GENERATION OF FREE-RADICALS DURING ANOXIA AND REOXYGENATION IN PERFUSED OSTEOBLAST-LIKE CELLS

Sensitivity to ischemia and reperfusion injury is a main problem affli cting tissues exposed to a prolonged period of oxygen deprivation, The generation of oxygen free radicals, in particular, is considered a ma jor cause of postischemic reperfusion injury, However, studies on the mechanisms of production of free radicals are limited by the difficult y to measure in real time their formation and to discriminate between the different oxyradical species, The aim of this study was to determi ne whether the formation of oxygen free radicals occurs in murine oste oblastlike cells (MC3T3-E1) exposed to anoxia and reoxygenation and to explore its relation to the reoxygenation injury, Cells were cast in agarose and perfused with oxygenated Krebs-Henseleit bicarbonate buffe r, Anoxia was obtained by shifting the gas phase of the media to 95% N -2-5% CO2. Oxygen free radicals were detected by enhanced chemilumines cence: anion superoxide or hydrogen peroxide was measured by adding lu cigenin or luminol plus horseradish peroxidase to the media, respectiv ely, Cell injury was assessed by the rate of lactate dehydrogenase rel ease, During the control period, lucigenin and luminol plus horseradis h chemiluminescences were 15 +/- 1 nA per chamber and 20 +/- 2 nA per chamber, respectively, and lactate dehydrogenase release was 10 +/- 1 mU per minute, During anoxia, both chemiluminescences dropped to backg round levels, although lactate dehydrogenase release increased progres sively to 38 +/- 7 mU per minute, During reoxygenation, O-2 formation increased sharply to 45 +/- 6 nA and decreased to control levels; H2O2 production increased slowly, reaching 42 +/- 7 nA at the end of the r eoxygenation period; lactate dehydrogenase declined progressively to c ontrol values, These results show that osteoblastlike cells produce me asurable amounts of superoxide and hydrogen peroxide radicals during r eoxygenation, Because lactate dehydrogenase release did not appear to relate to chemiluminescence, oxyradical flux may serve as a signal for other events that eventually lead to cell injury.