The hypothesis this proposal will test is whether results previously generated for regulation of hepatic Cytochrome P4503A (3A) in rat and/or rabbit models can be used to predict human hepatic 3A regulation. This hypothesis will be tested by examining regulation of 3A in replicating and primary cultures of human hepatocytes by environmental agents (six non-planar polychlorinated biphenyls, five cyclodiene organochlorine pesticides) and hormones (sex steroids and the peptide hormones, growth hormone and thyroid hormone) which have previously been demonstrated to regulate hepatic 3A in rats and rabbits. By identifying similarities and differences between rodent and human hepatic 3A regulation, where they exist, will contribute to the broad, long-term objective of this proposal which is to improve interspecies extrapolations and predictive modeling for human risk from toxic or carcinogenic chemicals. The proposed studies are based on the establishment and validation of human hepatocyte culture models which retain expression of the individual members of the human 3A gene family. Specifically, expression of 3A3/4, 3A5 and 3A7 will be examined in primary human hepatocytes, TONG/HCC and HepG2 cells, respectively.
The Specific Aims of this proposal are to elucidate the controls regulating xenobiotic and hormonal hepatocellular expression of the human CYP3A family in the human hepatocyte models. This will be done by first, using standard enzyme activity assays, specific antibodies and cDNAs to examine the effect of the test agents on the activity of human hepatic 3A enzymes, the amount of human hepatic 3A mRNAs (assayed by northern blot or the polymerase chain reaction), respectively. Having characterized xenobiotic and hormonal regulation of hepatic 3A, the second aim will be to determine the mechanism of regulation of 3A gene expression by the test agents at any one of the various regulatory levels, i.e., transcription (using the nuclear run-off assay), post-transcriptional (mRNA stability), and protein degradation (by measuring the rate of 3A turnover).
The final aim will be to determine the mechanism of 3A gene activation by environmental agents and peptide hormones. This will be accomplished by transfecting recombinant chimeric 3A-CAT plasmids (provided in collaboration with Project #1 of this program project and containing putatively regulatory regions of the 3A genes upstream of the reporter gene chloramphenicol acetyltransferase) into human hepatocytes and examining the effect of the test agents on CAT activity. These studies in human hepatocytes will provide novel information on the molecular regulation of human hepatic 3A by environmental agents and hormones.
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