Influence of nitric oxide on the generation and repair of oxidative DNA damage in mammalian cells.

Abstract

We have analysed the effects of endogenously and exogenously generated nitric oxide (NO) in cultured mammalian fibroblasts on: (i) the steady-state (background) levels of oxidative DNA base modifications; (ii) the susceptibility of the cells to the induction of additional DNA damage and micronuclei by H(2)O(2); and (iii) the repair kinetics of various types of DNA modifications. Steady-state levels of oxidative DNA base modifications, measured by means of an alkaline elution assay in combination with the repair endonuclease Fpg protein, were similar in NO-overproducing B6 mouse fibroblasts stably transfected with an inducible NO synthase (iNOS) and in control cells. Increased oxidative damage was only observed after exposure to high (toxic) concentrations of exogenous NO generated by decomposition of dipropylenetriamine-NONOate (DPTA-NONOate). Under these conditions, the spectrum of DNA modifications was similar to that induced by 3-morpholinosydnonimine, which generates peroxynitrite. The repair rate of additional oxidative DNA base modifications induced by photosensitization was not affected by the endogenous NO generation in the iNOS-transfected cells. However, it was completely blocked after pre-treatment with DPTA-NONOate at concentrations that did not cause oxidative DNA damage by themselves. In contrast, the repair of DNA single-strand breaks, sites of base loss (AP sites) and UVB-induced pyrimidine photodimers, was not affected. The endogenous generation of NO in the iNOS-transfected fibroblasts was associated with a protection from DNA single-strand break formation and micronuclei induction by H(2)O(2). These results indicate that NO generates cellular DNA damage only inefficiently and can even protect from DNA damage by H(2)O(2), but it selectively inhibits the repair of oxidative DNA base modifications.