Abstract
Perfluorinated compounds (PFCs) are emerging compounds of concern. They are widely distributed in the environment, wildlife and human. Concern has been raised over their possible adverse effects on human health. This study was designed to determine cytotoxic effects of two important PFCs, perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS), in a single and a mixture of them exposure to Hep G2 cells. The results showed that PFOA and PFOS (50–200 μmol/l) induced production of reactive oxygen species (ROS), dissipation of mitochondria membrane potential and apoptosis of Hep G2 cells. Moreover, activities of superoxide dismutase, catalase and glutathione reductase were increased, whereas activities of glutathione-S-transferase and glutathione peroxidase were decreased. Glutathione content was reduced. Differential expression of genes, such as p53, Bcl-2, caspase-9, was evident in PFOA or PFOS exposure groups. The possible mechanism was that they could overwhelm homeostasis of antioxidative systems, boost ROS generation, impact mitochondria, and affect genes expression of apoptotic regulators, which resulted in start-ups of apoptosis program. Cells exposed to mixture of PFOA and PFOS and each of them showed non-apoptotic rate significant difference, which indicated that the combined effect of two compounds was summation effect, but neither synergistic nor antagonistic effect.
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References
Agarwal R, Talati M, Lambert W, Clark AF, Wilson SE, Agarwal N, Wordinger RJ (1999) Fas-activated apoptosis and apoptosis mediators in human trabecular meshwork cells. Exp Eye Res 68:583–590. doi:10.1006/exer.1998.0636
Bossi R, Riget FF, Dietz R, Sonne C, Fauser P, Dam M, Vorkamp K (2005) Preliminary screening of perfluorooctane sulfonate (PFOS) and other fluorochemicals in fish, birds and marine mammals from Greenland and the Faroe Islands. Environ Pollut 136:323–329. doi:10.1016/j.envpol.2004.12.020
Cai J, Wallace DC, Zhivotovsky B, Jones DP (2000) Separation of cytochrome c-dependent caspase activation from thiol-disulfide redox change in cells lacking mitochondrial DNA. Free Radic Biol Med 29:334–342. doi:10.1016/S0891-5849(00)00312-9
Cathcart R, Schwiers E, Ames BN (1983) Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Anal Biochem 134:111–116. doi:10.1016/0003-2697(83)90270-1
Chen YM, Guo LH (2009) Fluorescence study on site-specific binding of perfluoroalkyl acids to human serum albumin. Arch Toxicol 83(3):255–261. doi:10.1007/s00204-008-0359-x
Chen XY, Lv P, Liu J, Xu KS (2008) Apoptosis of human hepatocellular carcinoma cell (HepG2) induced by cardiotoxin III through S-phase arrest. Exp Toxicol Pathol. doi:10.1016/j.etp.2008.09.006
Choi J, Cinrad CC, Malakowsky CA, Talent JM, Yuan CS, Gracy RW (2002) Flavones from Scutellaria baicalensis Georgi attenuate apoptosis and protein oxidation in neuronal cell lines. Biochim Biophys Acta 1571:201–210
Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326:1–16
Dlamini Z, Mbita Z, Zungu M (2004) Genealogy, expression, and molecular mechanisms in apoptosis. Pharmacol Ther 101:1–15. doi:10.1016/j.pharmthera.2003.08.005
Ekert PG, Read SH, Silke J, Marsden VS, Kaufmann H, Hawkins CJ, Gerl R, Kumar S, Vaux DL (2004) Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die. J Cell Biol 165:835–842. doi:10.1083/jcb.200312031
Ericson I, Gomez M, Nadal M, van Bavel B, Lindstrom G, Domingo JL (2007) Perfluorinated chemicals in blood of residents in Catalonia (Spain) in relation to age and gender: A pilot study. Environ Int 33:616–623. doi:10.1016/j.envint.2007.01.003
Giesy JP, Kannan K (2001) Global distribution of perfluorooctane sulfonate in wildlife. Environ Sci Technol 35:1339–1342. doi:10.1021/es001834k
Gil J, Almeida S, Oliveira CR, Rego AC (2003) Cytosolic and mitochondrial ROS in staurosporine-induced retinal cell apoptosis. Free Radic Biol Med 35:1500–1514. doi:10.1016/j.freeradbiomed.2003.08.022
Gong JG, Costanzo A, Yang HQ, Melino G, Kaelin WG Jr, Levrero M, Wang JYJ (1999) The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399(6738):806–809. doi:10.1038/21690
Gorczyca W, Gong JP, Ardelt B, Traganos F, Darzynkiewicz Z (1993) The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res 53:3186–3192
Guo Q, Christakos S, Robinsen N, Mattson MP (1998) Calbindin D28k blocks the proapoptotic actions mutant presenilin 1: reduced oxidative stress and preserved mitochondrial function. Proc Natl Acad Sci USA 95:3227–3232. doi:10.1073/pnas.95.6.3227
Guruge KS, Yeung LWY, Yamanaka N, Miyazaki S, Lam PKS, Giesy JP, Jones PD, Yamashita N (2006) Gene expression profiles in rat liver treated with perfluorooctanoic acid (PFOA). Toxicol Sci 89:93–107. doi:10.1093/toxsci/kfj011
Haynes RL, Brune B, Townsend AJ (2001) Apoptosis in Raw 264.7 cells exposed to 4-hydroxy-2-nonenal: dependence on cytochrome c release but not P53 accumulation. Free Radic Biol Med 30(8):884–894. doi:10.1016/S0891-5849(01)00476-2
He P, Wang AG, Xia T, Gao P, Niu Q, Guo LJ, Xu BY, Chen XM (2009) Mechanism of the neurotoxic effect of PBDE-47 and interaction of PBDE-47 and PCB153 in enhancing toxicity in SH-SY5Y cells. Neurotoxicology 30:10–15. doi:10.1016/j.neuro.2008.10.004
Higgins CP, Field JA, Criddle CS, Luthy RG (2005) Quantitative determination of perfluorochemicals in sediments and domestic sludge. Environ Sci Technol 39:3946–3956. doi:10.1021/es048245p
Hu W, Jones PD, Upham BL, Trosko JE, Lau C, Giesy JP (2002) Inhibition of gap junctional intercellular communication by perfluorinated compounds in rat liver and dolphin kidney epithelial cell lines in vitro and Sprague-Dawley rats in vivo. Toxicol Sci 68:429–436. doi:10.1093/toxsci/68.2.429
Hu DC, Liu Q, Cui HR, Xu HB, Wang H, Han DX (2005) Effects of amino acids from selenium-rich silkworm pupas on human hepatoma cells. Life Sci 77:2098–2110. doi:10.1016/j.lfs.2005.02.017
Hu XZ, Xu Y, Hu DC, Hui Y, Yang FX (2007) Apoptosis induction on human hepatoma cells Hep G2 of decabrominated diphenyl ether (PBDE-209). Toxicol Lett 171:19–28. doi:10.1016/j.toxlet.2007.04.002
Hu XZ, Hu DC, Xu Y (2009) Effects of tetrabrominated diphenyl ether and hexabromocyclododecanes in single and complex exposure to hepatoma HepG2 cells. Environ Toxicol Pharmacol 27:327–337. doi:10.1016/j.etap.2008.11.014
Jing Y, Ohizumi H, Kawazoe N, Hashimoto S, Masuda Y, Nakajo S, Yoshida T, Kuroiwa Y, Nakaya K (1994) Selective inhibitory effect of bufalin on growth of human tutor cells in vitro: association with the induction of apoptosis in leukemia HL-60 cells. Jpn J Cancer Res 85:645–651
Jung U, Zheng XX, Yoon SO, Chung AS (2001) Se-Methylselenocysteine induces apoptosis mediated by reactive oxygen species in HL-60 cells. Free Radic Biol Med 31:479–489. doi:10.1016/S0891-5849(01)00604-9
Kannan K, Choi J, Iseki N, Senthilkumar K, Kim DH, Masunaga S, Giesy JP (2002) Concentrations of perfluorinated acids in livers of birds from Japan and Korea. Chemosphere 49:225–231. doi:10.1016/S0045-6535(02)00304-1
Key BD, Howell RD, Criddle CS (1997) Fluorinated organics in the biosphere. Environ Sci Technol 31:2445–2454. doi:10.1021/es961007c
Kleszczynski K, Skladanowski AC (2009) Mechanism of cytotoxic action of perfluorinated acids. I. Alteration in plasma membrane potential and intracellular pH level. Toxicol Appl Pharmacol 234:300–305. doi:10.1016/j.taap.2008.10.008
Kleszczynski K, Gardzielewski P, Mulkiewicz E, Stepnowski P, Składanowski AC (2007) Analysis of structure–cytotoxicity in vitro relationship (SAR) for perfluorinated carboxylic acids. Toxicol In Vitro 21:1206–1211. doi:10.1016/j.tiv.2007.04.020
Knowles B, Howem C, Aden D (1980) Human hepatocellular carcinoma cell lines (HepG2) secrete the major plasma proteins and hepatitis B surface antigen. Science 209:497–499. doi:10.1126/science.6248960
Liao CY, Cui L, Zhou QF, Duan SM, Jiang GB (2009) Effects of perfluorooctane sulfonate on ion channels and glutamate-activated current in cultured rat hippocampal neurons. Environ Toxicol Pharmacol 27:338–344. doi:10.1016/j.etap.2008.11.013
Liu CS, Yu K, Shi XJ, Wang JX, Lam PKS, Wu RSS, Zhou BS (2007) Induction of oxidative stress and apoptosis by PFOS and PFOA in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Aquat Toxicol 82:135–143. doi:10.1016/j.aquatox.2007.02.006
Livingstone DR (2001) Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar Pollut Bull 42:656–666. doi:10.1016/S0025-326X(01)00060-1
Ma Y, Cao L, Kawabata T, Yoshino T, Yang BB, Okada S (1998) Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells. Free Radic Biol Med 25:568–575. doi:10.1016/S0891-5849(98)00088-4
Martin JW, Smithwick MM, Braune BM, Hoekstra PF, Muir DCG, Mabury SA (2004) Identification of long-chain perfluorinated acids in biota from the Canadian arctic. Environ Sci Technol 38:373–380. doi:10.1021/es034727+
Martinou JC, Green DR (2001) Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2(1):63–67. doi:10.1038/35048069
McConnell BB, Vertino PM (2000) Activation of a caspase-9-mediated apoptotic pathway by subcellular redistribution of the novel caspase recruitment domain protein TMS1. Cancer Res 60:6243–6247
Panaretakis T, Shabalina IG, Grander D, Shoshan MC, De Pierre JW (2001) Reactive oxygen species and mitochondria mediate the induction of apoptosis in human hepatoma HepG2 cells by the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid. Toxicol Appl Pharmacol 173:56–64. doi:10.1006/taap.2001.9159
Radice S, Chiesara E, Fucile S, Marabini L (2008) Different effects of PCB101, PCB118, PCB138 and PCB153 alone or mixed in MCF-7 breast cancer cells. Food Chem Toxicol 46:2561–2567. doi:10.1016/j.fct.2008.04.012
Ricci JE, Gottlieb RA, Green DR (2003) Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. J Cell Biol 160:65–75. doi:10.1083/jcb.200208089
Roos PH, Angerer J, Dieter H, Wilhelm M, Wolfle D, Hengstler JG (2008) Perfluorinated compounds (PFC) hit the headlines: meeting report on a satellite symposium of the annual meeting of the German Society of Toxicology. Arch Toxicol 82(1):57–59. doi:10.1007/s00204-007-0225-2
Santos MA, Pacheco M, Ahmad I (2004) Anguilla anguilla L. antioxidants responses to in situ bleached kraft pulp mill effluent outlet exposure. Environ Int 30:301–308. doi:10.1016/S0160-4120(03)00178-8
Seacat AM, Thomford PJ, Hansen KJ, Olsen GW, Case MT, Butenhoff JL (2002) Subchronic toxicity studies on perfluorooctanesulfonate potassium salt in cynomolgus monkeys. Toxicol Sci 68:249–264. doi:10.1093/toxsci/68.1.249
Shabalina IG, Panaretakis T, Bergstrand A, De Pierre JW (1999) Effects of the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid, on apoptosis in human hepatoma HepG2 cells. Carcinogensis 20:2237–2346. doi:10.1093/carcin/20.12.2237
Shen Y, White E (2001) p53-dependent apoptosis pathways. Adv Cancer Res 82:55–84. doi:10.1016/S0065-230X(01)82002-9
Shen HM, Ong CN, Shi CY (1995) Involvement of reactive oxygen species in aflatoxin B1-induced cell injury in cultured rat hepatocytes. Toxicology 99:115–123. doi:10.1016/0300-483X(94)03008-P
Simizu S, Takada M, Umezawa K, Imoto M (1998) Requirement of caspase-3(-like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs. J Biol Chem 273:26900–26907. doi:10.1074/jbc.273.41.26900
So MK, Taniyasu S, Yamashita N, Giesy JP, Zheng J, Fang Z, Im SH, Lam PKS (2004) Perfluorinated compounds in coastal waters of Hong Kong, South China, and Korea. Environ Sci Technol 38:4056–4063. doi:10.1021/es049441z
So MK, Miyake Y, Yeung WY, Ho YM, Taniyasu S, Rostkowski P, Yamashita N, Zhou BS, Shi XJ, Wang JX, Giesy JP, Yu H, Lam PKS (2007) Perfluorinated compounds in the Pearl River and Yangtze River of China. Chemosphere 68:2085–2095. doi:10.1016/j.chemosphere.2007.02.008
Son HY, Kim SH, Shin HI, Bae HI, Yang JH (2008) Perfluorooctanoic acid-induced hepatic toxicity following 21-day oral exposure in mice. Arch Toxicol 82(4):239–246. doi:10.1007/s00204-007-0246-x
Vettori MV, Goldoni M, Caglieri A, Poli D, Folesani G, Ceccatelli S, Mutti A (2006) Antagonistic effects of methyl-mercury and PCB153 on PC12 cells after a combined and simultaneous exposure. Food Chem Toxicol 44:1505–1512. doi:10.1016/j.fct.2006.04.009
Volkel W, Genzel-Boroviczeny O, Demmelmair H, Gebauer C, Koletzko B, Twardella D, Raab U, Fromme H (2008) Perfluorooctane sulphonate (PFOS) and perfluorooctanoic acid (PFOA) in human breast milk: Results of a pilot. Int J Hyg Environ Health 211(3–4):440–446. doi:10.1016/j.ijheh.2007.07.024
Winston GW, Di Giulio RT (1991) Peroxidant and antioxidant mechanisms in aquatic organisms. Aquat Toxicol 19:137–161. doi:10.1016/0166-445X(91)90033-6
Yao X, Zhong L (2005) Genotoxic risk and oxidative DNA damage in HepG2 cells exposed to perfluorooctanoic acid. Mutat Res 587:38–44
Zamzami N, Kroemer G (2001) The mitochondrion in apoptosis: how Pandora’s box opens. Nat Rev Mol Cell Biol 2:67–71. doi:10.1038/35048073
Zhang CX, Huang KX (2006) Mechanism of apoptosis induced by a polysaccharide, from the loach Misgurnus anguillicaudatus (MAP) in human hepatocellular carcinoma cells. Toxicol Appl Pharmacol 210:236–245. doi:10.1016/j.taap.2005.04.019
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This is a part of the work in the research projects supported by General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (2008IK171 and 2009IK139).
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Hu, XZ., Hu, DC. Effects of perfluorooctanoate and perfluorooctane sulfonate exposure on hepatoma Hep G2 cells. Arch Toxicol 83, 851–861 (2009). https://doi.org/10.1007/s00204-009-0441-z
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DOI: https://doi.org/10.1007/s00204-009-0441-z