Reactive oxygen species and mitochondria mediate the induction of apoptosis in human hepatoma HepG2 cells by the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid.

We have previously shown that one of the most potent rodent hepatocarcinogens, perfluorooctanoic acid (PFOA), induces apoptosis in human HepG2 cells in a dose- and time-dependent manner. In this study we have investigated the involvement of reactive oxygen species (ROS), mitochondria, and caspase-9 in PFOA-induced apoptosis. Treatment with 200 and 400 microM PFOA was found to cause a dramatic increase in the cellular content of superoxide anions and hydrogen peroxide after 3 h. Measurement of the mitochondrial transmembrane potential (Delta Psi(m)) after PFOA treatment showed a dissipation of Delta Psi(m) at 3 h. Caspase-9 activation was seen at 5 h after treatment with 200 microM PFOA. In order to evaluate the importance of these events in PFOA-induced apoptosis, cells were cotreated with PFOA and N-acetylcysteine (NAC), a precursor of glutathione, or Cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MPT pore). NAC reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced ROS. In addition, CsA also reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced opening of the MPT pore. In summary, we have delineated a ROS and mitochondria-mediated pathway for induction of apoptosis by PFOA.

[1]  Gregor Rothe,et al.  Flow Cytometric Analysis of Respiratory Burst Activity in Phagocytes With Hydroethidine and 2′,7′‐Dichlorofluorescin , 1990, Journal of leukocyte biology.

[2]  C. Scotto,et al.  Comparative effects of clofibrate on peroxisomal enzymes of human (Hep EBNA2) and rat (FaO) hepatoma cell lines. , 1995, European journal of cell biology.

[3]  H Yamanaka,et al.  Inhibition of mitochondrial respiration and oxygen-dependent hepatotoxicity by six structurally dissimilar peroxisomal proliferating agents. , 1992, Toxicology.

[4]  M. Dempsey,et al.  Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria. , 1989, The Journal of biological chemistry.

[5]  J. Reddy,et al.  Hydrogen peroxide generation in peroxisome proliferator-induced oncogenesis. , 2000, Mutation research.

[6]  D. Jaeck,et al.  Comparison of the effects of various peroxisome proliferators on peroxisomal enzyme activities, DNA synthesis, and apoptosis in rat and human hepatocyte cultures. , 1999, Toxicology and applied pharmacology.

[7]  Javier Naval,et al.  Implication of Mitochondrial Hydrogen Peroxide Generation in Ceramide-induced Apoptosis* , 1997, The Journal of Biological Chemistry.

[8]  G. Kroemer,et al.  The mitochondrion in cell death control: certainties and incognita. , 2000, Experimental cell research.

[9]  J. Depierre,et al.  Hepatic oxidative stress and related defenses during treatment of mice with acetylsalicylic acid and other peroxisome proliferators. , 1995, Journal of biochemical toxicology.

[10]  J. Popp,et al.  Several nongenotoxic carcinogens uncouple mitochondrial oxidative phosphorylation. , 1992, Biochimica et biophysica acta.

[11]  W. Telford,et al.  Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. , 1992, Cytometry.

[12]  S. Srinivasula,et al.  Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade , 1997, Cell.

[13]  J. Martinou,et al.  Mitochondria as the central control point of apoptosis. , 2000, Trends in cell biology.

[14]  J. Robinson,et al.  Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells , 1994, Journal of leukocyte biology.

[15]  B Beije,et al.  On the mechanism of the hepatocarcinogenicity of peroxisome proliferators. , 1991, Chemico-biological interactions.

[16]  G. Kroemer,et al.  Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis , 1996, FEBS letters.

[17]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[18]  K. Kolaja,et al.  Oxidative stress in nongenotoxic carcinogenesis. , 1995, Toxicology letters.

[19]  S. Duclos,et al.  Peroxisome proliferation and beta-oxidation in Fao and MH1C1 rat hepatoma cells, HepG2 human hepatoblastoma cells and cultured human hepatocytes: effect of ciprofibrate. , 1997, European journal of cell biology.

[20]  J. Depierre,et al.  Effects of the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid, on apoptosis in human hepatoma HepG2 cells. , 1999, Carcinogenesis.

[21]  G. Kroemer,et al.  Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo , 1995, The Journal of experimental medicine.

[22]  G. Williams,et al.  Effect of rodent hepatocarcinogenic peroxisome proliferators on fatty acyl-CoA oxidase, DNA synthesis, and apoptosis in cultured human and rat hepatocytes. , 1998, Toxicology and applied pharmacology.

[23]  L. Richert,et al.  Peroxisome proliferators induce apoptosis and decrease DNA synthesis in hepatoma cell lines , 2000, Human & experimental toxicology.

[24]  J. Depierre,et al.  Perfluorooctane sulfonic acid is a potent inducer of peroxisomal fatty acid beta-oxidation and other activities known to be affected by peroxisome proliferators in mouse liver. , 1993, Pharmacology & toxicology.

[25]  M Tanaka,et al.  The induction of peroxisome proliferation in rat liver by perfluorinated fatty acids, metabolically inert derivatives of fatty acids. , 1985, Journal of biochemistry.

[26]  A. Halestrap,et al.  Oxidative Stress, Thiol Reagents, and Membrane Potential Modulate the Mitochondrial Permeability Transition by Affecting Nucleotide Binding to the Adenine Nucleotide Translocase* , 1997, The Journal of Biological Chemistry.

[27]  P. Bernardi,et al.  Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane , 1994, Journal of bioenergetics and biomembranes.

[28]  K. Wallace,et al.  The effect of peroxisome proliferators on mitochondrial bioenergetics. , 1999, Toxicological sciences : an official journal of the Society of Toxicology.

[29]  R. Thurman,et al.  Induction of peroxisomes by treatment with perfluorooctanoate does not increase rates of H2O2 production in intact liver. , 1992, Toxicology letters.

[30]  A. Vercesi,et al.  The participation of pyridine nucleotides redox state and reactive oxygen in the fatty acid‐induced permeability transition in rat liver mitochondria , 1999, FEBS letters.

[31]  P. Costelli,et al.  Peroxisome proliferators induce apoptosis in hepatoma cells. , 1998, Cancer detection and prevention.

[32]  A. Liss,et al.  Perfluorinated fatty acids alter merocyanine 540 dye binding to plasma membranes. , 1987, Journal of toxicology and environmental health.

[33]  B. Kalderon,et al.  Mitochondrial protonophoric activity induced by a thyromimetic fatty acid analogue. , 2000, Biochimica et biophysica acta.