Cytochrome P450-mediated metabolism and cytotoxicity in rat cultured hepatocytes
The aim of this investigation was to define the in vitro conditions necessary to support cytochrome P450-mediated metabolism in rat cultured hepatocytes, such that this system could then be used as an in vitro model in the study of cytochrome P450-mediated cytotoxicity. Maintenance of P450-dependent enzyme activities in culture was not affected by supplementation of culture medium with haem, but was markedly influenced by the age and sex of the hepatocyte donor animal. Induction in primary culture by phenobarbitone and beta-naphthoflavone was investigated, and found to be quantitatively and qualitatively different to the induction observed in vivo, hepatocytes in culture being particularly refractive to induction by phenobarbitone. The maintenance in primary culture of a range of enzyme activities was determined following treatment of rats in vivo with isoniazid and dexamethasone, in addition to phenobarbitone and beta-naphthoflavone, and in general, there was good maintenance of the induced activities. The activities were chosen as possible selective substrates for the different induced isozymes, with a view to using the activity profiles to characterise different classes of inducer; however, although selective induction was observed with isoniazid, beta-naphthoflavone and dexamethasone, all the chosen activities were induced by phenobarbitone. The final part of this work involved determining cytotoxicity in vitro, following induction of P450 in vivo with phenobarbitone and beta-naphthoflavone. Seven known hepatotoxins were investigated, and the results obtained agreed well with available in vitro and in vivo literature data. In summary, a range of constitutive and induced enzyme activities were maintained at high levels in hepatocytes cultured for twenty-four hours from adult male rats, and an induction in vivo/hepatocyte culture protocol shown to be a viable in vitro model for the study of metabolism-mediated toxicity, as an alternative to induction and detection of toxicity in vitro. NB. This ethesis has been created by scanning the typescript original and may contain inaccuracies. In case of difficulty, please refer to the original text.