EFFECTS OF SUPEROXIDE ON NITRIC OXIDE-DEPENDENT N-NITROSATION REACTIONS

Recent studies have demonstrated that nitric oxide (NO) in the presenc e of superoxide (O-2(-)) may mediate mutagenesis via the N-nitrosation of DNA bases followed by nitrosative deamination to yield their hydro xylated derivatives. We have found that phorbol myristate acetate (PMA )-activated extravasated rat neutrophils (PMNs) will N-nitrosate 2,3-d iaminonaphthalene (DAN) to yield its highly fluorescent nitrosation pr oduct 2,3- naphthotriazole (triazole) via the L-arginine dependent for mation of NO. Addition of SOD enhanced triazole formation suggesting t hat O-2(-) production may inhibit the N-nitrosating activity and thus the mutagenic activity of inflammatory PMNs. The objective of this stu dy was to assess the role of superoxide as a modulator of NO-dependent N-nitrosation reactions using PMA-activated PMNs as well as a chemica lly defined-system that generates both NO and superoxide. We found tha t PMA-activation of PMNs reduced the amount of N-nitrosation of DAN by approximately 64% when compared to non- stimulated cells (450 vs. 125 0 nM). Addition of SOD but not inactivated SOD or catalase to PMA-acti vated PMNs enhanced the formation of triazole by approximately 4-fold (1950 nM). In addition, we found that the NO-releasing spermine/NO add uct (Sp/NO; 50 mu M) which produces approximately 1.0 nmol NO/min gene rated approximately 8000 nM of triazole whereas the combination of Sp/ NO and a superoxide generator (hypoxanthine/xanthine oxidase) that pro duces approximately 1.0 nmol O-2(-)/min reduced triazole formation by 90% (790 nM). Addition of SOD but not catalase restored the N-nitrosat ing activity. We conclude that equimolar fluxes of superoxide react ra pidly with NO to generate products that have only limited ability to N -nitrosate aromatic amino compounds and thus may have limited ability to promote mutagenesis via the nitrosative deamination of DNA bases.