SOURCE OF OXYGEN-FREE RADICALS PRODUCED BY RAT HEPATOCYTES DURING POSTANOXIC REOXYGENATION

The aim of this study was to determine the cellular source of oxygen f ree radicals generated by isolated hepatocytes during post-anoxic reox ygenation. Superoxide anions (O-2(-)) were detected by lucigenin chemi luminescence. Cell damage was assessed by LDH release. During anoxia, the chemiluminescence decreased to background levels while LDH release increased 8-fold. During reoxygenation, O-2(-) formation increased 15 -fold within 15 min then declined towards control levels. LDH release increased from 161 to 285 mU/min in the first 30 min of reoxygenation, then declined toward the control rate. Allopurinol, an inhibitor of t he xanthine-xanthine oxidase system, did not inhibit O-2- formation no r LDH release. Antimycin, a mitochondrial complex III inhibitor that d oes not block O-2(-) formation, increased both O-2(-) generation and L DH release 82 and 133% respectively. Diphenyleneiodonium (DPI), a mito chondrial and microsomal NADPH oxidase inhibitor, reduced O-2(-) and L DH release 60-70%. SOD, which catalyzes the dismutation of O-2- to H2O 2, was without effect on O-2(-) and LDH release, but TEMPO, a stable n itroxide which mimics SOD and easily penetrates the cell membrane, dec reased O-2(-) 86% without affecting LDH. These results suggest that mi tochondria or microsomes are the principal sites of O-2(-) production during reoxygenation of isolated hepatocytes, whereas the cytosolic xa nthine/xanthine oxidase system is apparently not involved.