Faculté de Médecine de Brest, EA 948, Laboratoire de Biochimie, BP 815, 29285 Brest, and
1 Service d'Alcoologie, Hôpital de Bohars, CHU de Brest, France
Received 6 April 1999; in revised form 13 May 1999; accepted 14 June 1999
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ABSTRACT |
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INTRODUCTION |
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In experimental conditions, Morimoto et al. (1995) showed that CYP2E1 induction was related to alcoholic liver disease possibly by a link involving oxidative stress, because CYP2E1 inhibitors reduced both alcoholic liver disease and oxidative stress. In a previous study (Dupont et al., 1998), we showed that CYP2E1 activity greatly influenced the formation of hydroxyethyl radicals in alcoholics but no information was available on the oxidative stress in these patients. Therefore, the aim of the present study was to investigate in a new population of alcoholic patients the relationship between CYP2E1 activity, as assessed by chlorzoxazone metabolism (Girre et al., 1994
) and some blood markers of oxidative stress, namely oxidized proteins, and lipid peroxides (LPO). Antibodies against the hydroxyethyl radical (HER) or malondialdehyde (MDA) adducts were also evaluated as markers of the production of free radicals, and vitamin E levels were also used as a marker of antioxidant defences.
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PATIENTS AND METHODS |
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CYP2E1 activity.
This was assessed using chlorzoxazone metabolism as previously described (Girre et al., 1994). Briefly, after a 12 h fast, subjects were administered a 500 mg tablet of chlorzoxazone (Lemmon Company, Sellersville, PA, USA). A blood sample was withdrawn 2 h later to measure chlorzoxazone and 6-hydroxychlorzoxazone using a high power liquid chromatography (HPLC) method (Lucas et al., 1993
). The 6-hydroxychlorzoxazone:chlorzoxazone concentration ratio (CHZ-MR, or chlorzoxazone metabolic ratio) has been shown to reflect the rate of chlorzoxazone hydroxylation and thus CYP2E1 activity. In the days preceding the study, subjects had taken no medication which might interfere with CYP2E1 activity, such as paracetamol, disulfiram or isoniazid.
Alcoholics were tested for chlorzoxazone metabolism on the day following their admission to hospital. Blood ethanol was determined on their arrival and before each chlorzoxazone test to verify that they had no ethanol in the blood because of the possible competition between ethanol and chlorzoxazone for metabolism by CYP2E1. Control subjects were not submitted to the CHZ test as normal values were already available in our laboratory (Lucas et al., 1995b).
Parameters of oxidative stress. These were assessed in the samples of controls and alcoholic patients. Blood samples were taken after an overnight fast, just before the CHZ test for the alcoholic patients. Plasma lipid peroxides (LPO) were estimated by haemoglobin-catalysed oxidation of 10-N-methyl-carbamoyl-3,7-dimethylamino-10-phenothiazine after treament with lipoprotein lipase using cumene hydroperoxide as standard, using a kit supplied by Kamiya Biomedical Co. (Seattle, WA, USA). Oxidized proteins were determined by the 2,4-dinitrophenylhydrazine procedure according to Levine et al. (1990). All reagents were of analytical grade and were purchased from Sigma (St Quentin Fallavier, France).
Vitamin E.
This was measured using a reversed-phase HPLC method as described by Ganiere-Monteil et al. (1994). Determination of -tocopherol in plasma was performed after extraction by hexane.
-Tocopherol was added as internal standard. Tocopherol was detected by its native fluorescence (excitation 295 nm; emission 340 nm).
- and
-Tocopherols were purchased from Sigma.
All other biochemical parameters were determined using a multiparametric routine automat (Synchron Clinical System Myosotis, Beckman Instruments, Brea, USA). Reagents were used according to the manufacturer's recommendations for determining alkaline phosphatase, gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, cholesterol and triglycerides levels. Carbohydrate-deficient transferrin (CDT) was assessed using a kit supplied by Biorad (Ivry sur Seine, France).
Detection of antibodies against protein adducts.
Antigens consisting of HER and MDA adducts with bovine serum albumin (BSA) were immobilized on microplates as previously described (Dupont et al., 1998). Circulating IgGs against epitopes (HER or MDA) were determined in serum samples using an enzyme-linked immunosorbent assay (ELISA) method. Data were expressed as the ratio between the absorbance readings in the wells containing HER-BSA or MDA-BSA and those containing unmodified protein to avoid non-specific reactions to different antigen used. Ratios of ~1 indicate the lack of these antibodies.
Statistical analysis
Results were expressed as means ± SD and were compared between controls and alcoholics by Student's t-test. Linear regression analysis was performed to assess the relationship between CYP2E1 activity and other parameters (Graphpad Instat, Graphpad Software, San Diego, CA, USA).
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RESULTS |
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Induction of CYP2E1 in chronic alcoholics
CYP2E1 activity was assessed using the CHZ-MR 2 h after intake of 500 mg CHZ. The CHZ-MR was increased by 4-fold in alcoholics, compared to non-alcoholics (Table 2) as previously shown in other studies (Lucas et al., 1995a
). A weak but significant correlation was observed between CYP2E1 induction and the mean amount of alcohol ingested per day (r = 0.31, P < 0.05), the CDT level (r = 0.33, P < 0.05) and the blood alcohol level (BAL) at the time of admission to hospital (r = 0.41, P < 0.001).
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Relationship between CYP2E1 and parameters of the oxidative stress
When the alcoholic population was divided into two groups according to the characteristics given in Table 3, one group with low CYP2E1 induction (CHZ-MR <0.69, mean + 3 SD of the control population) and the other with high induction (CHZ-MR >0.69) parameters of oxidative stress, expressed as percentage of variation vs controls, were not significantly different between both groups, except for anti-HER adduct antibodies (Figure 1
). As both groups differed very significantly by their CYP2E1 activity (P < 0.0001) (Table 3
), this suggests that CYP2E1, although contributing to the oxidative stress, is only one of the factors involved in the oxidation of lipids or proteins by contrast with its main role in the formation of HER adducts. Accordingly, no significant correlation was found between CYP2E1 activity and parameters of oxidative stress, except the anti-HER adducts (r = 0.45, P < 0.001).
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DISCUSSION |
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CYP2E1 activity can be estimated in humans using chlorzoxazone metabolism. Recent pharmacokinetic studies validated a single point chlorzoxazone measure at 2 h as an acceptable phenotypic index of the 6-hydroxychlorzoxazone formation clearance (Digler et al., 1997; Frye et al., 1998
) and the selectivity of this probe for CYP2E1 in humans has recently been reassessed (Lucas et al., 1999
). Regulation of CYP2E1 by ethanol may involve protein stabilization which appeared dependent upon blood-alcohol levels (BAL) in the rat and increased mRNA synthesis at high ethanol levels (Badger et al., 1993
; Ronis et al., 1993
; Roberts et al., 1994
) or by long-term ethanol consumption (Takahashi et al., 1993
). In our study, we observed for the first time a weak but significant correlation between CYP2E1 activity and the mean daily alcohol consumption, assessed by using a face-to-face interview, the CDT level (a biological marker of alcohol consumption) or BAL at admission to hospital. BAL might also regulate CYP2E1 activity in humans, but the poor correlation observed could be explained by the fact that level at admission does not necessarily reflect the mean daily BAL. Because of the rapid turnover of the enzyme (2.5 days in humans, Lucas et al., 1995a), CYP2E1 activity was determined on the day following the entry, as it was necessary to wait for the elimination of ethanol from the blood. However, CYP2E1 activities have been shown to differ greatly between individuals, at the basal level as after ethanol intoxication (Lucas et al., 1995b
). Known genetic polymorphism reported for 2E1 (Hayashi et al., 1991
; Hirvonen et al., 1993
) does not appear to be involved (Lucas et al., 1995a
; Carriere et al., 1996
), whereas environmental factors, such as physiological or hormonal status and diet, might be more determinant (Lucas et al., 1998
). McCarver et al. (1998) recently reported a new genetic polymorphism in the regulatory sequences of CYP2E1 associated with an increased hydroxylation of CHZ in the presence of obesity or ethanol intake. The frequency of this polymorphism was estimated to be 7% in Caucasians.
Ethanol administration has been shown to induce an oxidative stress either by enhancing the production of oxygen reactive species and/or by decreasing the level of endogenous antioxidants. In our study, blood markers of oxidative stress, such as oxidized proteins and lipid peroxides, were increased by ~20% in alcoholic patients, whereas plasma concentrations of -tocopherol, a marker of antioxidant defence, were depressed. These results are consistent with several previous reports showing increased lipoperoxidation and reduced plasma vitamin E levels in alcoholics entering a rehabilitation programme (Girre et al., 1990
; Lecomte et al., 1994
). Depletion in vitamin E might reflect the pro-oxidant action of alcohol in the liver and facilitate the development of lipid peroxidation in some patients, as supplementation of diet with vitamin E is known to reduce on-going in vivo lipid peroxidation and ethanol-induced liver damage (Bondy et al., 1996
). However, increased lipid peroxidation and decreased vitamin E levels are far from being constant after chronic ethanol administration (Fernandez-Solà et al., 1998
) and during the initial period, an adaptative enhancement in some elements of the antioxidant defence, such as an increase in glutathione peroxidase activity, may prevent the occurrence of oxidative stress and decrease in vitamin E. Tobacco smoking, which is very often associated with alcohol consumption, also produces free radicals and thus participates in the generation of oxidative stress (Khan et al., 1998
).
In order to analyse the relationship between CYP2E1 activity and oxidative stress in alcoholics, blood parameters of oxidative stress were compared in patients with low and high induction of CYP2E1. Patients with low CYP2E1 induction drank significantly less alcohol and had lower CDT levels than those with high CYP2E1 induction, but did not differ significantly by their smoking habits. Although low-induced patients displayed lower mean values than high-induced patients for all parameters, no significant difference was observed except for autoantibodies raised against HER adducts. CYP2E1 has been shown to be the major target protein involved in HER adducts (Clot et al., 1996) and generation of 1-hydroxyethyl radicals from ethanol involves at least two pathways: one with OH radicals produced in a Fenton-type reaction from endogenously formed hydroperoxide, and the other is cytochrome P-450-mediated and apparently independent from OH (Albano et al., 1991
; Reinke et al., 1997
). Thus, CYP2E1 may play a key role in the formation of these adducts and this is evidenced in our study. By contrast, no significant difference was observed with anti-MDA adduct antibodies, which reflect MDA formation, or the other parameters measuring oxidative stress, namely oxidized proteins, lipid hydroperoxides and vitamin E. During ethanol intoxication, pro-oxidant species are generated in hepatic tissue at various subcellular sites: in the endoplasmic reticulum by CYP2E1, in the cytosol by xanthine oxidase and in the mitochondria by the respiratory chain (Nordmann, 1994
). In addition, phagocytes recruited and activated by tissue injury will also contribute to this production. Many reports show that various experimental conditions of CYP2E1 induction may lead to overt lipid peroxidation. For example, combined treatment with chronic intragastric ethanol feeding and unsaturated fat-rich diets results in dramatically increased CYP2E1 activity and associated lipid peroxidation which can be prevented by CYP2E1 inhibitors in rats (Morimoto et al., 1995
). Due to its potential for free radical generation, CYP2E1 is thought to contribute to overall production of reactive oxygen species more significantly than do other enzymes. However, our study, which is the first to examine CYP2E1 activity in connection with oxidative stress in humans, does not show evident correlation between CYP2E1 induction and different parameters of oxidative stress. Our data thus suggest that CYP2E1 does not play a prominent role in the oxidative stress which occurs during ethanol intoxication, even if its participation is undoubted.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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* Author to whom correspondence should be addressed.
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REFERENCES |
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Albano, E., Clot, P., Morimoto, M., Tomasi, A., Ingelman-Sundberg, M. and French, S. (1995) Role of cytochrome P450 2E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol. Hepatology 23, 155163.[ISI]
Badger, T. M., Huang, J., Ronis, M. and Lumpkin, C. K. (1993) Induction of cytochrome P450 2E1 during chronic ethanol exposure occurs via transcription of the CYP2E1 gene when blood alcohol concentrations are high. Biochemical and Biophysical Research Communications 190, 780785.[ISI][Medline]
Bondy, S., Guo, S. and Adams, J. D. (1996) Prevention of ethanol-induced changes in reactive oxygen parameters by a-tocopherol. Alcohol and Alcoholism 31, 403410.[Abstract]
Carriere, V., Berthou, F., Baird, S., Belloc, C., Beaune, P. H. and de Waziers, I. (1996) Human cytochrome CYP2E1: from genotype to genotype. Pharmacogenetics 6, 203211.[ISI][Medline]
Cederbaum, A. I. (1989) Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity. Free Radical Biology and Medicine 7, 559567.[ISI][Medline]
Clot, P., Bellomo, G., Tabone, M., Arico, S. and Albano, E. (1995) Detection of antibodies against proteins modified by hydroxyethyl free radicals in patients with alcoholic cirrhosis. Gastroenterology 108, 201207.[ISI][Medline]
Clot, P., Albano, E., Eliasson, E., Tabone, M., Arico, S., Israel, Y., Moncada, C. and Ingelman-Sundberg, M. (1996) Cytochrome P450 2E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics. Gastroenterology 111, 206216.[ISI][Medline]
Clot, P., Parola, M., Bellomo, G., Dianzani, U., Carini, R., Tabone, M., Arico, S., Ingelman-Sundberg, M. and Albano, E. (1997) Plasma membrane hydroxyethyl radical adducts cause antibody-dependent cytotoxicity in rat hepatocytes exposed to alcohol. Gastroenterology 113, 265276.[ISI][Medline]
Digler, K., Metzler, J., Bode, J. C. and Klotz, V. (1997) CYP2E1 activity in patients with alcoholic liver disease. Journal of Hepatology 27, 10091014.[ISI][Medline]
Dupont, I., Lucas, D., Clot, P., Ménez, C. and Albano, E. (1998) Cytochrome P450 2E1 inducibility and hydroxyethyl radical formation among alcoholics. Journal of Hepatology 28, 564571.[ISI][Medline]
Eckström, G. and Ingelman-Sundberg, M. (1989) Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P450 (P450 IIE1). Biochemical Pharmacology 38, 13131319.[ISI][Medline]
Fernandez-Solà, J., Villegas, E., Nicolàs, J. M., Deulofeu, R., Antùnez, E., Sacanella, E., Estruch, R. and Urbano-Màrquez A. (1998) Serum and muscle levels of a-tocopherol, ascorbic acid and retinol are normal in chronic alcoholic myopathy. Alcoholism: Clinical and Experimental Research 22, 422427.[ISI][Medline]
Frye, R. F., Adenoyin, A., Maurio, K., Matzke, G. R. and Branch, R. A. (1998) Use of chlorzoxazone as in vivo probe of CYP2E1: choice of dose and phenotypic trait measure. Journal of Clinical Pharmacology 38, 8289.
Ganiere-Monteil, C., Kergueris, M. F., Pineau, A, Blanchard, B., Azoulay, C. and Larousse, C. (1994) Dosage du retinol et de l'alpha-tocophérol plasmatique par CLHP. Annales de Biologie Clinique 52, 547553.[ISI][Medline]
Girre, C., Hispard, E., Therond, P., Guedj, S., Bourdon, R. and Dally, S. (1990) Effect of abstinence from alcohol on the depression of glutathione peroxidase activity and selenium and vitamine E levels in chronic alcoholic patients. Alcoholism: Clinical and Experimental Research 14, 909912.[ISI][Medline]
Girre, C., Lucas, D., Hispard, E., Ménez, C., Dally, S. and Ménez, J-F. (1994) Assessment of cytochrome P450 2E1 induction in alcoholic patients by chlorzoxazone pharmacokinetics. Biochemical Pharmacology 47, 15031508.[ISI][Medline]
Hayashi, S. I., Watanabe, J. and Kawajiri, K. (1991) Genetic polymorphism in 5'-flanking region change transcriptional regulation of the human Cytochrome P450 IIE1 gene. Journal of Biochemistry (Tokyo) 110, 559565.[Abstract]
Hirvonen, A., Husgafvel-pursiainen, K., Antilla, S., Karjailanen, A. and Vainio, H. (1993) The human CYP2E1 gene and lung cancer: Dra I and Rsa I restriction length polymorphisms in a Finnish population. Carcinogenenesis 14, 8588.[Abstract]
Ingelman-Sundberg, M., Johansson, I., Yin, H., Terelius, Y., Eliasson, E., Clot, P. and Albano, E. (1993) Ethanol-inducible cytochrome P4502E1: Genetic polymorphism, regulation and possible role in the etiology of alcohol-induced liver disease. Alcohol 10, 447452.[ISI][Medline]
Khan, A. R., Mir, M. M., Wani, J. I., Siddiqi, M. A. and Salhuddin, M. (1998) Superoxide production by resting and activated polymorphonuclear leucocytes from smokers and non-smokers. Medical Science Research 26, 471473.[ISI]
Kurose, I., Higuchi, H., Kato, S., Miura, S. and Ishii, H. (1996) Ethanol-induced oxidative stress in the liver. Alcohol: Clinical and Experimental Research 20 (Suppl. 1), 77A85A.
Lecomte, E., Herbeth, B., Pirollet, P., Chancerelle, Y., Arnaud, J., Musse, N., Paille, F., Siest, G. and Artur, Y. (1994) Effect of alcohol consumption on blood antioxidant nutrients and oxidative stress indicators. American Journal of Clinical Nutrition 60, 255261.[Abstract]
Levine, R. L., Garland, D., Oliver, C. N., Amici, A., Climent, I., Lenz, A. G., Ahn, B. W., Shaltiel, S. and Stadtman, E.R. (1990) Determination of carbonyl content in oxidatively modified proteins. Methods in Enzymology 186, 346357.
Lucas, D., Ménez, C., Girre, C., Berthou, F. and Ménez, J.-F. (1993) High-performance liquid chromatographic determination of chlorzoxazone and 6-hydroxychlorzoxazone in serum: a tool for indirect evaluation of cytochrome P450 2E1 activity in humans. Journal of Chromatography B622, 7986.[Medline]
Lucas, D., Ménez, C., Girre, C., Bodénez, P., Hispard, E. and Ménez, J-F. (1995a) Decrease in cytochrome P4502E1 as assessed by the rate of chlorzoxazone hydroxylation in alcoholics during the withdrawal phase. Alcohol: Clinical and Experimental Research 19, 362366.[ISI][Medline]
Lucas, D., Ménez, C., Girre, C., Berthou, F., Bodénez, P., Joannet, I., Hispard, E., Bardou, L-G. and Ménez J-F. (1995b) Cytochrome P4502E1 genotype and chlorzoxazone metabolism in healthy and alcoholic Caucasian subjects. Pharmacogenetics 5, 298304.[ISI][Medline]
Lucas, D., Farez, C., Bardou, L. G., Vaisse, J., Attali, J. R. and Valensi, P. (1998) Cytochrome P450 2E1 activity in diabetic and obese patients as assessed by chlorzoxazone hydroxylation. Fundamental and Clinical Pharmacology 12, 553558.[ISI][Medline]
Lucas, D., Ferrara, R., Gonzales, E., Bodenez, P., Albores, A., Manno, M. and Berthou, F. (1999) Chlorzoxazone, a selective probe for phenotyping CYP2E1 in humans. Pharmacogenetics 9, 377388.[ISI][Medline]
McCarver, D. G., Byun, R., Hines, R. N., Hichme, M. and Wegenek, W. (1998) A genetic polymorphism in the regulatory sequences of human CYP2E1: Association with increased chlorzoxazone hydroxylation in the presence of obesity and ethanol intake. Toxicology and Applied Pharmacology 152, 276281.[ISI][Medline]
Morimoto, M., Hagbjörk, A. L., Wan, Y. J., Fu, P. C., Clot, P., Albano, E., Ingelman-Sundberg, M. and French, S. W. (1995) Modulation of experimental alcohol-induced liver disease by cytochrome P450 2E1 inhibitors. Hepatology 21, 16101617.[ISI][Medline]
Nordmann, R. (1994) Alcohol and antioxidant systems. Alcohol and Alcoholism 29, 513522.[Abstract]
Parola, M., Pinzani, M., Casini, A., Albano, E., Poli, G., Gentilini, A., Gentilini, P. and Dianzani, M. U. (1993) Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen a1 (I) gene expression in human liver fat storing cells. Biochemical and Biophysical Research Communications 194, 10441050.[ISI][Medline]
Reinke, L. A., Moore, D. R. and McCay, P. B. (1997) Free radical formation in livers of rats treated acutely and chronically with alcohol. Alcohol: Clinical Experimental Research 21, 642646.[ISI][Medline]
Roberts, B. J., Shoaf, S. E., Jeong, K. S. and Song, B. J. (1994) Induction of CYP2E1 in liver, kidney, brain and intestine during chronic ethanol administration and withdrawal. Biochemical and Biophysical Research Communications 205, 10641071.[ISI][Medline]
Ronis, M. J., Huang, J., Crouch, J., Mercado, C., Irby, D., Valentine, C. R., Lumpkin, C. K., Ingelman-Sundberg, M. and Badger, T. M. (1993) Cytochrome P450 2E1 induction during chronic alcohol exposure occurs by a two-step mechanism associated with blood alcohol concentration in rats. Journal of Pharmacology and Experimental Therapeutics 264, 944950.[Abstract]
Takahashi, T., Lasker, J. M., Rosman, A. S. and Lieber, C. S. (1993) Induction of cytochrome P-4502E1 in the human liver by ethanol is caused by a corresponding increase in encoding messenger RNA. Hepatology 17, 236245.[ISI][Medline]
Tsukamoto, H., Rippe, R., Niemelä, O. and Lin, M. (1995) Roles of oxidative stress in activation of Kupffer and Ito cells in liver fibrogenesis. Journal of Gastroenterology and Hepatology 10, S50S53.[ISI][Medline]