MUTAGENESIS ASSOCIATED WITH NITRIC-OXIDE PRODUCTION IN MACROPHAGES

To better understand the mechanisms through which persistent infection s/inflammation increase cancer risks, we assessed the potential genoto xic properties of NO produced by macrophages, We recently showed that mouse macrophage RAW264.7 cells were capable of resuming exponential g rowth after stimulation for NO production by interferon-gamma (IFN-gam ma) and/or lipopolysaccharide, Here, we report that increases in mutan t fraction (MF) in the endogenous, X-linked, hprt gene of the cells ar e associated with NO exposure. Cells stimulated with 100 units/ml IFN- gamma continuously for 14 and 23 days produced a total of 9.8 and 14 m u mol of NO per 10(7) cells, respectively. MFs in the hprt gene of NO producing cells were 16.6 and 31.3 x 10(-5), respectively, compared wi th 2.2 and 2.5 x 10(-5) in untreated cells. Addition of an NO synthase inhibitor, N-monomethyl-L-arginine, to the culture medium decreased N O production and MF by 90% and 85%, respectively. Reverse transcriptio n-PCR and DNA sequencing revealed that NO-associated hprt mutations di d not differ significantly from those arising spontaneously, with the exception that certain small deletions/insertions and multiple exon de letions were observed only in the former. MF also increased significan tly in cells stimulated for only 4 days with lipopolysaccharide plus I FN-gamma for higher rates of NO production. The types and proportion o f hprt mutations induced under these conditions were strikingly simila r to those associated with long-term NO exposure. These results indica te that NO exposure results in gene mutations in RAW264,7 cells throug h mechanisms yet to be identified and may also contribute to spontaneo us mutagenesis.