The obese male Zucker rat (fa/fa), a single locus mutant, is defective (non-responsive) to the inducing effects of phenobarbital (PB). This induction defect is not global but appeared to be limited to the PB- inducible isozymes of cytochrome P-450 (P-450 + P-450e) since other enzymes, namely glutathione transferase and apolipoprotein A-1, respond to PB-treatment. However, we have new information to suggest that this defect may also involve the regulation of at least one glucuronosyltransferase protein. Our evidence for the lack of inducibility of P-450b + P-450e in this model is based on P-450b & e mRNA, anti P-450b - reactive protein and P-450b related activities (including the oxidation of testosterone via the 16beta pathway. We propose to use the non-responsive fa/fa Zucker rodent model to probe the mechanisms of regulation of cytochrome P-450. We have also observed that the fa/fa Zucker rat is resistant to the hepatotoxic effects of acetaminophen and the PB-treatment enhances the protective f effect. We therefore propose to investigate the consequences of the induction defect in the context of e xenobiotic (acetaminophen) metabolism and hepatotoxicity.
The specific aims of the project are: a) to investigate the enzyme induction defect in the fa/fa rodent, to determine if the defect is the result of an inheritable defect in the PB induction mechanism and independent of hormonal control and lipid influences and b) to elucidate the fundamental mechanism by which the fa/fa Zucker rat is resistant to acetaminophen hepatotoxicity by investigating the balance between competing activation and detoxification reactions. The methods to be employed, standard in the field of xenobiotic metabolism and molecular biology, include cDNA probes to analyze cytochrome P-450 and apolipoprotein A-I mRNA, isolated hepatocyte cultures, mRNA analysis with oligonucleotide probes, Western blot analysis with monoclonal antibodies against specific P-450 isozymes (b, e, and h) and HPLC techniques. Additionally, we propose to assess lipid metabolism as it relates to membrane lipid composition (analysis of neutral and phospholipids and fatty acids) utilizing standard HPLC and GC methods. FActors which influence the activation of foreign compounds, bear directly on risk of xenobiotic exposure to human health.
