Age-dependent change of metabolic capacity and genotoxic injury in rat intestine

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Abstract

The ontogeny of intestinal phase I and II xenobiotic metabolising enzymes and influence on susceptibility to genotoxic injury, are unclear. This study assessed expression of cytochrome P450 monooxygenases (CYP1A, CYP2B, CYP2C, CYP3A, CYP4A), glutathione-S-transferase (GSTA1/2, GSTA3, GSTA4 and GSTM1), and uridine diphosphate glucuronosyl transferase (UGT) in rat intestine, between fetal life and maturity. Enzyme induction and DNA adduct formation were assessed after 3-methylcholanthrene (MC) exposure. Untreated rat intestine expressed CYP2B, GSTA1/2, GSTA4 and UGT at all stages of maturation, although CYP2B and GSTA1/2 increased in postnatal life. MC induced new expression of CYP1A, GSTA3 and enhanced expression of GSTA1/2 and UGT. Age-dependent differences of enzyme induction and DNA adduct formation between pre- and postnatal intestine and during postnatal maturation, were observed. Rat intestinal epithelium shows variable competence for MC metabolism and sustains disparate levels of DNA adducts during pre- and postnatal development.

Introduction

The intestine is exposed to potentially mutagenic substances from fetal development 1, 2to maturity. Polycyclic aromatic hydrocarbons (PAH) are found in tobacco smoke as well as in barbecued or smoked food [3]and have been shown to cause DNA adduct formation in human colon [4]. A relationship between cigarette smoking and colorectal cancer incidence has been demonstrated [5].

PAH induce a metabolic response, regulated by the aryl hydrocarbon receptor (Ahr) which involves cytochrome P450 CYP1A1, CYP1A2, alpha class glutathione-S-transferases (GSTs), uridine diphosphate glucuronosyl transferases (UGTs), as well as other enzymes [6]. This response produces a metabolic equilibrium, involving the generation of reactive electrophiles by cytochrome P450 monooxygenases on one hand and detoxification by phase II enzymes on the other. It determines the effective yield of PAH reactive intermediates which are capable of covalent binding with DNA, formation of adducts and initiation of genotoxic damage. Altered expression or induction of any bioactivation or detoxification enzymes could thus have profound influence on the toxicological outcome.

Certain phase I and II enzymes are implicated in tissue growth and development 7, 8. Conversely, little information is available relating to age dependent changes of metabolic capacity and their relationship to genotoxic injury in the intestine. Methylcholanthrene (MC) is a potent PAH carcinogen which may cross the placenta and shows a consistently positive correlation between Ahr responsiveness and tumorogenicity [9]. This study assessed the metabolic response and genotoxic injury induced by MC, in rat intestine at various developmental stages between fetal life and maturity. Expression and induction of cytochrome P450 monooxygenases (CYP1A, CYP2B, CYP2C, CYP3A, CYP4A), glutathione-S-transferases (GSTA1/2, GSTA3, GSTA4 and GSTM1), and uridine diphosphate glucuronosyl transferase (UGT) were assessed by immunoblot while MC induced DNA-adduct formation was measured by 32P-postlabelling.

Section snippets

Chemicals

All chemicals were of analytical grade and readily available commercially.

Animals

Inbred AO/Ola Hsd rats (Olac, Manchester, UK) were kept in a 12-h light-dark cycle and allowed standard diet (rat and mouse number1 diet, SDS Ltd.) and water ad libitum. All animals were weaned at 21 days. Postnatal studies were performed using male (150–250 g) animals only. For fetal studies, timed-matings were performed and gestational age was measured from the time a vaginal mucus plug first appeared. Fetal sex-typing

Expression of Phases I and II metabolising enzymes in maturing rat intestine before and after MC exposure.

Phases I and II enzymes and DNA adduct formation were assayed in a total of 120 postnatal and 72 fetal rat intestines.

Cytochrome P450 monooxygenases (CYP)

CYP 2B was expressed in untreated intestine throughout maturation. Expression levels were low in fetal and early postnatal intestine, but showed a modest increase at later stages of postnatal maturation. Intraperitoneal MC had minimal effects on CYP2B expression, but induced expression of CYP1A. Induction of CYP1A was greater in postnatal, than fetal intestine (Fig. 1). CYP2C,

Discussion

Adaptive changes in the intestinal digestive and other functions during development, enable transition to independent life [18]. The intestine is an important site of biotransformation and first pass metabolism 19, 20, 21, yet data relating to developmental change of intestinal phase I and II xenobiotic metabolising enzymes are scant. De Waziers et al. (1988) [22]observed no differences in rat intestine P450 content between 18 and 28 days of postnatal life but demonstrated a 4-fold increase of

Acknowledgements

Dr H.R.H. Patel was funded by a Medical Research Council Clinical Training Fellowship (G84/3286). Dr D.H. Phillips and Mr A. Hewer are supported by the Cancer Research Campaign. This financial support is gratefully acknowledged. We also thank Professors C. Roland Wolf, Biomedical Research Centre and Brian Burchell, Department of Biochemical Medicine, Ninewells Hospital, Dundee, Scotland for the kind provision of cytochrome P450, GST and UGT antibodies, respectively.

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