title: Arsenite-induced apoptosis is prevented by antioxidants in zebrafish liver cell line.
abstract: 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.
authors.affiliation: Department of Laboratory Animal Medicine, College of Veterinary Medicine and KRF Zoonotic Disease Priority Research Institute, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Seung-Hyeok
authors.initials: SH
authors.lastname: Seok
authors.forename: Min-Won
authors.initials: MW
authors.lastname: Baek
authors.forename: Hui-Young
authors.initials: HY
authors.lastname: Lee
authors.forename: Dong-Jae
authors.initials: DJ
authors.lastname: Kim
authors.forename: Yi-Rang
authors.initials: YR
authors.lastname: Na
authors.forename: Kyoung-Jin
authors.initials: KJ
authors.lastname: Noh
authors.forename: Sung-Hoon
authors.initials: SH
authors.lastname: Park
authors.forename: Hyun-Kyoung
authors.initials: HK
authors.lastname: Lee
authors.forename: Byoung-Hee
authors.initials: BH
authors.lastname: Lee
authors.forename: Doug-Young
authors.initials: DY
authors.lastname: Ryu
authors.forename: Jae-Hak
authors.initials: JH
authors.lastname: Park
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D018053:Arsenites / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000473:pathology* / Q000517:prevention & control*; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D006801:Humans; D008099:Liver / Q000473:pathology*; D018384:Oxidative Stress / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014343:Trypan Blue; D015027:Zebrafish
chemical_list: D000975:Antioxidants; D018053:Arsenites; D018840:HSP70 Heat-Shock Proteins; D049452:Green Fluorescent Proteins; D014343:Trypan Blue; C015001:arsenite; D004229:Dithiothreitol; D000111:Acetylcysteine