Elsevier

Toxicology

Volume 131, Issue 1, 2 November 1998, Pages 9-20
Toxicology

Comparison of the effects of some CYP3A and other enzyme inducers on replicative DNA synthesis and cytochrome P450 isoforms in rat liver

https://doi.org/10.1016/S0300-483X(98)00085-7Get rights and content

Abstract

The aim of this study was to investigate the mitogenic effects of some inducers of cytochrome P450 (CYP) isoforms in rat liver. Female Sprague-Dawley CD rats were treated with 100 mg/kg per day of either sodium phenobarbitone (NaPB), barbituric acid (BA), isoniazid (ISN), β-naphthoflavone (BNF), pregnenolone-16α-carbonitrile (PCN), miconazole (MIC) or clotrimazole (CLOT), 75 mg/kg per day methylclofenapate (MCP), 50 mg/kg per day dexamethasone (DEX) and 500 mg/kg per day troleandomycin (TAO) by daily oral gavage for four days. Treatment with all compounds except BA, ISN and MIC, significantly increased relative liver weight. Administration of NaPB, PCN, DEX, MIC, CLOT and TAO all induced total CYP content, and by Western immunoblotting, levels of CYP3A isoforms in hepatic microsomal fractions. Apart from CLOT, all these compounds induced microsomal testosterone 6β-hydroxylase activity. By measurement of marker enzyme activities and Western immunoblotting with antibodies to CYP1A2, CYP2B1/2 and CYP2E1, BNF, NaPB, ISN and MCP were shown to induce CYP1A2, CYP2B1/2, CYP2E and CYP4A isoforms, respectively. Replicative DNA synthesis was studied by implanting osmotic pumps containing 5-bromo-2′-deoxyuridine 1 day before the commencement of treatment with the enzyme inducers. Hepatocyte labelling index values were significantly increased by treatment with NaPB, PCN, MCP, CLOT and TAO, but not by BA, ISN, BNF, DEX and MIC. These studies demonstrate that while CYP2B and CYP4A enzyme inducers may stimulate replicative DNA synthesis, only some CYP3A enzyme inducers are mitogenic agents in rat liver.

Introduction

The cytochrome P450 (CYP)-dependent mixed function oxidase system comprises a superfamily of enzymes that play a major role in the metabolism of both xenobiotics and endogenous chemicals (Conney, 1986, Nelson et al., 1996, Parkinson, 1996). Many chemicals including therapeutic agents and other xenobiotics have been shown to either induce or inhibit CYP isoform-dependent enzyme activities (Conney, 1986, Okey, 1990, Parkinson, 1996). In the development of a new therapeutic agent, it is important to ascertain whether the compound will be either an inducer or an inhibitor of xenobiotic metabolising enzymes in order to minimise potential drug interactions. The induction of CYP3A isoforms is particularly important, because CYP3A isoforms constitute 30–50% of the total CYP content of human liver, and many drugs are substrates for CYP3A isoforms (Wrighton et al., 1993, Shimada et al., 1994, Maurel, 1996, Parkinson, 1996).

In rodent liver, the administration of CYP2B enzyme inducers, such as sodium phenobarbitone (NaPB), and CYP4A enzyme inducers (peroxisome proliferators), such as methylclofenapate (MCP), is known to result in an increase in liver weight as well as in CYP isoform induction (Grasso and Hinton, 1991, Lake, 1995, Nims and Lubet, 1996). These compounds produce an increase in replicative DNA synthesis during the first few days of treatment and liver enlargement is due to both hyperplasia and hypertrophy. Although considered non-genotoxic agents, chemicals such as NaPB and peroxisome proliferators can increase the incidence of liver tumours after chronic administration to rats and/or mice and may also act as promoters of liver tumour formation in rodents (Loury et al., 1987, Nims et al., 1987, Grasso and Hinton, 1991, Popp and Cattley, 1993, Rice et al., 1994, Lake, 1995, Whysner et al., 1996).

Less information appears to be available on the mitogenic effects in rodent liver of CYP3A isoform inducers. Cyproterone acetate, dexamethasone (DEX) and pregnenolone-16α-carbonitrile (PCN) are all known to induce CYP3A isoenzymes in rat liver (Schulte-Hermann and Parzefall, 1980, Wrighton et al., 1985, Cooper et al., 1993) and cyproterone acetate has been shown to induce replicative DNA synthesis in rat hepatocytes both in vivo (Schulte-Hermann et al., 1980) and in vitro (Parzefall et al., 1989). In contrast, Thatcher and Caldwell (1994) reported that neither DEX nor PCN induced replicative DNA synthesis after short term treatment of female Sprague-Dawley rats.

The aim of the present study was to obtain further information on the mitogenic effects of some CYP3A isoform inducers in rat liver. The compounds chosen for study were PCN, DEX, miconazole (MIC), clotrimazole (CLOT) and troleandomycin (TAO). For purposes of comparison, rats were also treated with some other enzyme inducers, namely NaPB, barbituric acid (BA), isoniazid (ISN), β-naphthoflavone (BNF) and MCP, which are known to induce CYP isoforms in other subfamilies including CYP1A, CYP2B, CYP2E and CYP4A.

Section snippets

Materials

NaPB was purchased from BDH Chemicals (Poole, Dorset, UK), MCP from Lancaster Synthesis (Morecambe, Lancs., UK) and BA, BNF, ISN, PCN, DEX, MIC, CLOT, TAO, 5-bromo-2′-deoxyuridine (BRDU), unlabelled testosterone and metabolites, enzyme cofactors etc. from Sigma–Aldrich Chemical (Poole, Dorset, UK). 7-Ethoxy-4-trifluoromethylcoumarin and 7-hydroxy-4-trifluoromethylcoumarin were obtained from Enzyme Systems Products (Livermore, CA, USA). [4-14C]Testosterone (specific activity 56 mCi/mmol) and

Results

The dose levels of the test compounds were based on those employed in previous studies on rat hepatic xenobiotic metabolising enzymes (Wrighton et al., 1985, Nims et al., 1987, Ritter and Franklin, 1987a, Ritter and Franklin, 1987b, Hostetler et al., 1989, Cooper et al., 1993, Japenga et al., 1993, Lake et al., 1995). The target dose level for all compounds was 100 mg/kg per day, but due to compound potency or toxicity, MCP, DEX and TAO were administered at other dose levels. Because of the

Discussion

Treatment with NaPB, BNF, PCN, MCP, DEX, CLOT and TAO, but not BA, ISN and MIC, for 4 days significantly increased relative liver weight in female Sprague–Dawley CD rats. Administration of BNF, ISN and MCP resulted in induction of CYP isoforms in the CYP1A, CYP2E and CYP4A subfamilies, respectively. NaPB induced CYP isoforms in the CYP2A, CYP2B and CYP3A subfamilies, whereas in keeping with previous work (Nims et al., 1987), BA produced only minor effects on the parameters of xenobiotic

Acknowledgements

The authors are grateful to MSD Research Laboratories (Harlow, Essex, UK) for the provision of a research grant. They thank Professor A.R. Boobis (Department of Clinical Pharmacology, Royal Postgraduate Medical School, Hammersmith, London, UK) for the gift of the CYP1A2 antibody.

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