Cytotoxicity of an organic hydroperoxide and cellular antioxidant defense system against hydroperoxides in cultured mammalian cells.

The cytotoxicity of an organic hydroperoxide, tert-butyl hydroperoxide (t-BHP), was evaluated by inhibition of cell growth and of colony formation in cultured Chinese hamster V79 cells depleted or not depleted of cell glutathione (GSH). The hydroperoxide was cytostatic as shown by a marked decrease of mitotic cells, hence with a very low ability to form a colony. By depletion of GSH in the cells a killing effect of the hydroperoxide appeared. Iron chelation by O-phenanthroline markedly suppressed both inhibition of cell growth and of colony formation by t-BHP, indicating participation of divalent iron in the induction of cytotoxicity. Likewise, the pretreatment of GSH-depleted cells with cysteamine, an effective radical scavenger but without action as an electron donor in GSH peroxidase (GSH-Px) reaction, partly suppressed t-BHP-induced cell growth inhibition. These results suggest involvement of iron-catalyzed radical reaction in the induction of cytotoxicity by t-BHP. The inhibition of incorporation of tritiated thymidine and uridine by t-BHP was a little more sensitive than inhibition of leucine incorporation. GSH depletion evenly enhanced inhibition of incorporation of those precursors by t-BHP. The sensitivity to t-BHP of the cells with increased activity of GSH-Px by 1.5-2 times of control, which was achieved by treatment with 60 nM Na2SeO3, was not different from that of the cells with normal activity of the enzyme. On the other hand, t-BHP selectively inhibited GSH-Px activity without effect on glutathione reductase activity. These observations suggest that the cellular antioxidant system can not efficiently play a protective role against exogenous hydroperoxides and also that the increased cytotoxicity of t-BHP by GSH depletion is due strongly to a decrease of cell GSH as a radical scavenger than as an electron donor in GSH-Px reaction.