Liver nuclear and microsomal CYP2E1-mediated metabolism of xenobiotics in rats chronically drinking an alcohol-containing liquid diet

Abstract

In previous studies from our laboratory, the presence in highly purified liver nuclei of metabolic pathways for processing ethanol (EtOH), N-nitrosodimethylamine (NDMA), carbon tetrachloride and chloroform was reported. All these chemicals are known to be metabolized in liver microsomes, via cytochrome P450 2E1 (CYP2E1)-mediated processes. In the present work we checked whether rat liver nuclei from rats chronically drinking an alcohol-containing liquid diet exhibited an enhanced ability to metabolize chemicals known to require CYP2E1 participation for given metabolic transformations. The nicotinamide adenosine dinucleotide phosphate (NADPH)-requiring metabolism of p-nitrophenol to p-nitrocathecol; the activation of carbon tetrachloride to trichloromethyl radicals, covalently binding to proteins; and the ring hydroxylation of aniline and o-toluidine were studied. Comparison of the obtained nuclear activities against the one present in the microsomal counterpart, and their respective response to the EtOH inductive effect after repetitive exposure to it, was studied. The obtained results showed that rat liver nuclei exhibited less pnitrophenol hydroxylase activity than microsomes, but it was inducible by repetitive alcohol drinking to equivalent levels of those of microsomes from control animals. Nuclei exhibited the ability to activate CCl₄, which was significantly enhanced by alcohol drinking. Aniline was ring hydroxylated in liver microsomes but not in nuclei from either control or EtOH-treated animals. In contrast, nuclei and microsomes metabolized o-toluidine to ring hydroxylated products. They are considered less toxic in nature but other authors reported a genotoxic effect for one of them. The production of the ring hydroxylated metabolites was enhanced by repetitive EtOH drinking. Results suggest that nuclear metabolism of xenobiotics might be relevant for either activations or detoxications mediated by CYP2E1 and that repetitive exposure to EtOH might significantly modulate those processes. Toxicology and Industrial Health 2006; 22: 367-374.

Keywords

alcohol aniline carbon tetrachloride CYP2E1 liver N-nitrosodimethylamine nuclear nuclei o-toluidine

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References

Bolt, H.M. , Roos, P.H. and Their, R. 2003: The cytochrome P450 isoenzyme CYP2E1 in the biological processing of industrial chemicals: consequences for occupational and environmental medicine . International Archives of Occupational and Environmental Health 7, 174-85 .

Castro, G.D. , Costantini, M.H. , Delgado de Layño, A.M.A. and Castro, J.A. 2002: Rat liver microsomal and nuclear activation of methanol to hydroxymethyl free radicals . Toxicology Letters 129, 227-36 .

Castro, G.D. , Delgado de Layño, A.M.A. and Castro, J.A. 1998: Liver nuclear ethanol metabolizing system (NEMS) producing acetaldehyde and 1-hydroxyethyl free radicals . Toxicology 129, 137-44 .

Castro, G.D. , Díaz Gómez, M.I. and Castro, J.A. 1989: Species differences in the interaction between CCl 4 reactive metabolites and liver DNA and nuclear protein fractions . Carcinogenesis 10, 289-94 .

Castro, G.D. , Díaz Gómez, M.I. and Castro, J.A. 1990: Biotransformation of carbon tetrachloride and lipid peroxidation promotion by liver nuclear preparations from different animal species . Cancer Letters 53, 9-15 .

Danford, N. 1991: The genetic toxicology of ortho-toluidine . Mutation Research 258, 207-36 .

Díaz Gómez, M.I. and Castro, J.A. 1980a: Covalent binding of carbon tetrachloride metabolites to liver nuclear DNA, proteins and lipids . Toxicology and Applied Pharmacology 56, 199-206 .

Díaz Gómez, M.I. and Castro, J.A. 1980b: Covalent binding of chloroform metabolites to nuclear proteins. No evidence for binding to nucleic acids . Cancer Letters 9, 213-18 .

Díaz Gómez, M.I. and Castro, J.A. 1980c: Nuclear activation of carbon tetrachloride and chloroform . Research Communications in Chemical Pathology and Pharmacology 27, 191-94 .

10.

Díaz Gómez, M.I. , Fanelli, S.L. , Castro, G.D. et al. 1999: A liver nuclear ethanol metabolizing system. Formation of metabolites that bind covalently to macromolecules and lipids . Toxicology 138, 19-28 .

11.

Díaz Gómez, M.I. , Valles, E.G. , Fanelli, S.L. et al. 2002: Alcohol induction of liver nuclear ethanol and Nnitrosodimethylamine metabolism to reactive metabolites . Teratogenesis Carcinogenesis and Mutagenesis 22, 139-45 .

12.

Ekström, G. , Von Bahr, C. and Ingelman-Sundberg, M. 1989: Human liver microsomal cytochrome P450IIE1. Immunological evaluation of its contribution to microsomal ethanol oxidation, carbon tetrachloride reduction and NADPH oxidase activity . Biochemical Pharmacology 38, 689-93 .

13.

Fanelli, S.L. and Castro, J.A. 1993: Carbon tetrachloride promoted malondialdehyde formation in liver microsomal and nuclear preparations from Sprague Dawley or Osborne Mendel male rats . Research Communications in Chemical Pathology and Pharmacology 82, 233-36 .

14.

Gad, S.C. 2001: Statistics for toxicologists. In Hayes, A.W. , editor, Principles and methods in toxicology , fourth edition. Taylor & Francis , 273-320.

15.

González, F.J. 2005: Role of cytochromes P450 in chemical toxicity and oxidative stress: studies with CYP2E1 . Mutation Research 569, 101-10 .

16.

Guenguerich, F.P. 1995: Human cytochrome P450 enzymes. In Ortiz de Montellano, P.R. , editor, Cytochrome P450 structure. Mechanism and biochemistry, second edition. Plenum Press , 473-535.

17.

Gupta, R.L. , Gupta, A.K. , Pathak, D.P. and Jumeja, T.R. 1987: Mutagenic studies of ortho-toluidine and its potential metabolites. Indian Journal in Experimental Biology 25, 618-22 .

18.

Gut, I. , Nedelcheva, V. , Soucek, P. et al. 1996: The role of CYP2E1 and 2B1 in metabolic activation of benzene derivatives . Archives of Toxicology 71, 45-56 .

19.

Hasumura, Y. , Teschke, R. , Lieber, C.S. 1974: Increased carbon tetrachloride hepatotoxicity, and its mechanism, after chronic ethanol consumption . Gastroenterology 66, 226-34 .

20.

Iarc 1982: Some aromatic amines, anthraquinones and nitroso compounds, and inorganic fluorides used in drinking-water and dental preparations. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Volume 27. IARC , 155-75.

21.

Ingelman-Sundberg, M. , Johansson, I. , Penttila, K.E. et al. 1988: Centrilobular expression of ethanol inducible cytochrome P450(IIE1) in rat liver . Biochemical Biophysical Research Communications 157, 55-60 .

22.

Jodynis-Liebert, J. and Bennasir, H.A. 2000: Gas chromatographic method for the determination of toluidines in spiked urine samples . Journal of Chromatography B Biomedical Science Application 738, 427-30 .

23.

Koop, D.R. and Coon, M.J. 1986: Ethanol oxidation and toxicity: role of alcohol P-450 oxygenase . Alcohol Clinical Experimental Research 10, 44s-49s .

24.

Lieber, C.S. 2004: The discovery of the microsomal ethanol oxidizing system and its physiologic and pathologic role . Drug Metabolism Reviews 36, 511-29 .

25.

Lieber, C.S. and De Carli, L.M. 1982: The feeding of alcohol in liquid diets: two decades of applications and 1982 update . Alcohol Clinical and Experimental Research 6, 523-31 .

26.

Lieber, C.S. and De Carli, L.M. 1989: Liquid diet technique of ethanol administration: 1989 update . Alcohol and Alcoholism 24, 197-211 .

27.

Lowry, O.H. , Rosebrough, N.J. , Farr, A.L. and Randall, R.J. 1951: Protein measurement with the Folin phenol reagent . Journal of Biological Chemistry 193, 265-75 .

28.

Mishin, V.M. , Koivisto, T. and Lieber, C.S. 1996: The determination of cytochrome P4502E1-dependent p-nitrophenol hydroxylation by high-performance liquid chromatography with electrochemical detection . Analytical Biochemistry 233, 212-15 .

29.

Morimoto, M. , Hagbjörk, A.L. , Wan, Y.J. et al. 1995: Modulation of experimental alcohol-induced liver disease by cytochrome P4502E1 inhibitors . Hepatology 21, 1610-17 .

30.

Ohkuma, Y. , Hiraku, Y. , Oikawa, S. et al. 1999: Distinct mechanisms of oxidative DNA damage by two metabolites of carcinogenic o-toluidine. Archives of Biochemistry and Biophysics 372, 97-106 .

31.

Stillwell, W.G. , Bryant, M.S. and Wishnok, J.S. 1987: GC-MS analysis of biologically important aromatic amines. Application to human dosimetry. Biomedical Environmental Mass Spectrometry 14, 221-27 .

32.

Tsutsumi, M. , Lasker, J.M. , Shimizu, M. et al. 1989: The intralobular distribution of ethanol-inducible P4502E1 in rat and human liver . Hepatology 10, 437-46 .

33.

Viviani, A. , Lutz, W.K. and Schlatter, C. 1978: Time course of the induction of aryl hydrocarbon hydroxylase in rat liver nuclei and microsomes by phenobarbital, 3-methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, dieldrin and other inducers . Biochemical Pharmacology 27, 191-94 .