Effects of the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid, on apoptosis in human hepatoma HepG2 cells

The effects of perfluorooctanoic acid (PFOA), a potent hepatocarcinogen and peroxisome proliferator in rodents, on human cells have not yet been exami ned. In the present study we demonstrate that treatment of human hepatoblas toma HepG2 cells with PFOA induces apoptosis, as well as perturbs the cell cycle. This apoptosis was characterized by electron microscopy, which revea led typical nucleosomal fragmentation (also observed as a 'DNA ladder' upon electrophoresis on agarose) and was quantitated using propidium iodide sta ining of cellular DNA and the terminal deoxynucleotidyl transferase-mediate d dUTP nick end labeling (TUNEL) assay. This process was dose- and time-dep endent: apoptosis became manifest with 200 mu M and maximal (45% of the cel ls) upon exposure to 450 mu M PFOA for 24 h, Electrophoresis of the DNA fro m HepG2 cells exposed to 500 mu M PFOA for 24 h or to 400 mu M PFOA for 48 h revealed a smear typical of non-specific degradation. These findings indi cate that in the presence of high concentrations of PFOA for long times, He pG2 cells undergo primary and secondary necrosis. Quantitation of trypan bl ue exclusion supported this conclusion, Flow cytometric analysis revealed t hat the cell cycle of HepG2 cells was perturbed by exposure to 50-150 mu M PFOA, A 50 mu M concentration resulted in a significant increase in the pro portion of G(2)/M cells and, simultaneously, a decrease in the number of ce lls in the S phase, whereas treatment with 100 or 150 mu M PFOA increased t he proportion of cells in the G(0)/G(1) phase and decreased the number of c ells in the G(2)/M and S phases. Simultaneous how cytometric analysis of ap optosis-associated DNA strand breaks using the TUNEL procedure and of propi dium iodide staining of cellular DNA revealed DNA breaks in HepG2 cells exp osed to 150 mu M PFOA, prior to nuclear fragmentation.