of Work: Primary rat hepatocyte (HPC) cultures and Hepatocyte/nonparenchymal cell (HPC/ NPC) cocultures were treated with Wyeth-14,643 (WY), a potent rodent carcinogen, and replicative DNA synthesis and specific mRNA transcript levels were determined by reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting. An increase in HPC replicative DNA synthesis was detected at 48 hours in both WY- treated HPC and HPC/NPC cocultures relative to controls. This increase was approximately 3 and 6 fold in HPC and HPC/NPC cultures, respectively, and was WY concentration-dependent. The levels of PPARa-transcriptionally dependent genes (cytochrome P4504A1, acyl-CoA oxidase (AOxase), and liver-fatty acid binding protein (L-FABP)) transcripts were determined as indicators of PPARa activation. These transcripts increased dose- dependently at 48 hours in both HPC and HPC/NPC cultures up to 10 mM WY. We also investigated the mechanisms of induction of replicative DNA synthesis by WY. WY-induced replicative DNA synthesis was concentration-dependent and was significantly greater in HPC incubated with NPC compared to HPC cultured separately. WY-induced DNA synthesis was inhibited by 10-undecynoic acid (4504A1 inhibitor) in a dose-dependent manner, with complete inhibition achieved at 250 mM. Further, WY-induced DNA synthesis was inhibited by indomethacin (cyclooxygenase inhibitor) in a dose- dependent with complete inhibition observed at 10 mM. In conclusion, an active P4504A1 and COXII are required for the induction of replicative DNA synthesis by WY, and since both HPC and NPC are required for an optimal response, we hypothesized that COXII and P4504A1 are localized in NPC and HPC, respectively. Work is currently in progress to examine this hypothesis by assessing the effects of WY and other peroxisome proliferators on HPC and NPC isolated from COXI and COXII knockout mice both in vivo and in vitro.
