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 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 microM and maximal (45% of the cells) upon exposure to 450 microM PFOA for 24 h. Electrophoresis of the DNA from HepG2 cells exposed to 500 microM PFOA for 24 h or to 400 microM 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 microM PFOA. A 50 microM concentration resulted in a significant increase in the proportion of G(2)/M cells and, simultaneously, a decrease in the number of cells in the S phase, whereas treatment with 100 or 150 microM PFOA increased the proportion of cells in the G(0)/G(1) phase and decreased the number of cells in the G(2)/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 microM PFOA, prior to nuclear fragmentation.
Shabalina
I G
IG
Unit of Biochemical Toxicology, Department of Biochemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.
Panaretakis
T
T
Bergstrand
A
A
DePierre
J W
JW
D017209:Apoptosis / Q000187:drug effects*; D002210:Caprylates / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000473:pathology* / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D005466:Fluorocarbons / Q000494:pharmacology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D008113:Liver Neoplasms / Q000473:pathology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D020025:Peroxisome Proliferators / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002210:Caprylates; D002273:Carcinogens; D005466:Fluorocarbons; D020025:Peroxisome Proliferators; C023036:perfluorooctanoic acid