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 ar senic 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 cell s. Following phosphate-citric acid buffer extraction, apoptotic cells with lower DNA content than the G1 cells were detected by flow cytomet ry. Immediately after 4 h of 40 mu M arsenite treatment, no appreciabl e 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 intrace llular peroxide level as measured by the fluorescent intensity of 2',7 '-dichlorofluorescein increased immediately following a 4-h arsenite t reatment. Simultaneous treatment with arsenite plus antioxidant (N-ace tyl-cysteine, Trolox, and Tempo); copper ion chelator (neocuproine); p rotein kinase inhibitor (H-7) or protein synthesis inhibitor (cyclohex imide) reduced the fraction of sub-G1 cell and internucleosomal DNA de gradation. 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 ra dicals, which selectively activates protein kinase through de novo syn thesis of macromolecules. (C) 1996 Wiley-Liss, Inc.