Arsenite induces apoptosis in Chinese hamster ovary cells by generation of reactive oxygen species.
Arsenic, a human carcinogen, possesses a serious environmental threat but the mechanism of its toxicity remains unclear. Knowledge of how arsenic induces cell death and how cells escape the death path may help to understand arsenic carcinogenesis. We have investigated the nature of sodium arsenite-induced cell death in Chinese hamster ovary K1 cells. Following phosphate-citric acid buffer extraction, apoptotic cells with lower DNA content than the G1 cells were detected by flow cytometry. Immediately after 4 h of 40 microM arsenite treatment, no appreciable fraction of cells with sub-G1 DNA content was detected; however, the sub-G1 cell fraction increased with postarsenite incubation time, and detectable increase started at 8 h of incubation, whereas the intracellular peroxide level as measured by the fluorescent intensity of 2',7'-dichlorofluorescein increased immediately following a 4-h arsenite treatment. Simultaneous treatment with arsenite plus antioxidant (N-acetyl-cysteine, Trolox, and Tempo); copper ion chelator (neocuproine); protein kinase inhibitor (H-7) or protein synthesis inhibitor (cycloheximide) reduced the fraction of sub-G1 cell and internucleosomal DNA degradation. Trolox, neocuproine, or cycloheximide given after arsenite treatment also effectively reduced apoptosis. These results lead to a working hypothesis that arsenite-induced apoptosis in CHO-K1 cells is triggered by the generation of hydrogen peroxide, followed by a copper-mediated Fenton reaction that catalyzes the production of hydroxyl radicals, which selectively activates protein kinase through de novo synthesis of macromolecules.
Wang
T S
TS
Institute of Radiation Biology, National Tsing-Hua University, Hsinchu, Taiwan, Republic of China.
Kuo
C F
CF
Jan
K Y
KY
Huang
H
H
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D018053:Arsenites / Q000494:pharmacology* / Q000633:toxicity; D016466:CHO Cells; D002452:Cell Count / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D002839:Chromans; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D016193:G1 Phase / Q000187:drug effects; D016195:G2 Phase / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*
D000975:Antioxidants; D018053:Arsenites; D002614:Chelating Agents; D002839:Chromans; D005452:Fluoresceins; D017382:Reactive Oxygen Species; C037631:2',7'-dichlorofluorescein; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D003513:Cycloheximide; D006861:Hydrogen Peroxide; C015001:arsenite; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid