Arsenite-induced apoptosis is prevented by antioxidants in zebrafish liver cell line.

This study evaluated oxidative stress-induced apoptosis as a possible mechanism of arsenite toxicity in zebrafish liver cell line (ZFL cells). The heat shock protein 70 (HSP70), a chaperone protein, appears to provide protection against oxidative stress and apoptosis. Using the MTT assay, we demonstrated that survival of ZFL cells treated with arsenite for 24h decreased in a dose-dependent manner. The possible mechanisms that promote the cytotoxicity of arsenite were addressed. Cell viability assays revealed that arsenite caused a dose-dependent increase in cell death, and pretreatment of the ZFL cells with antioxidants blunted these effects. Antioxidants such as N-acetyl-cysteine (NAC, 5 mM) and dithiothreitol (DTT, 80 microM) significantly prevented ZFL cells from arsenite-induced death. Nuclear staining was performed using 1 microg/ml Hoechst, and cells were analyzed with a fluorescent microscope. Arsenite (30 microM) induced massive apoptosis that was identified by morphology and condensation and fragmentation of the nuclei of the ZFL cells. Pretreatment with NAC or DTT before arsenite insult effectively protected the cells against oxidative stress-induced apoptosis from the arsenite. Using a transfected human hsp 70 promoter-enhanced green fluorescent protein (EGFP) reporter, pHhsp70-EGFP, the induction of HSP70 against oxidative stress-induced apoptosis by arsenite was observed. The induction of HSP70 by arsenite increased in a dose-dependent manner, and pretreatment of transfected ZFL cells with NAC or DTT before arsenite insult reduced EGFP expression. Taken together, our results provide evidence that stimulation of the heat shock response is a sensitive biomarker of arsenic exposure and that arsenite causes oxidative stress-induced apoptosis in ZFL cells.

[1]  K. Kitchin.  Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. , 2001, Toxicology and applied pharmacology.

[2]  P. Collodi,et al.  Derivation and characterization of a zebrafish liver cell line , 1994, Cell Biology and Toxicology.

[3]  C. Rice-Evans,et al.  Oxidative stress and heat shock protein induction in human cells. , 1987, Free radical research communications.

[4]  M. Edwards,et al.  Induction of heat shock proteins as a measure of chemical cytotoxicity. , 1990, Toxicology in vitro : an international journal published in association with BIBRA.

[5]  K. Nakamuro,et al.  Comparative studies of chromosomal aberration induced by trivalent and pentavalent arsenic. , 1981, Mutation research.

[6]  J F Barrett,et al.  Arsenic inhibition of telomerase transcription leads to genetic instability. , 2001, The Journal of clinical investigation.

[7]  B. Sanders,et al.  Stress proteins in aquatic organisms: an environmental perspective. , 1993, Critical reviews in toxicology.

[8]  D. R. Lewis,et al.  Drinking water arsenic in Utah: A cohort mortality study. , 1999, Environmental health perspectives.

[9]  T. Kuo,et al.  Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. , 1992, British Journal of Cancer.

[10]  K. Jan,et al.  Arsenite induces oxidative DNA adducts and DNA-protein cross-links in mammalian cells. , 2001, Free radical biology & medicine.

[11]  L. Hightower,et al.  Evaluation of heavy‐metal ion toxicity in fish cells using a combined stress protein and cytotoxicity assay , 1994 .

[12]  S. Toyokuni,et al.  The role of oxidative DNA damage in human arsenic carcinogenesis: detection of 8-hydroxy-2'-deoxyguanosine in arsenic-related Bowen's disease. , 1999, The Journal of investigative dermatology.

[13]  Jae-Hak Park,et al.  Specific activation of the human HSP70 promoter by copper sulfate in mosaic transgenic zebrafish. , 2006, Journal of biotechnology.

[14]  D. Ryu,et al.  β-Naphthoflavone Caused Up-Regulation of AhR Regulated GFP in Transgenic Zebrafish , 2004 .

[15]  L. Ryan,et al.  Risk of internal cancers from arsenic in drinking water. , 2000, Environmental health perspectives.

[16]  W. Goessler,et al.  Uptake and transformation of arsenosugars in the shrimp Crangon crangon , 1999 .

[17]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[18]  R. Morimoto,et al.  The Chaperone Function of hsp70 Is Required for Protection against Stress-Induced Apoptosis , 2000, Molecular and Cellular Biology.

[19]  B. Fowler,et al.  Alteration in protein synthesis in primary cultures of rat kidney proximal tubule epithelial cells by exposure to gallium, indium, and arsenite. , 1990, Toxicology and applied pharmacology.

[20]  D. Powers.  Fish as model systems. , 1989, Science.

[21]  L. Sciola,et al.  Role of oxidative stress in the manganese and 1-methyl-4-(2′-ethylphenyl)-1,2,3,6-tetrahydropyridine-induced apoptosis in PC12 cells , 1997, Neurochemistry International.

[22]  D. Menzel,et al.  Arsenic disrupts cellular levels of p53 and mdm2: a potential mechanism of carcinogenesis. , 1999, Biochemical and biophysical research communications.

[23]  L. Hightower,et al.  Recent applications of fish cell culture to biomedical research. , 1988, The Journal of experimental zoology.

[24]  R. Mehra,et al.  Preliminary morphological and histochemical changes in rat spinal cord neurons following arsenic ingestion. , 2005, Neurotoxicology.

[25]  P. Goering,et al.  Relationship between stress protein induction in rat kidney by mercuric chloride and nephrotoxicity. , 1992, Toxicology and applied pharmacology.

[26]  D. Dixon,et al.  The use of fish cell cultures as an indication of contaminant toxicity to fish , 1985 .

[27]  D. Sheehan,et al.  Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei. , 1987, Biochimica et biophysica acta.

[28]  Helmut Segner,et al.  The use of Fish Cells in Ecotoxicology: The Report and Recommendations of ECVAM Workshop 471, 2 , 2003, Alternatives to laboratory animals : ATLA.

[29]  Yu-Chieh Wang,et al.  Comparison of the cytotoxicity induced by different exposure to sodium arsenite in two fish cell lines. , 2004, Aquatic toxicology.

[30]  Qing‐Yu He,et al.  A proteome analysis of the arsenite response in cultured lung cells: evidence for in vitro oxidative stress-induced apoptosis. , 2004, The Biochemical journal.

[31]  B. Fowler,et al.  Evidence for oxidative damage to red blood cells in mice induced by arsine gas. , 1990, Toxicology.

[32]  T. Nakajima,et al.  Studies on the accumulation and transformation of arsenic in freshwater organisms II. Accumulation and transformation of arsenic compounds by Tilapia mossambica. , 2002, Chemosphere.