Research SectionEffect of some Indole Derivatives on Xenobiotic Metabolism and Xenobiotic-induced Toxicity in Cultured Rat Liver Slices
Introduction
Cytochrome P-450 (CYP) isoforms have a major role in the metabolism of xenobiotics and certain endogenous chemicals (Nelson et al., 1996; Parkinson, 1996). A wide variety of chemicals have been shown to modulate CYP isoform activities in both experimental animals and humans (Conney, 1986; Okey, 1990; Parkinson, 1996). For example, certain cruciferous vegetables (e.g. cabbage, Brussels sprouts, broccoli) can induce xenobiotic metabolism in humans (Kall et al., 1996; McDanell et al., 1992; Pantuck et al., 1979, Pantuck et al., 1984; Verhoeven et al., 1997). The feeding of diets containing cruciferous vegetables has also been shown to induce xenobiotic metabolizing enzymes in rat liver and intestine (McDanell et al., 1987; Vang et al., 1991; Verhoeven et al., 1997; Wortelboer et al., 1992b). It is now well established that cruciferious vegetables contain a variety of chemicals which can modulate the activities of both phase I and phase II xenobiotic metabolizing enzymes (McDanell et al., 1988; Verhoeven et al., 1997; Zhang et al., 1992). Some compounds are termed bifunctional inducers since they can induce both phase I and II xenobiotic metabolizing enzymes, whereas others which only induce phase II enzymes are termed monofunctional inducers (Prestera et al., 1993).
One important class of enzyme inducers derived from cruciferous vegetables are indole derivatives (for structures see Fig. 1), including indole-3-carbinol (I3C) and indole-3-acetonitrile (3-ICN), which are formed from the breakdown of the glucosinolate glucobrassicin (McDanell et al., 1988; Verhoeven et al., 1997). Under acidic conditions, I3C can form a number of condensation products (Bjeldanes et al., 1991; de Kruif et al., 1991; McDanell et al., 1988; Verhoeven et al., 1997) including the dimer 3,3′-diindolylmethane (DIM). The effect of I3C, 3-ICN and DIM on rat hepatic xenobiotic metabolizing enzymes has been studied after in vivo administration and in rat hepatocyte cultures in vitro (de Kruif et al., 1991; McDanell et al., 1987; Staack et al., 1998; Verhoeven et al., 1997; Wortelboer et al., 1992a, Wortelboer et al., 1992c).
The induction of phase I and II xenobiotic metabolizing enzymes by cruciferous vegetables and component chemicals has been shown to enhance the metabolic detoxification of a number of carcinogens and to reduce DNA adduct formation (McDanell et al., 1988; Verhoeven et al., 1997). For example, the formation of DNA adducts in rat colon by the cooked food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine was reduced by the administration of I3C (Guo et al., 1995). In some, but not all, studies treatment with cruciferous vegetables or chemicals derived from cruciferous vegetables has been shown to reduce subsequent tumour formation following administration of genotoxic carcinogens to experimental animals (McDanell et al., 1988; Stoewsand, 1995; Verhoeven et al., 1997).
Several studies have demonstrated that precision-cut liver slices may be employed in an in vitro model system for investigations of xenobiotic metabolism, xenobiotic-induced toxicity and the effects of xenobiotics on enzyme activities (Bach et al., 1996; Parrish et al., 1995; Lake et al., 1993). The objective of this study was to evaluate the effects of some indole derivatives on CYP isoforms in cultured rat liver slices. In addition, the effect of pretreatment of rat liver slices with one indole derivative, namely DIM, on the toxicity of aflatoxin B1 (AFB1) and monocrotaline was also studied. Both AFB1 and monocrotaline are known to require metabolic activation in order to exert their toxic effects (Dueker et al., 1992; Eaton and Gallagher, 1994; Huxtable, 1997; Imaoka et al., 1992; Zimmerman, 1978).
Section snippets
Materials
Aflatoxin B1, monocrotaline, I3C, 3-ICN, enzyme substrates and cofactors were purchased from Sigma-Aldrich Company Ltd (Poole, Dorset, UK). 3,3′-Diindolylmethane (DIM) was generously provided by Dr B.J. Blaauboer, whereas Aroclor 1254 (ARO) was the gift of the Monsanto Chemical Co. (St Louis, MO, USA). Western immunoblotting kits, antibodies to rat CYP isoforms CYP1A1, CYP2B1/2 and CYP3A and l-[1-14C]leucine (sp. act. 54 mCi/mmol) were obtained from Amersham International plc (Little Chalfont,
In vivo studies
Male Sprague–Dawley rats were treated with 25 and 100 mg/kg/day I3C for 3 days. I3C produced dose-related increases in relative liver weight and in microsomal protein and CYP content (Table 1). Marked increases were observed in 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-depentylase activities, which are considered markers for CYP1A and CYP2B isoforms, respectively (Burke et al., 1985; Nerurkar et al., 1993; Parkinson, 1996). The induction of 7-ethoxyresorufin and 7-pentoxyresorufin
Discussion
While rat hepatocyte cultures have been used extensively for xenobiotic metabolizing enzyme induction studies, fewer investigations have been performed with liver slices (Bach et al., 1996; LeCluyse et al., 1996). Rat liver slices have been employed to study the induction of peroxisomal enzymes (Beamand et al., 1993) and CYP isoforms in the CYP1A, CYP2B, CYP3A and CYP4A subfamilies (Drahushuk et al., 1996; Gokhale et al., 1997; Lake et al., 1993, Lake et al., 1996; Müller et al., 1996). As
Acknowledgements
We thank Dr B. J. Blaauboer, Dr C. A. de Kruif and Dr H. M. Wortelboer (RITOX, University of Utrecht, The Netherlands) for the gift of a sample of DIM, and Professor A.R. Boobis (Imperial College School of Medicine, Hammersmith Hospital, London, UK) for the gift of the CYP1A2 antibody. This work forms part of a research project sponsored by the UK Ministry of Agriculture, Fisheries and Food to whom our thanks are due. The results of the research are the property of the Ministry of Agriculture,
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