Mechanism of oxidative DNA damage induction in a strict anaerobe, Prevotella melaninogenica

Abstract

We investigated the mechanism of the oxidative DNA damage induction by exposure to O 2 in Prevotella melaninogenica, a strict anaerobe. Flow cytometry with hydroethidine and dichlorofluorescein diacetate showed that O 2 exposure generated O 2 − and H 2O 2. Results of electron spin resonance with α-(4-pyridyl-1-oxide)- N-tert-butylnitrone and ethanol showed that O 2 exposure also induced OH radical generation in P. melaninogenica loaded with FeCl 2 but not in samples without FeCl 2 loading. In P. melaninogenica, O 2 exposure increased 8-hydroxydeoxyguanosine (8OHdG), typical of oxidative DNA damage. Catalase inhibited the increase, but the OH radical scavengers did not. Phenanthroline, a membrane-permeable Fe and Cu chelator, increased the 8OHdG induction. In FeCl 2-loaded samples, induction of 8OHdG decreased. Addition of H 2O 2 markedly increased 8OHdG levels. These results indicate that in P. melaninogenica, exposure to O 2 generated and accumulated O 2 − and H 2O 2, and that a crypto-OH radical generated through H 2O 2 was the active species in the 8OHdG induction.