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.
Karam
W G
WG
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, RTP, NC 27709, USA.
Ghanayem
B I
BI
D000818:Animals; D002273:Carcinogens / Q000633:toxicity*; D002478:Cells, Cultured; D018920:Coculture Techniques; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D011743:Pyrimidines / Q000633:toxicity*; D051381:Rats; D011916:Rats, Inbred F344; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects*
D002273:Carcinogens; D011743:Pyrimidines; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; C006253:pirinixic acid; D004247:DNA; D013936:Thymidine