Research Section
Effect of some Indole Derivatives on Xenobiotic Metabolism and Xenobiotic-induced Toxicity in Cultured Rat Liver Slices

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Abstract

In this study the effect of some indole derivatives on xenobiotic metabolizing enzymes and xenobiotic-induced toxicity has been examined in cultured precision-cut liver slices from male Sprague–Dawley rats. While treatment of rat liver slices for 72 hours with 2–200 μm of either indole-3-carbinol (I3C) or indole-3-acetonitrile (3-ICN) had little effect on cytochrome P-450 (CYP)-dependent enzyme activities, enzyme induction was observed after in vivo administration of I3C. The treatment of rat liver slices with 50 μm 3,3′-diindolylmethane (DIM; a dimer derived from I3C under acidic conditions) for 72 hours resulted in a marked induction of CYP-dependent enzyme activities. DIM appears to be a mixed inducer of CYP in rat liver slices having effects on CYP1A, CYP2B and CYP3A subfamily isoforms. Small increases in liver slice reduced glutathione levels and glutathione S-transferase activity were also observed after DIM treatment. While aflatoxin B1 and monocrotaline produced a concentration-dependent inhibition of protein synthesis in 72-hour-cultured rat liver slices, cytotoxicity was markedly reduced in liver slices cultured with 50 μm DIM. These results demonstrate that cultured rat liver slices may be employed to evaluate the effects of chemicals derived from cruciferous and other vegetables on CYP isoforms. In addition, liver slices can also be utilized to examine the ability of such chemicals to modulate xenobiotic-induced toxicity.

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,

References (48)

  • O.H. Lowry et al.

    Protein measurement with the Folin phenol reagent

    Journal of Biological Chemistry

    (1951)
  • R. McDanell et al.

    Differential induction of mixed-function oxidase (MFO) activity in rat liver and intestine by diets containing processed cabbage: correlation with cabbage levels of glucosinolates and glucosinolate hydrolysis products

    Food and Chemical Toxicology

    (1987)
  • R. McDanell et al.

    Chemical and biological properties of indole glucosinolates (glucobrassicins): a review

    Food and Chemical Toxicology

    (1988)
  • D. Müller et al.

    Monooxygenation, cytochrome P4501A1 and P4501A1–mRNA in rat liver slices exposed to beta-naphthoflavone and dexamethasone in vitro

    Experimental Toxicology and Pathology

    (1996)
  • P.V. Nerurkar et al.

    Methoxyresorufin and benzyloxyresorufin : substrates preferentially metabolized by cytochromes P4501A2 and 2B, respectively, in the rat and mouse

    Biochemical Pharmacology

    (1993)
  • A.B. Okey

    Enzyme induction in the cytochrome P-450 system

    Pharmacology and Therapeutics

    (1990)
  • A.R. Parrish et al.

    Precision-cut tissue slices : applications in pharmacology and toxicology

    Life Sciences

    (1995)
  • R. Staack et al.

    A comparison of the individual and collective effects of four glucosinolate breakdown products from Brussels sprouts on induction of detoxification enzymes

    Toxicology and Applied Pharmacology

    (1998)
  • G.S. Stoewsand

    Bioactive organosulfur phytochemicals in Brassica oleracea vegetables—a review

    Food and Chemical Toxicology

    (1995)
  • O. Vang et al.

    Induction of cytochrome P-450IA1, IA2, IIB1, IIB2 and IIE1 by broccoli in rat liver and colon

    Chemico–Biological Interactions

    (1991)
  • D.T.H. Verhoeven et al.

    A review of mechanisms underlying anticarcinogenicity by brassica vegetables

    Chemico–Biological Interactions

    (1997)
  • H.M. Wortelboer et al.

    Acid reaction products of indole-3-carbinol and their effects on cytochrome P450 and phase II enzymes in rat and monkey hepatocytes

    Biochemical Pharmacology

    (1992)
  • H.M. Wortelboer et al.

    Effects of cooked Brussels sprouts on cytochrome P-450 profile and phase II enzymes in liver and small intestinal mucosa of the rat

    Food and Chemical Toxicology

    (1992)
  • H.M. Wortelboer et al.

    Effects of indole-3-carbinol on biotransformation enzymes in the rat: in vivo changes in liver and small intestinal mucosa in comparison with primary hepatocyte cultures

    Food and Chemical Toxicology

    (1992)
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