title: Effects of superoxide on nitric oxide-dependent N-nitrosation reactions.
abstract: Recent studies have demonstrated that nitric oxide (NO) in the presence of superoxide (O2-) may mediate mutagenesis via the N-nitrosation of DNA bases followed by nitrosative deamination to yield their hydroxylated derivatives. We have found that phorbol myristate acetate (PMA)-activated extravasated rat neutrophils (PMNs) will N-nitrosate 2,3-diaminonaphthalene (DAN) to yield its highly fluorescent nitrosation product 2,3-naphthotriazole (triazole) via the L-arginine dependent formation of NO. Addition of SOD enhanced triazole formation suggesting that O2- production may inhibit the N-nitrosating activity and thus the mutagenic activity of inflammatory PMNs. The objective of this study was to assess the role of superoxide as a modulator of NO-dependent N-nitrosation reactions using PMA-activated PMNs as well as a chemically defined-system that generates both NO and superoxide. We found that PMA-activation of PMNs reduced found that PMA-activation of PMNs reduced the amount of N-nitrosation of DAN by approximately 64% when compared to non-stimulated cells (450 vs. 1250 nM). Addition of SOD but not inactivated SOD or catalase to PMA-activated PMNs enhanced the formation of triazole by approximately 4-fold (1950 nM). In addition, we found that the NO-releasing spermine/NO adduct (Sp/NO; 50 microM) which produces approximately 1.0 nmol NO/min generated approximately 8000 nM of triazole whereas the combination of Sp/NO and a superoxide generator (hypoxanthine/xanthine oxidase) that produces approximately 1.0 nmol O2-/min reduced triazole formation by 90% (790 nM). Addition of SOD but not catalase restored the N-nitrosating activity. We conclude that equimolar fluxes of superoxide react rapidly 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.
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D008297:Male; D009504:Neutrophils / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D015538:Nitrosation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000494:pharmacology*
chemical_list: D009566:Nitrates; D009573:Nitrites; D013481:Superoxides; C026373:2,3-diaminonaphthalene; D009569:Nitric Oxide; D015081:2-Naphthylamine