Carcinogenesis, Vol. 20, No. 12, 2237-2246,
December 1999
© 1999 Oxford University Press
Cancer Biology |
Effects of the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid, on apoptosis in human hepatoma HepG2 cells
Unit of Biochemical Toxicology, Department of Biochemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm and
1 Research Center, Huddinge Hospital, Karolinska Institute, S-141 86 Stockholm, Sweden
The effects of perfluorooctanoic acid (PFOA), a potent hepatocarcinogen and peroxisome proliferator in rodents, on human cells have not yet been examined. In the present study we demonstrate that treatment of human hepatoblastoma HepG2 cells with PFOA induces apoptosis, as well as perturbs the cell cycle. This apoptosis was characterized by electron microscopy, which revealed typical nucleosomal fragmentation (also observed as a `DNA ladder' upon electrophoresis on agarose) and was quantitated using propidium iodide staining of cellular DNA and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. This process was dose- and time-dependent: apoptosis became manifest with 200 µM and maximal (45% of the cells) upon exposure to 450 µM PFOA for 24 h. Electrophoresis of the DNA from HepG2 cells exposed to 500 µM PFOA for 24 h or to 400 µM PFOA for 48 h revealed a smear typical of non-specific degradation. These findings indicate that in the presence of high concentrations of PFOA for long times, HepG2 cells undergo primary and secondary necrosis. Quantitation of trypan blue exclusion supported this conclusion. Flow cytometric analysis revealed that the cell cycle of HepG2 cells was perturbed by exposure to 50–150 µM PFOA. A 50 µM concentration resulted in a significant increase in the proportion of G2/M cells and, simultaneously, a decrease in the number of cells in the S phase, whereas treatment with 100 or 150 µM PFOA increased the proportion of cells in the G0/G1 phase and decreased the number of cells in the G2/M and S phases. Simultaneous flow cytometric analysis of apoptosis-associated DNA strand breaks using the TUNEL procedure and of propidium iodide staining of cellular DNA revealed DNA breaks in HepG2 cells exposed to 150 µM PFOA, prior to nuclear fragmentation.
Abbreviations: DMSO, dimethyl sulfoxide; EDTA, ethylenediamine tetraacetic acid; PBS, phosphate-buffered saline; PFOA, perfluorooctanoic acid.
2 To whom correspondence should be addressed Email: aris{at}tuborg.biokemi.su.se
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