences in the proportions of extensive metabolisers to poor metabolisers in various ethnic groups, but there are also pronounced variations in the metabolic capacity among those classified as extensive metabolisers. 2. The mean debrisoquine metabolic ratio of Caucasian extensive metabolisers is lower than that for a number of African populations. In the present study, we have searched for novel CYP2D6 mutations to explain the diminished enzyme activity in African populations. 3. Three Zimbabwean Shona subjects with EM phenotypes (metabolic ratios for debrisoquine of 0.4, 1.5 and 10.5 respectively) were selected and the open reading frame of the CYP2D6 gene of each was sequenced. 4. The subject with metabolic ratio of 10.5 was found to be homozygous for an allele with a nucleotide exchange in exon 2, 1111C-->T causing a 107Thr-->Ile amino acid exchange in a conserved region of the enzyme. In addition, he was homozygous for the 2938C-->T and 4268G-->C mutations causing 296Arg-->Ser and 486Ser-->Thr amino acid substitution found in the CYP2D6*2 allele. 5. Seventy-six Zimbabwean Shona subjects were subsequently genotyped for the 1111C-->T mutation and for the intron 1 gene conversion present in the CYP2D6*2 gene. The 1111C-->T mutation was found at an allele frequency of 34% and was only present in alleles carrying the gene conversion in intron 1 indicative for the CYP2D6*2 gene. 6. This allele (CYP2D6*17), containing the 1111C-->T, 2938C-->T and 4268G-->C mutations, was found to be strongly associated with lower capacity for debrisoquine hydroxylation. We therefore postulate that the CYP2D6*17 allele might contribute to the molecular basis of the previously established diminished debrisoquine hydroxylase activity in African Bantu populations.

Black People↗
PubMed1996

Expression, catalytic activity, and inducibility of cytochrome P450 2E1 (CYP2E1) in the rat central nervous system.

Cytochrome P450 2E1 (CYP2E1) metabolizes several neuroactive substrates, including exogenous compounds such as anesthetics, organic solvents, and muscle relaxants as well as endogenous substrates such as arachidonic acid. CYP2E1 and its mRNA were found to be expressed in the rat hippocampus, where the enzyme was localized mainly to the microsomal fraction. Chlorzoxazone (CZN), a CYP2E1 substrate, was 6-hydroxylated in hippocampal homogenates with a K(m) of 25.5 microM and a Vmax of 0.22 pmol/mg/min. CYP2E1 was also expressed in vitro in cortical glial cultures, where CYP2E1 mRNA levels were found to be 1,000-fold lower than in rat liver. Exposure of cortical glial cultures to 25 or 100 mM ethanol for 24 h caused a fourfold and sixfold increase, respectively, in the rate of CYP2E1-dependent 6-hydroxylation of CZN. After a continuous exposure to 100 mM ethanol for 48 or 72 h, however, the hydroxylation rate was down-regulated. Chlormethiazole, a potent inhibitor of hepatic CYP2E1 transcription, inhibited the ethanol-dependent induction of CYP2E1 by 50%. In vivo, acute ethanol treatment of rats (24 h, 3 g/kg) resulted in a 1.8-fold increase in the rate of CZN 6-hydroxylation in hippocampal homogenates. It is concluded that CYP2E1 is expressed and catalytically active in the rat CNS, and that CYP2E1 can be induced by a relatively low concentration of ethanol in cortical glial cultures. It is suggested that CYP2E1 substrates may be metabolically activated in situ in the CNS.

Animals↗
PubMed1996

Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics.

BACKGROUND & AIMS: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. METHODS: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. RESULTS: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively, The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. CONCLUSIONS: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease.

Adult↗
PubMed1996

Nomenclature for human CYP2D6 alleles.

To standardize CYP2D6 allele nomenclature, and to conform with international human gene nomenclature guidelines, an alternative to the current arbitrary system is described. Based on recommendations for human genome nomenclature, we propose that alleles be designated by CYP2D6 followed by an asterisk and a combination of roman letters and arabic numerals distinct for each allele with the number specifying the key mutation and, where appropriate, a letter specifying additional mutations. Criteria for classification as a separate allele and protein nomenclature are also presented.

Alleles↗
PubMed1996

PCR-based genotyping for duplicated and deleted CYP2D6 genes.

The debrisoquine hydroxylase (CYP2D6), which metabolizes more than 30 different drugs, is highly polymorphic. In subjects having either very low or very high enzyme activity, drug therapy at recommended doses using CYP2D6 substrates may lead to either increased risk of side effects or therapeutic failure. We here describe PCR-based methods for detection of alleles having either duplicated, multiduplicated or deleted active CYP2D6 genes. As a control reaction, the entire coding region of the CYP2D6 gene is amplified. In conjunction with analysis of common mutations using this product as a template, the methods described can be used for genotyping of individuals being either poor, intermediate rapid, normal or ultrarapid metabolizers and provides an efficient tool for individualization of drug therapy.

Cytochrome P-450 CYP2D6↗
PubMed1996

Debrisoquine and S-mephenytoin hydroxylation phenotypes and genotypes in a Korean population.

One hundred and fifty-two healthy Korean volunteers were phenotyped with debrisoquine and mephenytoin and genotyped with respect to CYP2D6. The debrisoquine metabolic ratio (MR) varied between 0.09 and 6.3, and all subjects were thus classified as extensive metabolizers of debrisoquine. Polymerase chain reaction (PCR)-based amplification of genomic DNA with primers specific for the C188-->T mutation present in exon 1 of the CYP2D6*10B allele was performed and revealed an allele frequency of 0.51 in this Korean population. Forty-three subjects (28%) were homozygous for CYP2D6*10B, 69 subjects (45%) were heterozygous for this allele, while in 40 subjects (26%) no exon 1 mutation could be found. All subjects except one homozygous for the wild type allele had MRs below 0.75 whereas the MR was higher than 0.99 in all subjects homozygous for the CYP2D6*10B allele. The MRs in the three genotype groups were significantly different (p < 0.0001; Kruskal-Wallis test). Eco RI RFLP analysis of DNA from six subjects with debrisoquine MRs < or = 0.11 revealed that only one (MR 0.09) carried a duplicated CYP2D6*Z-gene (CYP2D6*2X2) as indicated by the Eco RI 12.1 kb haplotype. It is concluded that, as shown earlier for Chinese and Japanese populations, the CYP2D6*10B-allele containing the C188-->T mutation is the major cause of diminished CYP2D6 activity in Koreans. In this Korean population, the MR of debrisoquine was shifted towards higher values (lower CYP2D6 activity) compared with Caucasian populations but the shift appeared to be less pronounced than earlier shown for Chinese. Twenty-four subjects (16%) were poor metabolizers of S-mephenytoin as indicated by the S/R mephenytoin ratio of about 1. Twenty-three of these were genotyped with respect to the defect CYP2C19-alleles CYP2C19*2 and CYP2C19*3. Of the 46 poor metabolizer alleles, 32 (70%) were CYP2C19*2 and the remaining 14 (30%) were CYP2C19*3. Thus, the defect CYP2C19*2 and CYP2C19*3-alleles explained 100% of the 23 Korean poor metabolizers of S-mephenytoin.

Adult↗
PubMed1996

S-mephenytoin hydroxylation phenotype and CYP2C19 genotype among Ethiopians.

The polymorphic metabolism of S-mephenytoin and the distribution of two known deleterious mutant CYP2C19 alleles was determined among 114 healthy unrelated black Ethiopians. Six subjects (5.2%) were poor metabolizers (PMs) of S-mephenytoin. The frequencies of the defective CYP2C19*2 (CYP2C19m1) and CYP2C19*3 (CYP2C19m2) alleles were 0.14 and 0.02, respectively. Three of the PMs were homozygous for the CYP2C19*2 allele and the remaining three PMs were heterozygous for both the CYP2C19*2 and CYP2C19*3 mutant alleles. It is concluded that the frequency of PMs for S-mephenytoin is similar in Ethiopians, Zimbabweans and Caucasians and that the CYP2C19*3 allele, for the first time identified in a black population, together with the CYP2C19*2 allele account for all of the defective CYP2C19 alleles among the Ethiopian PMs.

Alleles↗
PubMed1996

Phase II metabolism of benzene.

The hepatic metabolism of benzene is thought to be a prerequisite for its bony marrow toxicity. However, the complete pattern of benzene metabolites formed in the liver and their role in bone marrow toxicity are not fully understood. Therefore, benzene metabolism was studied in isolated rodent hepatocytes. Rat hepatocytes released benzene-1,2-dihydrodiol, hydroquinone (HQ), catechol (CT), phenol (PH), trans-trans-muconic acid, and a number of phase II metabolites such as PH sulfate and PH glucuronide. Pretreatment of animals with 3-methylcholantrene (3-MC) markedly increased PH glucuronide formation while PH sulfate formation was decreased. Likewise, V79 cells transfected with the 3-MC-inducible rat UGT1.6 cDNA showed a considerable rate of PH and HQ glucuronidation. In addition to inducing glucuronidation of phenols, 3-MC treatment (reported to protect rats from the myelotoxicity of benzene) resulted in a decrease of hepatic CYP2E1. In contrast, pretreatment of rats with the CYP2E1-inducer isopropanol strongly enhanced benzene metabolism and the formation of phenolic metabolites. Mouse hepatocytes formed much higher amounts of HQ than rat hepatocytes and considerable amounts of 1,2,4-trihydroxybenzene (THB) sulfate and HQ sulfate. In conclusion, the protective effect of 3-MC in rats is probably due to a shift from the labile PH sulfate to the more stable PH glucuronide, and to a decrease in hepatic CYP2E1. The higher susceptibility of mice toward benzene may be related to the high rate of formation of the myelotoxic metabolite HQ and the semistable phase II metabolites HQ sulfate and THB sulfate.

Animals↗
PubMed1996

Frequent distribution of ultrarapid metabolizers of debrisoquine in an ethiopian population carrying duplicated and multiduplicated functional CYP2D6 alleles.

The debrisoquine hydroxylase (CYP2D6) catalyzes the oxidative metabolism of more than 40 different clinically important drugs. The CYP2D6 gene is highly polymorphic. Defect alleles, causing the poor metabolizer phenotype, and also alleles with duplicated or multiduplicated active genes, causing ultrarapid metabolism, have been described. In the current investigation, we have evaluated the CYP2D6 phenotype (n = 115) and genotype (n = 122) among healthy Ethiopians. Only two subjects (1.8%) exhibited metabolic reaction (MR) for debrisoquine > 12.6 and were classified as poor metabolizers. A mutation in exon 1 causing a 34Pro --> Ser amino acid exchange, typical of the Chinese CYP2D6*10B (Ch1) allele and yielding an unstable enzyme, was present among 16% of the population and the carriers exhibited a high MR (0.9-5.0). Increased MR was also found among 18% of the subjects with a 107Thr --> Ile mutation associated to the CYP2D6*17(Z) allele causing diminished activity of CYP2D6 in vivo. Interestingly, 29% of the population investigated carried alleles with duplicated or multiduplicated CYP2D6 genes, indicative of ultrarapid metabolism. Xbal and EcoRI RFLP analyses identified individuals having new alleles with four or five CYP2D6*2(L) genes. Subjects with duplicated or multiduplicated CYP2D6*2 genes exhibited the lowest MR. These results suggest that the Ethiopian population, in comparison to Caucasian, Oriental and other Black populations, is genetically different with respect to the constitution of the CYP2D locus. The results also show that subjects carrying duplicated or multiduplicated active CYP2D6 genes are very common in certain ethnic groups, implicating this issue of potential global importance.

Alleles↗
PubMed1996

Induction of cytochrome P450 2E1 expression in rat and gerbil astrocytes by inflammatory factors and ischemic injury.

Hepatic cytochromes P450 are known to be down-regulated by cytokines, lipopolysaccharide, Gram-positive bacteria, and viruses. Little is known, however, about the regulation by inflammation of cytochromes P450 in other tissues. We have found that lipopolysaccharide and interleukin-1 beta stimulate the expression of catalytically active CYP2E1 (but not CYP1A1 or CYP2B) up to 7-fold in rat brain primary cortical glial cultures. The induction reached a maximum after 24 hr and was accompanied by an increase in CYP2E1 mRNA. Chlormethiazole, a specific inhibitor of hepatic CYP2E1 transcription, completely inhibited the induction of CYP2E1 at the mRNA and enzyme levels. Immunofluorescence studies showed CYP2E1 to be expressed in a subset of astrocytes in the lipopolysaccharide-stimulated cortical glial cultures. Using a model of global ischemic injury in the gerbil, we found CYP2E1 to be induced in vivo in astrocytes in the inflammatory phase, 1-3 weeks after the lesion. Likewise, CYP2E1 was induced in the rat cortex 1 week after a focal ischemic injury. Our results suggest tissue-specific regulation of CYP2E1 by inflammatory factors and that CYP2E1 may play a role in astrocytes during inflammation in the brain.

Animals↗
PubMed1995

Induction mechanisms of cytochrome P450 2E1 in liver: interplay between ethanol treatment and starvation.

Chronic ethanol exposure causes marked induction of the ethanol-inducible cytochrome P450 (CYP) 2E1 isozyme in the centrilobular liver region, where alcoholic damage commonly is initiated. In contrast to most other CYP forms, which are ligand-activated at the transcriptional level, ethanol induction of CYP2E1 has been found to be post-translational. However, transcriptional activation of the CYP2E1 gene was recently described in fed animals maintained at very high ethanol levels. To further evaluate mechanisms of ethanol-mediated CYP2E1 induction we compared the effect of short-term heavy-ethanol treatment and fasting on CYP2E1 mRNA, protein and catalytic activity. High blood-ethanol levels (20-70 mM) were maintained for 3 days by regular alcohol intubations to fed or fasted rats. During this period, the amount of liver CYP2E1 apoprotein increased a maximum of 20-fold and catalytic activity 16-fold, both in fed and fasted animals, whereas starvation alone caused only a 4- to 5-fold increase. By comparison, the amount of CYP2E1 mRNA, as assayed both by Northern blot and slot blot, was significantly increased (5- to 6-fold) by ethanol only in fasted rats; this increase was smaller than that observed after fasting alone (8- to 9-fold). Analysis of cell lysates isolated from the periportal and perivenous region revealed that the increase in CYP2E1 mRNA by fasting occurred in the perivenous region. Thus no evidence was obtained for an increased pretranslational CYP2E1 gene expression as a consequence of the continuous presence of ethanol at intoxicating levels for 3 days. CYP2E1 mRNA elevation seems to be strongly associated with starvation while alcohol treatment increases the amount of enzyme, primarily by ligand-dependent stabilization of the synthesized protein. Our results indicate that transcriptional activation of CYP2E1 requires the long-term presence of highly intoxicating ethanol levels. It is conceivable that such activation occurs via indirect physiological responses related to those triggered by starvation.

Animals↗