Genetic Polymorphism of Cytochrome P-4502E1 and Mitochondrial Aldehyde Dehydrogenase in a Korean Population

Mitochondrial aldehyde dehydrogenase (ALDH2) is mainly responsible for the oxidation of acetaldehyde generated during alcohol oxidation in vivo. Cytochrome P-4502E1 (CYP2E1), a liver microsomal enzyme, also metabolizes acetaldehyde and ethanol. Genetic polymorphism of ALDH2 and CYP2E1 was investigated among 481 Korean adults. A new restriction fragment-length polymorphism method was developed to determine the genotype of the ALDH2 alleles. This method proved to be simpler and faster than the hybridization method using allele-specific oligonucleotide probes and polymerase chain reaction-directed mutagenesis. The allele frequencies of ALDH2¹ and ALDH2² were 0.840 and 0.160, respectively. This allele frequency of ALDH2² is less than in Japanese people. Genetic polymorphism of CYP2E1 was investigated using polymerase chain reaction and restriction fragment-length polymorphism. The estimated allele frequencies for C1 and c2 were 0.808 and 0.192.     

Respective Roles of Human Cytochrome P-4502E1, 1A2, and 3A4 in the Hepatic Microsomal Ethanol Oxidizing System

The microsomal ethanol oxidizing system comprises an ethanolinducible cytochrome P-4502E1, but the involvement of other P-450s has also been suggested. In our study, human CYP2E1, CYP1A2, and CYP3A4 were heterologously expressed in HepG2 cells, and their ethanol oxidation was assessed using a corresponding selective inhibitor: all three P-450 isoenzymes metabolized ethanol. Selective inhibitors-4-methylpyrazole (CYP2E1), furafylline (CYP1A2), and troleandomycin (CYP3A4)?also decreased microsomal ethanol oxidation in the livers of 18 organ donors. The P-450-dependent ethanol oxidizing activities correlated significantly with those of the specific monooxygenases and the immunochemically determined microsomal content of the respective P-450. The mean CYP2E1-dependent ethanol oxidation in human liver microsomes [1.41 ± 0.11 nmol min^-1 (mg protein)^-1] was twice that of CYP1A2 (0.61 ± 0.07) or CYP3A4 (0.73 ± 0.11) (p < 0.05). Furthermore, CYP2E1 had the highest (p < 0.05) specific activity [28 ± 2 nmol min^-1 (nmol CYP2E1)^-1 versus 17 ± 3 nmol min^-1 (nmol CYP1A2)^-1, and 12 ± 2 nmol min^-1 (CYP3A4)^-1, respectively]. Thus, in human liver microsomes, CYP2E1 plays the major role. However, CYP1A2 and CYP3A4 contribute significantly to microsomal ethanol oxidation and may, therefore, also be involved in the pathogenesis of alcoholic liver disease.     

Immunohistochemical Determination of Hepatic Cytochrome P-4502E1 in Formalin-Fixed, Paraffin-Embedded Sections

Cytochrome P-4502E1 (2E1) is inducible by chronic ethanol consumption that results in enhanced activation of anesthetics and commonly used drugs (such as acetaminophen) to hepatotoxins. Therefore, assessment of hepatic 2E1 is needed in prescribing these drugs for the management of alcoholic patients. Currently, measurement of 2E1 requires either immunohistochemistry on frozen sections or Western blot (WB) analysis of homogenized tissue in excess of that needed for pathology. To obtain a more widely applicable method, we developed a procedure to detect 2E1 by immunohistochemistry in formalin-fixed, paraffin-embedded liver biopsies obtained routinely for diagnosis. Data were collected from rats fed ethanol-containing or control liquid diets for 3 weeks. lmmunostaining was performed using anti-human rabbit 2E1 antibody as the primary antibody, and the immunoreaction was detected by the avidin-biotin immunoperoxidase method after treating sections with target unmasking fluid, an antigen retrieval buffer that enhanced the staining of 2E1. In control rats, 2E1 staining was weak and perivenular. After ethanol feeding, it showed a lobular gradient, strongest perivenular and weakest periportal, similar to that seen in frozen sections. The staining intensity was scored as: 0 (no staining) to 3 (strong staining). The zonal staining was scored as follows: 1 = perivenular zonal staining, 2 = midzonal, and 3 = panlobular. With the product of the two scores, a significant difference was found between alcohol-fed and control rats (5.1 ± 0.3 vs. 0.8 ± 0.2, p < 0.001). 2E1 assessments by WB were also significantly different for these rat pairs (68.5 ± 2.1 vs. 7.9 ± 0.8 arbitrary units/mg protein, p < 0.001), with a parallel increase of immunostaining scores and WB measurement of 2E1 content. This immunohistochemical method was then validated in 14 paraffin-embedded percutaneous human liver biopsy samples. In livers of nonalcoholics, 2E1 staining was seen in the perivenular zone only, whereas in samples of alcoholics, the staining was perivenular to midzonal and sometimes periportal. A significant correlation between the zonal staining scores (r_s= 0.67, p < 0.005) or intensity ± zonal staining scores (r_s= 0.79, p < 0.001) and WB analysis was found. The immunohistochemical assessments of 2E1 expression in formalin-fixed, paraffin-embedded sections from livers of alcoholics was found to correlate with WB analysis, and lobular distribution was consistent with that seen in frozen sections. The proposed method should therefore be useful for the assessment of 2E1 content in paraffin-embedded liver samples, thereby aiding in the management of heavy drinkers.     

Evidence that Cytochrome P-4502E1 Contributes to Ethanol Elimination at Low Doses: Effects of Diallyl Sulfide and 4-Methyl Pyrazole on Ethanol Elimination in the Perfused Rat Liver

The roles of cytochrome P-4502E1 and alcohol dehydrogenase (ADH) on ethanol (EtOH) hepatic elimination was examined in the perfused rat liver. EtOH concentration-time curves of outflow after instantaneous administration (0.46 mg) through the portal vein with or without perfusion of diallyl sulfide (DAS), a selective cytochrome P-4502E1 inhibitor, and/or 4-methyl pyrazole (4-MP), a classical ADH inhibitor, were analyzed by the statistical moment analysis and the compartment dispersion model. Recovery ratios obtained by moment analysis significantly changed with perfusion of inhibitors ( p < 0.01). Values of the hepatic volume of distribution and the relative dispersion were significantly higher by the perfusion of DAS and 4-MP ( p < 0.01). In the two-compartment dispersion model, the partition ratio ( K ') and the first-order elimination constant ( K _e) were decreased significantly by DAS ( p < 0.05). By the addition of 4-MP, the blood volume of distribution ( V _B ) and the backward partition rate constant ( k ₂₁) were increased significantly ( p < 0.05). K _e values were decreased significantly to 0 ( p < 0.001). The decrease of elimination rates by DAS and/or 4-MP shows the inhibition of metabolic pathways. The change of V _B and k ₂₁ caused by DAS and 4-MP indicates that EtOH taken into hepatic tissues was not metabolized and flowed out into the perfusates. Inhibition rates calculated from the efficiency number with addition of DAS and DAS + 4-MP were 40.7 and 99.3%. Therefore, cytochrome P-4502E1 and ADH accounted for 40 and 60% of the hepatic EtOH elimination at low doses.     

Induction of Cytochrome P-4502E1 by Ethanol in Rat Kupffer Cells

Ethanol has been shown to affect several Kupffer cell functions, but the mechanisms underlying these changes are unknown. One possible mediator is cytochrome P-4502E1 (CYP2E1), an ethanol-inducible enzyme that has been associated with toxic effects in the liver, as well as in many extrahepatic organs. To assess whether CYP2E1 can be induced by ethanol in Kupffer cells, male rats pair-fed ethanol-containing or control Lieber-DeCarli diets for 3 weeks were studied. Immunoblotting experiments showed that ethanol-treatment caused a 7-fold increase in CYP2E1 content both in Kupffer cells and hepatocytes. When expressed per milligram of S9 protein, the content of CYP2E1 in Kupffer cells was, however, 10 times lower than in hepatocytes. Immunohistochemical studies revealed that CYP2E1 is located in the endoplasmic reticulum of Kupffer cells in vivo and that it is also present in isolated Kupffer cells. In both Kupffer cells and hepatocytes, ethanol feeding increased the hydroxylation of p -nitrophenol, a relatively specific substrate for CYP2E1, demonstrating that the induced CYP2E1 was catalytically active. This reaction was significantly inhibited by anti-CYP2E1 IgG in both types of cells. Although CYP2E1 may not be the predominant pathway for ethanol metabolism in hepatocytes, it is possibly the major one in Kupffer cells. Thus, the induction of CYP2E1 by ethanol in these cells could cause significant changes in intracellular acetaldehyde concentrations which, together with increased lipid peroxidation, may contribute to the development of alcoholic liver injury.     

Characteristics of Japanese Alcoholics with the Atypical Aldehyde Dehydrogenase 2*2.I. A Comparison of the Genotypes of ALDH2, ADH2, ADH3, and Cytochrome P-4502E1 Between Alcoholics and Nonalcoholics

We examined the genotypes of the aldehyde dehydrogenase (ALDH)-2 , alcohol dehydrogenase (ADH)-2, ADH3 , and P-4502E1 loci of 53 alcoholics and 97 nonalcoholics. All of the subjects fulfilled the DSM-III-R criteria for alcohol dependence. The control group consisted of 97 subjects who were either hospital staff or students. We also compared the frequencies of homozygous ALDH2*1/1 and heterozygous ALDH2*1/2 genotypes in alcoholics. Our study revealed differences in the allelic frequencies of the ALDH2, ADH2 , and ADH3 loci between alcoholics and nonalcoholics. For alcoholics with both homozygous ALDH2*1/1 and heterozygous ALDH2*1/2 genotypes, it was found that ADH2 and ADH3 played important roles. Alcoholics with the heterozygous ALDH2*1/2 genotype showed a significantly higher frequency of ADH2*1/1 than ones with the homozygous ALDH2*1/1 genotype. We assume ADH2*1 plays an important role in the development of alcoholism in alcoholics with the heterozygous ALDH2*1/2 genotype.     

Decrease in Cytochrome P4502E1 as Assessed by the Rate of Chlorzoxazone Hydroxylation in Alcoholics during the Withdrawal Phase

To evaluate cytochrome P4502E1 (CYP2E1) induction in alcoholics, the ratio of the concentrations of 6-hydroxychlorzoxazone (6-OH-CHZ) and chlorzoxazone (CHZ) was measured in blood 2 hr after CHZ ingestion using a HPLC method. This ratio was determined in controls and in alcoholic patients after 1, 2, 3, 4, 5, 8, and 21 days withdrawal. It was found to be 0.34 ± 0.03 in 30 controls and 1.05 ± 0.14 in 41 alcoholic patients within 2 days following ethanol withdrawal. This ratio decreased rapidly during withdrawal as attested by the short half-life of CYP2E1, which was found to be 2.5 days. Patients tested for CHZ metabolism after 8 or 21 days alcohol abstinence displayed the same ratio as controls [0.35 ± 0.03 ( n = 28) and 0.31 ± 0.03 ( n = 34), respectively]. No correlation was observed between γ-glutamyltransferase, carbohydrate-deficient transferrin values, the amount of alcohol consumed/day, and the 6-OH-CHZ/CHZ ratio. There was no influence of smoking on the rate of CHZ hydroxylation, because smokers displayed the same ratio as nonsmokers [0.33 ± 0.025 ( n = 62) and 0.33 ± 0.02 ( n = 30), respectively]. The CHZ hydroxylation ratio seems to be a good reflection of the hepatic and extrahepatic CYP2E1 activity in humans.     

Acetaminophen Metabolism in Patients with Different Cytochrome P-4502E1 Genotypes

Cytochrome P-450 (CYP) 2E1 is the major ethanol-oxidizing enzyme of the nonalcohol dehydrogenase metabolic pathway in the liver. Recently, the presence of genetic polymorphisms of this enzyme was confirmed. In this study, to clarify the influence of CYP2E1 genotype on alcohol metabolism, we analyzed acetaminophen metabolism in subjects with different CYP2E1 genotypes. In normal subjects, a half-life of acetaminophen from blood was the longest in type A ( c1/c1 ) and was the shortest in type C ( c2/c2 ). The elimination rate in type C was more than twice that of type A and type B ( c1/c2 ). In type A, both half-life and elimination rate of acetaminophen were not different between patients with noncirrhotic alcoholic liver disease within 1 week after abstinence and in normal subjects. In one patient with minimal change, there were no differences in both half-life and elimination rate within 1 and 6 weeks after abstinence. On the other hand, in type B, half-life was shorter and the elimination rate was greater in alcoholic noncirrhotic patients within 1 week after abstinence than in alcoholic patients with type A and in normal subjects with type B. In type B, half-lives were shorter, and the elimination rates were greater in patients with alcoholic liver disease within 1 week after abstinence than 4 to 6 weeks after abstinence. These results suggest the possibility that alcohol metabolism in individuals with the c2 gene may be greater than those with the c1 gene, and that the induction of CYP2E1 by ethanol in type B may occur more markedly than that in type A, although the sample number is too small to obtain final conclusions.     

Polyenylphosphatidylcholine opposes the increase of cytochrome P-4502E1 by ethanol and corrects its iron-induced decrease

Dietary iron overload damages membrane phospholipids and decreases microsomal cytochromes P-450. We wondered whether this might also pertain to cytochrome P-4502El (2E1) and whether polyenyiphosphatidylcholine (PPC), a 94-96% pure mixture of linoleate-rich polyunsaturated phosphatidylcholines that protects against alcohol-induced liver injury, also affects 2E1, either in the presence or absence of iron. Accordingly, rats were fed for 8 weeks our standard liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either PPC (3 g/1000 Cal) or equivalent amounts of linoleate (as safflower oil). 2E1 was assessed by Westem blots and by two of its characteristic enzyme activities: the microsomal ethanol oxidizing system (MEOS), evaluated by the conversion of ethanol to acetaldehyde (determined by head space GC), and p-nitrophenolhydroxylase (PNP) activity, measured by HPLC with W detection of 4nitrocatechol. With ethanol (36% of energy) replacing carbohydrates, 2E1 content increased 10-fold, with a corresponding increase in PNP and MEOS activities, but when carbonyl iron (5 g/1000 Cal) was added, the induction was significantly reduced. This iron-induced decrease was corrected by PPC. PPC is rich in linoleate, but when the latter was given as triglycerides (safflower oil), there was no effect, whereas hepatic nonheme iron content was the same in both these groups. It also was found that in the absence of iron, the ethanol-mediated induction of 2E1 and its corresponding enzyme activities were significantly less with PPC ( p < 0.001) than with safflower oil. In addition, in alcohol-fed animals, PPC decreased the oxidative stress (as determined by F₂-isoprostanes), which reflects yet another hepatoprotective effect of PPC.     

Use of Lymphocytes for Assessing Ethanol-Mediated Alterations in the Expression of Hepatic Cytochrome P4502E1

The ethanol-inducible cytochrome P4502E1 (2E1) is involved in the bioactivation of numerous hepatotoxins and hepatocarcinogens. Because high levels of expression may enhance the degree and severity of hepatotoxicity from exposure to chemicals metabolized by this enzyme, a relatively noninvasive method to phenotypically distinguish those individuals exhibiting elevated concentrations of 2E1 may be useful. With this in mind, we examined whether ethanol exposure could alter 2E1 in rabbit white blood cells and liver in a similar manner. Microsomes prepared from freshly isolated, rather than cultured cells, were used to immunochemically detect 2E1. The enzyme was found in lymphocytes and neutrophils. Lymphocytes, which comprise the majority of the white cell population in rabbits, were monitored for changes in 2E1 protein levels after ethanol exposure and compared with alterations of the hepatic enzyme. Results presented herein demonstrate that the degree of enhancement in 2E1 expression of lymphocytes and liver was dependent on the length and dose of alcohol exposure. Indeed, correlations were observed between blood alcohol concentrations and 2E1 content in lymphocytes (r= 0.65, p < 0.01) and liver (r= 0.60, p < 0.01). The greatest increase in 2E1 (6- to 10-fold) occurred in both liver and lymphocytes at a dose of 15% ethanol for 12 days of treatment. This induction was evident regardless of whether blood was taken from treated and compared with untreated rabbits or if white cells were obtained from the same animal before and after ethanol exposure. The latter findings demonstrate that changes in lymphocyte 2E1 were caused by ethanol exposure and not to variability in enzyme expression among rabbits. Interestingly, at the 10% dose, elevation of 2E1 was noted as early as 3 days, declined at 6 days, and at 12 and 24 days returned to slightly higher levels than those seen at the 3-day exposure period. This pattern of 2E1 elevation was observed in both the liver and lymphocytes, in fact, at all exposure periods and at the two doses of alcohol examined, a correlation (r= 0.70, p < 0.01) was observed between lymphocyte and liver 2E1 content. Collectively, these studies show that induction of 2E1 in lymphocytes and liver occurs in a parallel fashion. Furthermore, results suggest that blood 2E1 may be used in humans as a phenotypic marker for xenobiotic-promoted alterations in the expression of the liver enzyme. These findings should have a significant impact on in vivo monitoring of this P450 enzyme.     

Ethanol-induced apoptosis to stable HepG2 cell lines expressing human cytochrome P-4502E1

In a previous study (Wu and Cederbaum, J. Biol. Chem. 271:23914-23919, 1996), ethanol was shown to be cytotoxic to HepG2 cells, which were transduced to express human cytochrome P-4502E1 (CYP2E1) but not to control HepG2 cells. The goal of the current study was to evaluate whether this toxicity was apoptotic in nature. Incubation of CYP2E1-expressing HepG2 cells with 100 mM ethanol for 2 days produced morphological changes and DNA fragmentation (in situ labeling, flow cytometry, and DNA ladder formation) indicative of apoptosis. No changes were observed in the control HepG2 cells that do not express CYP2E1. Ethanol-induced apoptosis was also observed in HepG2 cells transiently transfected to express CYP2E1. The ethanol-induced apoptosis was prevented by 4-methylpyrazole, an inhibitor of ethanol oxidation by CYP2E1, and by trolox, an antioxidant that prevents lipid peroxidation. Ethanol treatment of the cells expressing CYP2E1 resulted in increased activities of caspases 1 and 3. An inhibitor of these caspases prevented the ethanol-induced apoptosis in the stable cell lines and the transiently transfected cell lines. Ethanol did not cause apoptosis in a HepG2 cell line overexpressing bcl-2 plus CYP2E1, but did cause apoptosis in cell lines expressing CYP2E1 in the absence of bcl-2. These experiments demonstrate that ethanol can produce apoptosis in HepG2 cells that express CYP2E1. Increased production of reactive oxygen species and lipid peroxidation can be associated with apoptotic cell death. The prevention of the ethanol-induced apoptosis by 4-methylpyrazole and by trolox suggests that production of a prooxidative state as a consequence of ethanol oxidation by CYP2E1 results in eventual activation of caspases such as caspases 1 and 3, which can trigger the apoptotic process.     

Genetic Polymorphism of Aldehyde Dehydrogenase 2 in Patients With Upper Aerodigestive Tract Cancer

Background : Alcohol consumption is one of the major risk factors of the upper aerodigestive tract (UADT) cancers, and combined cancers are frequently discovered in the patients with UADT cancer. The association between esophageal cancer and alcohol-related metabolizing enzymes is well studied, but only a few examinations about the association between head and neck cancer and the enzymes were performed.
Methods : Fifty-two patients with UADT cancer (head and neck cancer in 25, esophageal cancer in 19, and multiple cancers in 8) were examined in the alcohol habit and in the polymorphisms of aldehyde dehydrogenase 2 (ALDH2) and cytochrome P-4502E1.
Results : Patients with multiple cancers had significantly higher ethanol consumption than the other two groups ( p < 0.001). The frequency of ALDH2* 1/2*2 heterozygote was significantly lower ( p = 0.009) in patients with head and neck cancer (5/25) than patients with esophageal cancer (11/19). The allele frequency of P-4502E1 did not show a significant difference between the groups ( p = 0.700).
Conclusions : These results demonstrated the difference in the frequency of ALDH2 heterozygote between the patients with esophageal cancer and patients with head and neck cancer.     

Effect of the Cytochrome P-1450IIE1 Genotype on Ethanol Elimination Rate in Alcoholics and Control Subjects

We studied an influence of genetic polymorphisms in the cytochrome P-450IIE1 (CYP2E1) gene on ethanol elimination rate in alcoholic patients and healthy subjects. The CYP2E1 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism method for 124 alcoholics and 54 healthy subjects. There was no significant difference in the gene frequency of CYP2E1 between alcoholics and healthy control subjects. Blood ethanol concentrations in the 65 alcoholics on admission ranged from 0.32 to 4.22 mg/ml. In the patients with the c1/c2 genotype, the elimination rate was significantly correlated with blood ethanol concentration. In each of the three genotypes of CYP2E1, the patients were divided into three groups based on ethanol concentrations. The average of the ethanol elimination rate in the patients with c1/c2 having blood ethanol levels of ≧2.5 mg/ml was significantly higher than the rates in the two other groups of c1/c2. When blood ethanol levels were ≧2.5 mg/ml, the elimination rate in the patients with c1/c2 was significantly higher than that in those with c1/cl. Regardless of the CYP2E1 genotype, the elimination rate in the alcoholics was higher than that in the control subjects when blood ethanol levels were < 1.0 mg/ml. These results suggest the possibility that the c2 allele of CYP2E1 influences the rate of ethanol elimination at high ethanol levels. The rate of ethanol elimination was independent of liver disorder judged by serum total bilirubin values.     

Enzymic catalysis of the accumulation of acetaldehyde from ethanol in human prenatal cephalic tissues: evaluation of the relative contributions of CYP2E1, alcohol dehydrogenase, and catalase/peroxidases

BACKGROUND: The human prenatal brain is very sensitive to the toxic effects of ethanol, but very little information is available concerning the conversion of ethanol to the highly cytotoxic metabolite, acetaldehyde, in that organ. Thus, experiments were designed to investigate rates of accumulation of acetaldehyde from ethanol in the prenatal human brain. METHODS: Prenatal human cephalic tissue homogenates were used as enzyme sources and were compared with analogous preparations of adult rat livers. Generated acetaldehyde was derivatized with cyclohexane-1,3-dione to yield fluorescent decahydroacrizine-1,8-dione, which was readily separated, detected, and quantitated with HPLC. RESULTS: Detected rates of accumulation were unexpectedly high, even in the absence of added NADPH, NAD+, or H2O2, which are cofactors/cosubstrates for cytochrome P-450-, alcohol dehydrogenase- and catalase/peroxidase-catalyzed reactions, respectively. Without added cofactors/cosubstrates or other components and under linear reaction conditions, rates in human prenatal cephalic preparations were approximately 20% of those observed with analogous preparations of adult rat livers. Cofactor/cosubstrate-independent reactions were localized in the cytosolic (soluble) fraction and were strongly dependent on molecular oxygen (O2). They were not inhibited substantially by carbon monoxide (CO:O2 = 80:20 vs N2:O2 = 80:20) or by pyrazole in concentrations up to 10 mM and were only weakly inhibited by azide. Preincubations with excess catalase did not result in decreased activity. Reactions exhibited substrate saturation and heat inactivation indicating enzymic catalysis. CONCLUSIONS: Experiments indicated a relatively rapid accumulation of acetaldehyde from ethanol in human prenatal brain tissues and suggested that the observed cofactor/cosubstrate-independent reactions were largely independent of P-450 cytochromes, alcohol dehydrogenases, or catalase/peroxidases. Results were consistent with catalysis by an as yet unidentified cytosolic oxidase(s).     

Changes in Microsomal Phospholipases and Arachidonic Acid in Experimental Alcoholic Liver Injury: Relationship to Cytochrome P-450 2E1 Induction and Conjugated Diene Formation

We evaluated the role of changes in microsomal phospholipases (A and C) and arachidonic acid in the intragastric rat feeding model. The experimental animals (male Wistar rats), divided into 4–5 rats/group, were fed the following diets: corn oil and ethanol and corn oil plus dextrose. One set of groups was killed after 2 weeks of feeding, and the second set was killed after 1 month. For each animal, microsomal analysis of cytochrome P-450 2E1 (CYP 2E1) and fatty acids was done. Fourteen animals had analyses of phospholipase C (PLC) and phospholipase A (PLA), and 10 animals had measurements of conjugated dienes. A significant correlation was obtained between the level of CYP 2E1 and the decrease in arachidonic acid (AA) from baseline levels ( r = 0.69, p < 0.01). The decrease in AA also correlated with the increase in conjugated dienes ( r = 0.70, p < 0.05). PLA and PLC activities were both significantly increased in the corn oil and ethanol groups. The activity of PLC correlated with the decline in AA ( r = 0.69, p < 0.01). The correlations noted between the decrease in microsomal AA and CYP 2E1 induction and conjugated diene formation suggest that these processes may be interlinked especially in regard to generation of lipid peroxides that may play a role in alcoholic liver injury.     

Inducibility of Cytochromes P-4502E1 and P-4501A1 in the Rat Pancreas

Cytochrome P-450 (CYP) isoenzymes have been incriminated in the toxicity and carcinogenicity of various xenobiotics in different tissues, but prior measurements of their activity in pancreatic microsomes have been disappointing. We now applied new isolation methods and a highly sensitive procedure to assay for the metabolism of p -nitrophenol and 7-ethoxyresorufin, specific substrates for CYP2E1 (2E1) and CYP1A1 (1A1), respectively. 2E1 and 1A1 content was estimated with high-resolution chemiluminescent Western blots using recombinant 2E1 and 1A1 as standards. We found that p-nitrophenol hydroxylase activity was 5.07 ± 0.66 and 1.50 ± 0.26 pmol/ min/mg of protein in pancreatic microsomes of ethanol-fed and control rats, respectively. Chronic ethanol treatment increased 2E1 content in pancreatic microsomes 3.6-fold. Activity and content of 2E1 were also assessed in hepatic microsomes: specific activity (expressed per 2E1 content) was similar in pancreatic and hepatic microsomes. There was also an inductive effect of 3-methylcholan-threne (MC) on 1A1 in pancreatic microsomes. Pancreatic microsomal 7-ethoxyresorufin-O-dealkylation activity in MC-treated rats was 19.6 ± 1.7 pmol/min/mg of protein, 61-fold higher than in controls. MC treatment increased the 1A1 content in pancreatic microsomes 42-fold. These results demonstrate that, in pancreatic microsomes, ethanol and MC exert striking inductive effects on 2E1 and 1A1 activities, which could play a role in the pathogenesis of pancreatitis and/or pancreatic cancer.