Induction of replicative DNA synthesis and PPAR alpha-dependent gene transcription by Wy-14 643 in primary rat hepatocyte and non- parenchymal cell co-cultures

Peroxisome proliferators (PP) are known hepatocarcinogens in rats and mice. We have investigated the ability of Wyeth-14 643 (Wy), a PP and potent rodent carcinogen, to induce replicative DNA synthesis and to modulate the levels of peroxisome proliferator activated receptor-alpha (PPAR alpha) transcriptionally-dependent genes in primary rat hepatocyte (HPC) cultures and hepatocyte/nonparenchymal cell (HPC/NPC) co-cultures maintained on Matrigel. Four days after plating, cells were treated with Wy and replicative DNA synthesis was quantitated using [3H]thymidine incorporation and specific mRNA transcript levels were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). An increase in HPC replicative DNA synthesis was detected at 48 h in both Wy-treated HPC and HPC/NPC co-cultures 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 PPAR alpha-transcriptionally dependent genes [cytochrome P4504A1, acyl-CoA oxidase (AOxase), and liver-fatty acid binding protein (L-FABP)] transcripts were determined as indicators of PPAR alpha activation. These transcripts increased dose-dependently at 48 h in HPC/NPC cultures up to 10 microM Wy. Similarly, RT-PCR product levels were also increased in HPC cultures with 10 microM Wy at 48 h. In conclusion, we have investigated the transcription of PPAR alpha-dependent genes and HPC replicative DNA synthesis by Wy in HPC/NPC co-cultures. Results of this work are clearly more reflective of the known in vivo effects of PP and suggest that HPC/NPC co-cultures are more appropriate than HPC cultures for such studies. The effect of PP on human HPC/NPC co-cultures is currently being investigated in our laboratory in an attempt to assess human risks to these chemicals more directly.