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.
Seok
Seung-Hyeok
SH
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.
Baek
Min-Won
MW
Lee
Hui-Young
HY
Kim
Dong-Jae
DJ
Na
Yi-Rang
YR
Noh
Kyoung-Jin
KJ
Park
Sung-Hoon
SH
Lee
Hyun-Kyoung
HK
Lee
Byoung-Hee
BH
Ryu
Doug-Young
DY
Park
Jae-Hak
JH
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
D000975:Antioxidants; D018053:Arsenites; D018840:HSP70 Heat-Shock Proteins; D049452:Green Fluorescent Proteins; D014343:Trypan Blue; C015001:arsenite; D004229:Dithiothreitol; D000111:Acetylcysteine