Peroxisome proliferators do not increase DNA synthesis in purified rat hepatocytes

induce peroxisomes in rodent liver increase DNA synthesis in isolated hepat ic parenchymal cells, but not as well in vitro as in vivo. It is also known that tumour necrosis factor ex (TNF alpha) is mitogenic in isolated hepato cytes. Since Kupffer cells are a major source of TNF alpha in the liver and have recently been shown to be activated by peroxisome proliferators, the possibility exists that the effect of peroxisome proliferators on DNA synth esis in parenchymal cells is via Kupffer cell contamination of isolated hep atocyte preparations. The purpose of this study was to evaluate this hypoth esis by studying the effect of model peroxisome proliferators on purified h epatocyte preparations. Hepatocytes were prepared from rat liver by standar d calcium-free and collagenase perfusion. Subsequently, cells were centrifu ged through Percoll to remove contaminating nonparenchymal cells. Cells wer e at least 99.9% pure as assessed by cell counting using specific markers f or hepatocytes (resorufin O-glucoside) and Kupffer cells (FITC-labelled lat ex beads). Hepatocytes were cultured in Williams medium + 10% fetal bovine serum for 24 h followed by culture for 48 h in Williams medium plus or minu s drug or mitogen additions. Under these conditions epidermal growth factor stimulated DNA synthesis assessed by incorporation of [H-3]thymidine -5-fo ld over control levels. The peroxisome proliferators WY,14-643 and nafenopi n, however, had no effect on DNA synthesis, although they did increase acyl -CoA oxidase as expected. In contrast, TNFa increased cell proliferation ne arly 10-fold in purified hepatocytes, an effect nearly doubled by WY-14,643 , Further, when conditioned medium from purified Kupffer cells incubated wi th WY-14,643 was added to pure hepatocytes, DNA synthesis was increased ove r 2-fold in a time-dependent manner. Collectively, these data support the h ypothesis that peroxisome proliferators do not influence DNA synthesis in i solated hepatocytes per se. Rather, they stimulate cytokine production by K upffer cells which in turn increases DNA synthesis in parenchymal cells. An increase in mitogenic cytokine production by Kupffer cells is necessary fo r stimulation of DNA synthesis in purified rat parenchymal cells.