Oxidative stress occurs in perfused rat liver at low oxygen tension by mechanisms involving peroxynitrite

Ethanol increases free radical formation; however, it was recently demonstr ated that it also causes extensive hypoxia in rat liver in vivo. To address this issue, it was hypothesized that peroxynitrite formed in normoxic peri portal regions of the liver lobule has its reactivity enhanced in hypoxic p ericentral regions where the pH is lower. Via this pathway, peroxynitrite c ould lead to free radical formation in the absence of oxygen. Livers from f ed vats were perfused at low flow rates for 75 min. Under these conditions, periportal regions were well oxygenated but pericentral areas became hypox ic. Low-flow perfusion caused a significant 6-fold increase in nitrotyrosin e accumulation in pericentral regions. During the last 20 min of perfusion, the spin-trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone was infused an d adducts were collected for electron-spin resonance analysis. A six-line r adical adduct signal was detected in perfusate. Direct infusion of peroxyni trite produced a radical adduct with identical coupling constants, and a si milar pattern of nitrotyrosine accumulation was observed. Retrograde perfus ion at low rates resulted in accumulation of nitrotyrosine in periportal re gions. Although the magnitude of the radical in perfusate was increased by ethanol, it was not derived directly from it. Both nitrotyrosine accumulati on and radical formation were reduced by inhibition of nitric oxide synthas e with N-nitro-L-arginine methyl ester, but not with the inactive D-isomer. Radical formation was decreased nearly completely by superoxide dismutase and N-nitro-L-arginine methyl ester, consistent with the hypothesis that th e final prooxidant is a derivative from both NO and superoxide (i.e., perox ynitrite). These results support the hypothesis that oxidative stress occur s in hypoxic regions of the liver lobule by mechanisms involving peroxynitr ite.