Genotoxicity associated with NO production in macrophages and co-cultured target cells.

We previously used mouse macrophage-like RAW264.7 cells as an experimental system for the study of nitric oxide (NO)-associated genotoxicity under physiologically relevant conditions, and characterized genotoxic effects in the NO-producing cells. Here we report experiments utilizing a co-culture system enabling parallel studies of cytotoxic and genotoxic responses in co-cultured target cells as well as in macrophages stimulated to produce NO. We found that co-cultivation with macrophages stimulated to produce NO for 38-42 h resulted in significant increases in mutant fraction in the endogenous genes of target human TK6 and hamster CHO-AA8 cells and in the macrophages themselves, accompanied by a substantial decrease in cell viability. Addition of N-methyl-L-arginine, an NO synthase inhibitor, abrogated much of the cytotoxicity and genotoxicity in both target and macrophages cells, verifying the role of NO in the induction of these responses. We also showed that NO-associated genotoxic response in macrophages could be influenced by culture medium. Collectively, these results support the hypothesis that NO production by activated macrophages may contribute to genotoxic risks associated with chronic inflammation.