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M Ingelman-Sundberg

Publications and source records attributed to M Ingelman-Sundberg.

At least 127 records · Page 7Linked to original sources
PubMed1995

Stable expression of human cytochrome P450 2E1 in V79 Chinese hamster cells.

A V79 Chinese hamster cell line was constructed for stable expression of human cytochrome P450 2E1 (CYP2E1) by integration of a SV40 Early promoter recombinant CYP2E1 cDNA into the chromosomal DNA. The cDNA encoded CYP2E1 was effectively expressed and enzymatically active, as shown by hydroxylation of chlorzoxazone and of p-nitrophenol, at rates of about 70 pmol x mg-1 total protein x min-1. CYP2E1 content and activity was increased upon cultivation in the presence of ethanol indicating a substrate mediated stabilization effect. A similar stabilizing effect was also observed for inhibitors of CYP2E1, e.g. imidazole, 4-methylpyrazole, and isoniazid. The feasibility of the newly established cell line V79MZh2E1 for toxicological studies was shown by CYP2E1-mediated activation of N-nitrosodimethylamine and p-nitrophenol and a dose-dependent cytotoxic and mutagenic effect.

Animals↗
PubMed1995

Inhibition of CYP2E1 activity does not abolish pulsatile urine alcohol concentrations during chronic alcohol infusions.

Continuous, long-term intragastric infusions of ethanol leads to a two-step induction of hepatic cytochrome P450 2E1 (CYP2E1) that is correlated with blood ethanol concentrations (BECs) and urine alcohol concentrations (UECs). In addition, long-term and continuous ethanol infusion does not produce a steady-state BEC, but results in pulsatile BECs and UECs having peak-peak duration of approximately 6 days and ethanol concentrations ranging from near zero to over 500 mg/dl. In the present study, rats were treated with ethanol (levels reaching 13 g.kg-1.day-1) for 38 days in the presence of compounds reported to block CYP2E1 activity or expression, to study the possible involvement of CYP2E1 in the pulsatile BECs. The inhibitors used were chlormethiazole (CMZ); diallysulfide (DAS); phenethyl isothiocyanate (PET) and dihydrocapsacin (DHC). Hepatic microsomal metabolism of carbon tetrachloride and p-nitrophenol, as well as mean CYP2E1 apoprotein levels, were significantly greater (P < or = 0.05) in ethanol-treated rats than in control rats, whereas rats treated with DAS, CMZ or PET had significantly (P < or = 0.05) reduced p-nitrophenol and carbon tetrachloride metabolism and lower CYP2E1 apoprotein levels compared to those of ethanol controls. UECs were variable in all ethanol-treated groups and there was a typical pulsatile pattern that had a mean interpulse interval (the number of days between the peaks of two consecutive pulses) ranging over 5.4 +/- 0.3-6.0 +/- 0.7 days and a mean amplitude (nadir to peak UEC) of 415 +/- 39-337 +/- 33 mg/dl. None of the putative CYP2E1 blockers altered the pulsatile nature of ethanol in urine. Our results suggest that pulsatile UECs are not the result of variations in the amount of CYP2E1.

Animals↗
PubMed1995

Activation of alkylhydrazines to free radical intermediates by ethanol-inducible cytochrome P-4502E1 (CYP2E1).

Electron spin resonance (EPR) spectroscopy analysis using the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) was used to measure the formation of free radical intermediates during NADPH-dependent oxidation of 1-methyl-, 1-ethyl-, and 1-isopropylhydrazine in rat liver microsomes and in reconstituted enzyme systems. The experiments in microsomes revealed that the specific activation of the hydrazines, as measured by the EPR signal intensities, was about two-fold higher, when expressed per nmol of P-450, in microsomes from rats treated with ethanol (EtOH) as compared to membranes isolated from either phenobarbital (PB)-, beta-naphthoflavone (beta-NF)-treated or control rats. Furthermore, kinetic experiments revealed that EtOH-microsomes had an apparent affinity for 1-ethylhydrazine about one order of magnitude higher than PB-microsomes. In reconstituted vesicular systems composed of phospholipids, NADPH cytochrome P-450 reductase and P-450, the intensities of EPR signals produced by the formation of the methyl-, ethyl- and isopropyl-free radicals, were 3- to 5-fold more intense in membrane vesicles containing ethanol-inducible CYP2E1 than phenobarbital-inducible CYP2B1. By contrast, CYP1A2, CYP2B4 and CYP2C4 were inefficient catalysts of radical formation. Desferrioxamine, catalase and superoxide dismutase did not influence the extent of ethyl radicals formed in EtOH-microsomes, indicating that hydroxyl radicals are not involved in the CYP2E1-dependent activation of 1-ethylhydrazine. Addition of cytochrome b5, an efficient donor of the second electron to P-450 and hence an inhibitor of the formation of the oxy-cytochrome P-450 complex, increased to be consistent with the results, did not influence the amount of ethyl radicals trapped. In liver microsomes from untreated rats selective substrates of CYP2E1, such as diethyl-dithiocarbamate and p-nitrophenol, as well as anti-CYP2E1-IgG, inhibited the free radical formation from 1-ethylhydrazine by about 60%. The anti-CYP2E1 IgG used significantly inhibited ethyl radical production also in human liver microsomes incubated with 1-ethylhydrazine and 4-POBN. Taken together, these results indicate that CYP2E1, as compared to other rat liver cytochromes P-450, is an efficient catalyst of transformation of alkylhydrazines to free radical intermediates, a finding that might be of importance in the development of the toxicity of these compounds.

Animals↗
PubMed1995

Effect of carrageenan-induced granuloma on hepatic cytochrome P-450 isozymes in rats.

Carrageenan-induced granuloma was used to study the apoprotein and RNA content, and catalytic activities of several cytochrome P-450 isozymes in liver. This model allowed discrimination between acute and chronic phases of experimental inflammation. The expression of most isozymes studied (CYP2D, CYP2E1, CYP3A1 and CYP4A) was reduced to 20% of the control level during the acute phase and partially recovered (30-60% of control group) during the chronic phase. CYP2B1 content was decreased to 65% of control during the acute and chronic phases of inflammation. RNA (CYP2B1 and CYP2E1) showed a strong depression during the acute phase and recovered during the chronic phase, without differences between isoenzymes. In most cases, there was a good correlation between the apoprotein content of isozymes and related activities. Our results show that the depletion of cytochrome P-450 induced by inflammation depends on the severity of the disease. Experimental inflammation equally affect the transcription of CYP2B1 and CYP2E1, so differences in apoprotein content and related activities between isozymes may due to differential posttranscriptional regulation.

Acute Disease↗
PubMed1995

Phenotyping and genotyping of S-mephenytoin hydroxylase (cytochrome P450 2C19) in a Shona population of Zimbabwe.

The S-mephenytoin hydroxylase has recently been identified as cytochrome P450 2C19 (CYP2C19). This enzyme metabolizes mephenytoin, diazepam, omeprazole, and citalopram and has been shown to be polymorphically distributed. One clinical implication of CYP2C19-dependent drug metabolism for persons who reside in tropical regions is in the use of the antimalarial drug chloroguanide hydrochloride, which is apparently biotransformed to its active metabolite by this isozyme. In this investigation we studied mephenytoin metabolism in 103 black Zimbabwean Shona subjects. Four were identified as poor metabolizers (4%). This prevalence is comparable to that in white subjects (2% to 5%) but lower than the 15% to 20% incidence of poor metabolizers among Oriental subjects. Of the subjects phenotyped, 84 were genotyped for the G-->A mutation in exon 5 of CYP2C19, which creates a cryptic splice site, causing the production of a nonfunctional protein. Three of the four poor metabolizers were homozygous for this mutation, whereas the fourth one was heterozygous. The G-->A mutation has been shown to predict the incidence more than 60% of poor metabolizers among white subjects and Japanese subjects, and in the current investigation we also obtained a similar relationship in the black population.

Adult↗
PubMed1995

CYP-2E1 inhibitors partially ameliorate the changes in hepatic fatty acid composition induced in rats by chronic administration of ethanol and a high fat diet.

The objective of this study was to determine if ethanol-induced cytochrome P450 2E1 (CYP2E1) was responsible for the changes in hepatic fatty acids observed in rats fed ethanol intragastrically. We hypothesized that if CYP2E1 was responsible for these changes then CYP2E1 inhibitors fed with ethanol should prevent the ethanol-induced changes in fatty acids. We compared the fatty acid composition of the liver in rats fed ethanol alone with that in rats fed ethanol with the CYP2E1 inhibitors, diallyl sulfide and phenethyl isothiocyanate. In each experiment, rats pair-fed isocaloric glucose were included to determine the effect of the inhibitors alone on the hepatic fatty acid composition. The lobular distribution of succinic dehydrogenase was determined histochemically because the lobular distribution of CYP2E1 shifts to the periportal area in livers of rats fed CYP2E1 inhibitors. The CYP2E1 inhibitors ameliorated both the ethanol-induced changes in fatty acids and the shift in succinic dehydrogenase. Rats fed ethanol but no inhibitors had significantly greater hepatic total fatty acids and triglyceride fractions than when inhibitors were fed ethanol. Ethanol altered the fatty acid composition compared with rats fed ethanol with CYP2E1 inhibitors. The ratio of 20:4/18:2 was significantly lower and that of 18:1/18:0 was greater in alcohol-fed rats compared with their pair-fed controls. The CYP2E1 inhibitors inhibited many of the above effects of alcohol. The data suggest that the changes in the fatty acid composition due to ethanol ingestion are the result of CYP2E1-dependent lipid peroxidation and fatty acid metabolism.

Allyl Compounds↗
PubMed1995

Genetic polymorphism of cytochromes P450 1A1, 2D6 and 2E1: regulation and toxicological significance.

Because of important roles of cytochromes P450 in the metabolic activation of many precarcinogens, extensive research in the past has focused on the relationship between the distribution of polymorphic variants of different isozymes of P450 and cancer susceptibility. In this respect three isozymes in particular have been studied, CYP1A1, CYP2D6, and CYP2E1. Both CYP1A1 and CYP2E1 participate in the metabolism of many suspected as well as established carcinogens, whereas essentially only one carcinogenic substrate has been identified for CYP2D6. Polymorphic sites for the three CYP genes have been identified both in the open reading frame as well as in introns and the regulatory 5' region. In the present contribution we summarize the molecular epidemiological research relating CYP polymorphism to cancer susceptibility and in some cases to toxicity. An interesting polymorphism has been described on the phenotypic level for the inducibility of CYP1A1, a polymorphism that in some studies has been related to a mutation in the 3' flanking region of the CYP1A1 gene. However, the genetic basis for this polymorphism might be inherited in the genes coding for proteins responsible for the induction of CYP1A1, ie, the Ah receptor or the ARNT protein. Data on lung cancer and CYP1A1 gene polymorphism indicate that carriers of genotypes associated with CYP1A1 inducibility are at higher risk for cancer, but that, at least for Caucasians, the recognized mutations probably identify only a fraction of the inducible individuals. The amount of DNA adducts correlates well in some studies to the individual activity registered for CYP1A1. CYP2D6 phenotype and genotype have mainly been related to the incidence of lung cancer, but results from 13 different studies now show an absence of any significant correlation between these parameters. In the case of CYP2E1, some studies indicate a relationship between lung cancer and the occurrence of a rare allele, although future research is needed in order to establish a significant relationship. It is concluded that, at the present stage, none of the polymorphic sites determined in the CYP genes can yet be used as markers for increased lung cancer risk. Future research in this field might be focused on the establishment of new polymorphic sites in the CYP genes, affecting inducibility or function, and on the molecular basis for the interesting differences in CYP1A1 inducibility.

Animals↗
PubMed1995

Phenethyl isothiocyanate, a new dietary liver aldehyde dehydrogenase inhibitor.

Phenethyl isothiocyanate (PEITC) is found in cabbages and other commonly ingested cruciferous vegetables. Isothiocyanates have anticarcinogenic properties, proposed to be mediated in part by their inhibition of several cytochrome P450 (CYP) forms. We administered PEITC to rats treated chronically with ethanol for 38 days by means of total enteral nutrition model to inhibit CYP2E1. We observed that PEITC not only efficiently prevented the ethanol-induced elevation of CYP2E1 apoprotein and mRNA, but also significantly elevated blood acetaldehyde levels. An elevation also was observed in naive animals acutely administered PEITC and ethanol, an effect found to be associated with marked inhibition of liver aldehyde dehydrogenase (ALDH). PEITC (1 mmol/kg) inhibited total liver ALDH activity by more than 70% and inhibition persisted for at least 24 hr. The inhibition was similar to that caused by an equimolar dose of disulfiram. Experiments using subcellular rat liver fractions revealed that both low- and high-Km ALDH forms were inhibited by low concentrations of PEITC (IC50 = 0.8-6.0 microM). Importantly, the mitochondrial low-Km ALDH activity, which is mainly responsible for detoxification of low aldehyde levels, was strongly inhibited (IC50 = 1.4 microM). In contrast, neither alcohol dehydrogenase nor lactate dehydrogenase activity was inhibited by PEITC. Thus, PEITC inhibits liver ALDH with a potency similar to that of disulfiram, suggesting that, in susceptible individuals, ingestion of large amounts of cruciferous vegetables in combination with alcohol could give rise to antabus-like symptoms. This property of PEITC must be taken into account in experimental alcohol research and in evaluating its proposed anticarcinogenic actions on chemical procarcinogens that are activated into potentially carcinogenic aldehydes.

Acetaldehyde↗
PubMed1995

Ultrarapid hydroxylation of debrisoquine in a Swedish population. Analysis of the molecular genetic basis.

Hydroxylation of debrisoquine, catalyzed by the cytochrome P450 CYP2D6 exhibits genetic polymorphism, with large inter-individual differences in metabolic capacity. About 7% of Caucasians carry deficient CYP2D6 alleles and lack the CYP2D6 enzyme (poor metabolizers). We have shown in two Swedish families, individuals carrying duplicated or amplified functional CYP2D6L-genes (CYP2D6L2), causing the opposite phenomenon, ultrarapid metabolism of debrisoquine. In the present study, the occurrence of extra copies of CYP2D6L-alleles was studied in relation to debrisoquine metabolic ratio (MR) in 270 Swedish Caucasians including 64 selected subjects with very rapid metabolism (MR < or = 0.2). Thirteen of the 64 subjects carried a duplicated CYP2D6-gene as identified by EcoRI and XbaI restriction fragment length polymorphism and allele-specific polymerase chain reaction-amplification of genomic DNA. A new allele with three active CYP2D6L-genes was identified, characterized by an XbaI 54 kilobase fragment. This indicates a preference of the CYP2D6L-gene to be amplified compared to other CYP2D6 genes. Only one subject with an MR higher than 0.2 carried the duplicated CYP2D6L-allele, also being heterozygous for the defect CYP2D6B-allele. The overall frequency of the duplicated/amplified CYP2D6-allele was about 1%, and was present in 40% of subjects with MRs < or = 0.1. Thus, other variant CYP2D6-genes may exist that cause increased CYP2D6 activity. In conclusion, a haplotype with duplicated or amplified functional CYP2D6 genes predicts, with high accuracy, ultrarapid metabolism of debrisoquine. Genotyping for this CYP2D locus variant might be of value in patients not responding to generally recommended doses of CYP2D6 substrates, to distinguish between high metabolic capacity and noncompliance.

Cytochrome P-450 CYP2D6↗
PubMed1995

Modulation of experimental alcohol-induced liver disease by cytochrome P450 2E1 inhibitors.

This study was done to determine if a relationship exists between CYP2E1 induction by ethanol, lipid peroxidation, and liver pathology in experimental alcohol-induced liver disease in the rat. Rats were fed ethanol with or without diallyl sulfide (DAS) or phenethyl isothiocyanate (PIC) intragastrically for 1 month. CYP2E1 induction by ethanol was correlated with lipid peroxidation, liver microsomal CYP2E1 hydroxylation of paranitrophenol, and the liver pathology score using the data from the PIC-fed rats. Some of the data from the ethanol and DAS-fed rats were not included here because they have been reported elsewhere. Microsomal CYP2E1 protein levels induction by ethanol was decreased by PIC ingestion. Similarly, PIC reduced the increase microsomal reduced form of nicotinamide-adenine dinucleotide (NADPH)-dependent lipid peroxidation and p-nitrophenol hydroxylase (PNPH) activity, induced by ethanol feeding. The lipid peroxidation was reduced to below control levels; however, the pathology score was partially but not significantly reduced by isothiocyanate feeding. CYP2E1 messenger RNA (mRNA) was decreased by both inhibitors of CYP2E1. Immunohistochemical staining of liver for CYP2E1 protein showed that the lobular distribution of the isozyme changed from the centrilobular to a diffuse pattern, with an increase in the periportal region when the CYP2E1 inhibitors were fed with ethanol, and that this change correlated with the change in the distribution of fat in the lobule. The data support the idea that there is a link between CYP2E1 induction by ethanol and the early phase of ethanol-induced liver injury in this rat model. This link may involve lipid peroxidation, but other factors related to CYP2E1 induction must also be involved.

Administration, Oral↗
PubMed1994

Induction, suppression and inhibition of multiple hepatic cytochrome P450 isozymes in the male rat and bobwhite quail (Colinus virginianus) by ergosterol biosynthesis inhibiting fungicides (EBIFs).

Ergosterol biosynthesis inhibiting fungicides (EBIFs) have complex effects on the hepatic microsomal monooxygenase systems of vertebrate species, having been described as mixed inducers and inhibitors of cytochrome P450. In the current study, we examined the effects of two EBIFs in clinical use, clotrimazole and ketoconazole, and two agricultural EBIFs, propiconazole and vinclozolin, on hepatic monooxygenase activities and P450 apoprotein expression in the male Sprague-Dawley rat and the male bobwhite quail. EBIFs produced Type II binding spectra with hepatic microsomes from both species and were effective inhibitors of methoxyresorufin O-demethylase, an activity selective for P450 isozymes in gene family 1. However, the EBIFs varied widely in their effectiveness as inducers of P450 isozymes in gene families 1, 2, 3 and 4, both within the same species and between species. In the rat, clotrimazole was the most effective inducer, increasing expression of CYP 3A isozymes over 450-fold, CYP 2B1/2 30-fold and CYP 1A1/2 12-fold and suppressing expression of CYP 2C11 nearly 70%. By contrast, in the quail, clotrimazole was the least effective inducer. In quail, vinclozolin and propiconazole elevated total P450 content 10- and 7-fold, respectively. The induction response also appeared to be mixed, but in this case consisted of a 5-fold induction of P450s in gene family 1A, a 3-fold induction of P450s in gene family 3A and 4A, and induction of protein(s) from gene family 2, cross-reactive with antisera against rat CYP 2C11 and CYP 2A1. A protein that was cross-reactive with antibodies raised against rat CYP 2B1 was decreased with EBIF treatment. In conclusion, EBIFs have complex patterns of induction, suppression and inhibition of cytochrome P450 isozymes in both mammals and birds, which vary according to both the fungicide and the species.

Animals↗
PubMed1994

Characterization of cytochrome P450 2E1 induction in a rat hepatoma FGC-4 cell model by ethanol.

The hepatic microsomal ethanol-oxidizing system (MEOS) has been well characterized as an important pathway in ethanol metabolism. Cytochrome P450 2E1 (CYP 2E1), the principal component of MEOS, is ethanol inducible and has been implicated in hepatotoxicity associated with alcohol abuse and exposure to organic solvents. Results of chronic in vivo experiments have shown that ethanol induction of hepatic CYP 2E1 occurs by a two-step mechanism. The first step of induction is associated with low blood alcohol concentrations (BACs) and appears to be post-transcriptional, whereas high BACs observed in step-two induction are associated with increased CYP 2E1 gene transcription. The mechanisms underlying these induction steps are under intense investigation. Progress in this area has been limited due to lack of hepatic cell culture models that express CYP 2E1. We report here an in vitro tissue culture cell model, the FGC-4 hepatoma cell line, that exhibits basal levels of CYP 2E1 apoprotein that are inducible by ethanol treatment. Total cellular RNA and microsomal fractions were isolated from control or ethanol-treated confluent cells, and CYP 2E1 mRNA and apoprotein levels were characterized by northern blot or immunoblot analysis, respectively. Initial experiments on isolated microsomes revealed detectable levels of CYP 2E1 apoprotein in control cells that were induced 5-fold in cells treated with 100 mM ethanol for 24 hr. Concentration-response experiments demonstrated that the maximal 24-hr induction in CYP 2E1 apoprotein level was 5-fold and was attained at a concentration of 10 mM ethanol. Interestingly, while the steady-state mRNA levels encoding CYP 2E1 were detectable, they remained unchanged in identically treated cells. Furthermore, there was no observed increase in CYP 2E1 mRNA levels in an extended time course to 72 hr or at higher alcohol concentrations (up to 1500 mM), providing preliminary evidence that the induction is post-transcriptional. The time course of CYP 2E1 apoprotein induction by exposure to 100 mM ethanol demonstrated maximal induction at 8 hr. Measurement of CYP 2E1 apoprotein levels after removal of ethanol from pretreated cells demonstrated the half-life of the apoprotein to be 12.7 hr, in good agreement with previous reports using primary hepatocytes. The half-life of the induced protein after ethanol removal in the presence of cyclohexamide (10 micrograms/mL) was biphasic with a rapid 1.8 hr first phase followed by a slower 44.7 hr second phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Substrate-regulated, cAMP-dependent phosphorylation, denaturation, and degradation of glucocorticoid-inducible rat liver cytochrome P450 3A1.

The major rat glucocorticoid-inducible cytochrome P450 (CYP3A1) is known to be regulated at a transcriptional level by glucocorticoids and at a post-translational level by substrate-dependent stabilization. We have investigated mechanisms of substrate/ligand stabilization using primary hepatocytes, isolated liver microsomes from dexamethasone-treated rats, and purified enzymes. Treatment of hepatocytes with glucagon caused a 3-fold increase in CYP3A1 phosphorylation as well as an enhanced degradation rate of the enzyme. Specific CYP3A1 substrates or ligands, such as erythromycin, triacetyloleandomycin, and clotrimazole (CTZ) protected the enzyme from degradation in hepatocytes and inhibited in a concomitant manner (r = 0.99) glucagon-induced phosphorylation of the enzyme. In vitro experiments with purified CYP3A1 and isolated liver microsomes revealed one major site (Ser393) phosphorylated by the catalytic subunit of cAMP-dependent kinase, a reaction inhibited by ligands. Experiments in microsomes showed the presence of an endogenous cAMP-dependent kinase active on CYP3A1. Addition of exogenous cAMP-dependent kinase increased the rate of microsomal CYP3A1 phosphorylation, a reaction further stimulated by NADPH, but inhibited by CTZ. The microsomal phosphorylation caused a pronounced denaturation of cytochrome P450, as revealed spectrophotometrically, whereas CTZ protected from this reaction. Similar effects were noted when the CYP3A1-dependent 6 beta-hydroxylation of testosterone was monitored. It is suggested that the cellular CYP3A1 level is regulated to a significant extent posttranslationally by substrate-regulated cAMP-dependent phosphorylation on Ser393, followed by denaturation and degradation in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1994

Genetic polymorphism of cytochrome P450. Functional consequences and possible relationship to disease and alcohol toxicity.

The hepatic cytochrome P450 system participates in the oxidative metabolism of numerous endogenous and exogenous compounds. In total several hundred different P450s have been cloned, but it appears that in humans only about 5-10 isoforms account for the major part of drug metabolism. Some of these are polymorphically distributed in the population. Cytochrome P450 2D6 catalyzes the oxidation of over 25 clinically important drugs, eg neuroleptics, antidepressants and lipophilic beta-blockers. Seven % of Caucasians and 1% of Orientals are defective in this enzyme and clearance of drugs metabolized by the enzyme may be substantially decreased in these individuals, with potentially increased risks for side effects caused by the drug treatment. Some individuals are ultrarapid metabolizers and do not achieve therapeutic drug levels at ordinary doses. The molecular genetic basis of these polymorphisms are presented. Methods for genotyping, which can be of predictive value for a more efficient drug therapy, are discussed. Ethanol-inducible cytochrome P450 2E1 (CYP2E1) oxidizes ethanol and acetaldehyde, in addition to over 80 toxicologically important xenobiotics. Furthermore, this isozyme produces reactive oxy radicals which are implicated in the aetiology of alcoholic liver disease. The gene is polymorphic and a mutation in a putative binding site for HNF1, described to affect gene expression, is more rare among subjects with lung cancer as compared to healthy controls. Further studies might give an answer as to whether any of the polymorphic CYP2E1 alleles is associated with the sensitivity to obtain alcoholic liver disease.

Animals↗
PubMed1994

Fish oil, alcohol, and liver pathology: role of cytochrome P450 2E1.

Rats were fed ethanol in combination with fish oil or a corn diet in order to evaluate the effect of fish oil feeding on liver injury, microsomal ethanol oxidation, and NADPH-dependent lipid peroxidation. The rats were maintained on the dietary regimen for 72 days, and for comparison, pair-fed controls were studied. The liver pathology score progressively worsened in rats fed alcohol, both in combination with fish oil and corn oil, but the severity of inflammation and focal fibrosis was greater in the ethanol fish oil fed rats as compared with the ethanol corn oil group, whereas the fatty change was greater in the ethanol corn oil fed rats. The alcohol treatment caused a 2-fold increase of the liver microsomal P450 content, and about a similar increase in the rate of microsomal NADPH oxidation. The amount of ethanol-inducible CYP2E1 was about 10-fold higher in alcohol-fed rats as compared with pair-fed controls. The NADPH-dependent lipid peroxidation in liver microsomes was about 10-fold higher in microsomes from alcohol-treated rats fed corn oil as compared with controls, but only 2- to 3-fold higher in alcohol-fed rats receiving fish oil than in pair-fed controls. This was due to a higher rate of NADPH-dependent lipid peroxidation in the control rats receiving fish oil. There was a pronounced correlation between the amount of CYP2E1 and the microsomal NADPH peroxidation in variously treated rats, and between the 2E1 levels and the pathology score. The data suggest that fish oil diet, like corn oil, supports ethanol-induced liver injury which is related to CYP2E1 induction and the presence of polyunsaturated fatty acids in the diet (i.e., either n-6 or n-3).

Alanine Transaminase↗
PubMed1994

Genetic analysis of the Chinese cytochrome P4502D locus: characterization of variant CYP2D6 genes present in subjects with diminished capacity for debrisoquine hydroxylation.

Cytochrome P4502D6 (CYP2D6) catalyzes the oxidative metabolism of several clinically important classes of drugs. Many of these have lower metabolic clearance rates among Chinese, compared with Caucasians, and are prescribed at lower doses for Asian patients. We have now evaluated the molecular genetic basis for this interethnic difference in drug metabolism. The CYP2D loci from two Chinese subjects, one homozygous for the XbaI 44-kilobase haplotype and one homozygous for the XbaI 29-kilobase haplotype, were cloned and characterized. Sequence analysis revealed two variant CYP2D6 genes, CYP2D6Ch1 and CYP2D6Ch2, having mutations yielding two and eight amino acid substitutions, respectively. Exon 9 of the CYP2D6Ch2 gene contained a sequence of 49 bases originating from the pseudogene CYP2D7P. In addition, mutations in the 5' flanking region common to both CYP2D6Ch genes were found. To evaluate the origin of the detrimental mutation in the genes, parts of the 5' flanking regions were introduced into a Hep G2/simian virus 40 expression system with chloramphenicol acetyltransferase as a reporter gene, and transfected cells were analyzed for activity. The ability of the upstream regions to bind nuclear factors was also evaluated using gel-shift analysis. Furthermore, several chimeric constructs of the CYP2D6wt and CYP2D6Ch genes were made, inserted into pCMV2 vectors, and expressed in COS-1 cells. A part of the upstream region of base pairs -1407 to -1068 was found to constitute an enhancer element, but the CYP2D6Ch-specific mutations did not influence the chloramphenicol acetyltransferase activity in the expression system. In contrast, expression of the chimeric genes revealed that the detrimental mutation of the CYP2D6Ch genes was C188-->T, causing a Pro34-->Ser amino acid substitution in a region that is a highly conserved in cytochromes P450 belonging to gene families 1 and 2. This substitution caused expression of a more unstable gene product, as evident from comparison of the relative levels of CYP2D6 mRNA, CYP2D6 protein, and bufuralol 1'-hydroxylase activities in pCMV2-CYP2D6-transfected COS-1 cells. Allele-specific polymerase chain reaction analysis of genomic DNA from 90 Chinese individuals revealed that the CYP2D6Ch1 allele was the most common one and its distribution correlated well with a higher metabolic ratio for debrisoquine. These data demonstrate that important interethnic differences exist in the structure of the CYP2D locus, and they suggest that the frequent distribution of the C188-->T mutation among the CYP2D6Ch genes explains the lower capacity among Chinese to metabolize drugs that are substrates of CYP2D6, such as antidepressants and neuroleptic agents.

Asian People↗
PubMed1994

Chlormethiazole as an efficient inhibitor of cytochrome P450 2E1 expression in rat liver.

Ethanol-inducible cytochrome P450 (CYP) 2E1 (CYP2E1) is responsible for the metabolism of many xenobiotics which exert toxic effects in humans. Specific inhibitors might constitute valuable tools in the elucidation of the pharmacological and toxicological roles of this isozyme in vivo. In the present investigation we have evaluated the effects of a drug used for treatment of ethanol withdrawal states, chloromethiazole (CMZ), on CYP2E1 expression in rat liver. A 4-fold induction of CYP2E1 was observed after 3 days of starvation, accompanied by a similar increase in the level of the corresponding mRNA. CMZ specifically inhibited the elevation of CYP2E1 mRNA and protein, but did not prevent CYP2B1 and CYP3A1 or CYP1A1 induction caused by treatment with phenobarbital or beta-naphthoflavone, respectively. From nuclear run-off experiments it was apparent that the rate of the CYP2E1 gene transcription was inhibited greatly by CMZ treatment. Rats treated with ethanol in a total enteral nutrition model had higher CYP2E1-dependent hepatic microsomal activities of p-nitrophenol hydroxylase and carbon tetrachloride-induced lipid peroxidation than controls, and simultaneous CMZ treatment abolished the ethanol-dependent induction. In vitro experiments with rat liver microsomes showed that CMZ did not act as an inhibitor of CYP2E1-dependent catalytic activities or as an inhibitor of microsomal NADPH and CYP2E1-dependent lipid peroxidation. In conclusion, we suggest that CMZ might constitute an efficient and specific inhibitor of CYP2E1 expression suitable for in vivo experiments.

Animals↗