Free radical production requires both inducible nitric oxide synthase and xanthine oxidase in LPS-treated skin.
Free radical formation has been investigated in diverse experimental models of LPS-induced inflammation. Here, using electron spin resonance (ESR) and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, we have detected an ESR spectrum of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts in the lipid extract of mouse skin treated with LPS for 6 h. The ESR spectrum was consistent with the trapping of lipid-derived radical adducts. In addition, a secondary radical-trapping technique using dimethyl sulfoxide (DMSO) demonstrated methyl radical formation, revealing the production of hydroxyl radical. Radical adduct formation was suppressed by aminoguanidine, N-(3-aminomethyl)benzylacetamidine (1400W), or allopurinol, suggesting a role for both inducible nitric oxide synthase (iNOS) and xanthine oxidase (XO) in free radical formation. The radical formation was also suppressed in iNOS knockout (iNOS(-/-)) mice, demonstrating the involvement of iNOS. NADPH oxidase was not required in the formation of these radical adducts because the ESR signal intensity was increased by LPS treatment in NADPH oxidase knockout (gp91(phox-/-)) mice as much as it was in the wild-type mouse. Nitric oxide (*NO) end products were increased in LPS-treated skin. As expected, the *NO end products were not suppressed by allopurinol but were by aminoguanidine. Interestingly, nitrotyrosine formation in LPS-treated skin was also suppressed by aminoguanidine and allopurinol independently. Pretreatment with the ferric iron chelator Desferal had no effect on free radical formation. Our results imply that both iNOS and XO, but neither NADPH oxidase nor ferric iron, work synergistically to form lipid radical and nitrotyrosine early in the skin inflammation caused by LPS.
Nakai
Kozo
K
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, MD F0-01, Research Triangle Park, NC 27709, USA.
Kadiiska
Maria B
MB
Jiang
Jin-Jie
JJ
Stadler
Krisztian
K
Mason
Ronald P
RP
D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D003872:Dermatitis / Q000201:enzymology / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism*; D006146:Guanidines / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000633:toxicity*; D051379:Mice; D008810:Mice, Inbred C57BL; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012867:Skin / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005609:Free Radicals; D006146:Guanidines; D008070:Lipopolysaccharides; D000493:Allopurinol; D052247:Nitric Oxide Synthase Type II; D014969:Xanthine Oxidase; C004479:pimagedine