DNA-DAMAGE AND OXIDATION OF THIOLS PEROXYNITRITE CAUSES IN RAT THYMOCYTES

We report here the ability of peroxynitrite to cause DNA strand breaks and to oxidize cellular thiol groups in viable rat thymocytes. Peroxy nitrite was added to rat thymocytes in a phosphate buffer and DNA dama ge was measured by the fluorescence analysis of DNA unwinding assay. P eroxynitrite causes DNA strand breaks in a dose-dependent fashion. Fou r hydroxyl radical scavengers, namely mannitol, dimethyl sulfoxide, so dium benzoate, and Trolox, were tested for their ability to protect DN A from oxidative damage by peroxynitrite. Mannitol failed to protect D NA at concentrations at which it would have conferred nearly complete protection from damage by the hydroxyl radical. Strikingly, dimethyl s ulfoxide and benzoate, which are more efficient hydroxyl radical scave ngers than mannitol, caused an increase in DNA damage. Trolox was the only scavenger, among the four tested here, that was able to protect D NA from oxidative damage by peroxynitrite. We have previously shown th at, among the scavengers tested, Trolox is the most effective scavenge r of HOONO, where HOONO* is a reactive form of HOONO that is a more s elective oxidant than is the hydroxyl radical (see W. A. Pryer, X. Jin , and G. L. Squadrito, 1994, Proc. Natl. Acad. Sci. USA 91, 11173-1117 7). Thus, these results are consistent with our earlier observations t hat oxidations by peroxynitrite involve a reactive intermediate, HOONO , rather than hydroxyl radicals. Peroxynitrite also oxidized cellular thiols in a dose-dependent fashion. Greater than 90% of the cells exp osed to peroxynitrite were still viable for up to 10 min after DNA dam age and thiol oxidation had occurred. In conclusion, DNA damage caused by peroxynitrite can be rationalized as caused by a powerful oxidant, HOONO, which is formed during the decomposition of peroxynitrite to nitrite. (C) 1995 Academic Press, Inc.