Vol. 55, Issue 4, 708-715, April 1999

Oxidative Stress Occurs in Perfused Rat Liver at Low Oxygen Tension by Mechanisms Involving Peroxynitrite

Gavin E. Arteel, Maria B. Kadiiska, Ivan Rusyn, Blair U. Bradford, Ronald P. Mason, James A. Raleigh, and Ronald G. Thurman

Laboratory of Hepatobiology and Toxicology, Department of Pharmacology (G.E.A., I.R., B.U.B, R.G.T.), Department of Radiation Oncology (G.E.A., J.A.R.), and Curriculum in Toxicology (G.E.A., I.R., R.P.M., J.A.R., R.G.T.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and Laboratory of Pharmacology and Chemistry, National Institutes of Environmental Health Sciences, Research Triangle Park, North Carolina (M.B.K.,R.P.M.)

Ethanol increases free radical formation; however, it was recently demonstrated that it also causes extensive hypoxia in rat liver in vivo. To address this issue, it was hypothesized that peroxynitrite formed in normoxic periportal regions of the liver lobule has its reactivity enhanced in hypoxic pericentral regions where the pH is lower. Via this pathway, peroxynitrite could lead to free radical formation in the absence of oxygen. Livers from fed rats were perfused at low flow rates for 75 min. Under these conditions, periportal regions were well oxygenated but pericentral areas became hypoxic. Low-flow perfusion caused a significant 6-fold increase in nitrotyrosine accumulation in pericentral regions. During the last 20 min of perfusion, the spin-trap -(4-pyridyl-1-oxide)-N-tert-butylnitrone was infused and adducts were collected for electron-spin resonance analysis. A six-line radical adduct signal was detected in perfusate. Direct infusion of peroxynitrite produced a radical adduct with identical coupling constants, and a similar pattern of nitrotyrosine accumulation was observed. Retrograde perfusion at low rates resulted in accumulation of nitrotyrosine in periportal regions. Although the magnitude of the radical in perfusate was increased by ethanol, it was not derived directly from it. Both nitrotyrosine accumulation and radical formation were reduced by inhibition of nitric oxide synthase 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 the final prooxidant is a derivative from both NO· and superoxide (i.e., peroxynitrite). These results support the hypothesis that oxidative stress occurs in hypoxic regions of the liver lobule by mechanisms involving peroxynitrite.