For many environmental chemicals that have genotoxic or cytotoxic potential, metabolism by the host organism is intimately linked both to the injurious actions of the chemical as well as to its ultimate detoxification and excretion. Since the liver is the primary organ of metabolism for foreign compounds as well as endobiotics such as steroids, there is considerable interest in developing primary hepatocyte cultures as in vitro model systems that can be used to study the regulation of enzymes involved in the metabolism of such compounds. Isom etal. (16), have shown that primary hepatocytes maintained culture in the presence of dimethyl sulfoxide (DMSO) retain several differentiated functions and normal morphology for at least 7 weeks. We have subsequently found that cells maintained under these conditions respond to growth hormone (GH), which is now thought to be the ultimate mediator of sex differences in the hepatic metabolism of many compounds. We propose to use these advances as the basis for an in vitro model system for the study of the regulation of xenobiotic metabolizing enzymes. We propose to characterize the activity of the cytochrome P-450 dependent pathways of metabolism (phase I pathways) and several conjugation reactions (phase II pathways) of hepatocytes derived from male and female rats maintained in DMSO supplimented media and to use this system to study the hormonal regulation of these pathways by GH alone and in combination with estradiol, testosterone, and triiodothyronine. The substrates selected for P- 450 activity are aldrin, ethoxyresorufin, and testosterone. This range of substrates will allow us to detect most, if not all, changes in P-450 isozyme composition that may develop in response to hormone actions. For conjugation reactions and 3H- benzo(a)pyrene 4-5 oxide for epoxide hydrolase and glutathione-S transferase assays. Binding of 125I GH to hepatocytes will be measured to determine if changes in enzyme activity reflect changes at the GH receptor level. Species and strain comparisons of the role of GH, sex steroids and other hormones as regulators of xenobiotic and steroid metabolism will be conducted in mice BALB/cJ and CRL:CD-1 strains, chosen for their responsiveness and nonresponsiveness to androgens, respectively.
| Kempe, K C; Isom, H C; Greene, F E (1995) Responsiveness of an SV40-immortalized hepatocyte cell line to growth hormone. Biochem Pharmacol 49:1091-8 |
