Mutagenesis of the supF Gene of pSP189 Replicating in AD293 Cells Cocultivated with Activated Macrophages: Roles of Nitric Oxide and Reactive Oxygen Species

Abstract

Dysregulated production of nitric oxide (NO*) and reactive oxygen species by inflammatory cells contributes to mutagenesis and carcinogenesis. We have characterized mutagenesis in the target supF gene of pSP189 replicating in AD293 cells cocultivated with mouse macrophage-like RAW264.7 cells activated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Activated macrophages produced substantial amounts of NO*, superoxide anion (O2*-), and hydrogen peroxide (H2O2) over 12-72 h periods. A time-dependent decrease in total cell number and a 3.7-fold increase in supF mutation frequency (MF), compared with unstimulated controls, were observed at 72 h. The increase in MF was effectively suppressed by N-methyl-L-arginine monoacetate (NMA), an NO* synthase inhibitor, and also by superoxide dismutase (SOD) and catalase (CAT); cotreatment with NMA and SOD/CAT suppressed mutagenesis by 87% at 72 h. Mutations in supF were mainly multiple sequence changes (47%) and single base pair substitutions (51%) following IFN-gammaLPS activation. Following cotreatment with NMA alone or together with SOD/CAT, however, single base pair substitutions were prevalent (70 and 85%); decreased multiple mutations were observed (24 and 11%). Almost all single base pair substitutions induced under all exposure conditions occurred at G:C base pairs (87.8-94.6%). Whereas those induced by all treatments consisted predominantly of G:C to T:A transversions, G:C to T:A and A:T to T:A transversions were less frequent following treatment with NMA alone or with SOD/CAT compared to those induced by activated macrophages without additional treatment. Our results strongly suggest that ONOO- or its derivatives generated by reaction of NO* with O2*- may have been a major contributor to the observed mutagenesis by the activated macrophages, and mitigating their effects might serve a preventive function in ameliorating cancer risks associated with prolonged inflammation.