Generation of free radicals during anoxia and reoxygenation in perfused osteoblastlike cells

Sensitivity to ischemia and reperfusion injury is a main problem afflicting tissues exposed to a prolonged period of oxygen deprivation. The generation of oxygen free radicals, in particular, is considered a major cause of postischemic reperfusion injury. However, studies on the mechanisms of production of free radicals are limited by the difficulty to measure in real time their formation and to discriminate between the different oxyradical species. The aim of this study was to determine whether the formation of oxygen free radicals occurs in murine osteoblastlike 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 buffer. Anoxia was obtained by shifting the gas phase of the media to 95% N2-5% CO2. Oxygen free radicals were detected by enhanced chemiluminescence: anion superoxide or hydrogen peroxide was measured by adding lucigenin or luminol plus horseradish peroxidase to the media, respectively. Cell injury was assessed by the rate of lactate dehydrogenase release. During the control period, lucigenin and luminol plus horseradish 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 background levels, although lactate dehydrogenase release increased progressively to 38 +/- 7 mU per minute. During reoxygenation, O2 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 reoxygenation period; lactate dehydrogenase declined progressively to control values. These results show that osteoblastlike cells produce measurable amounts of superoxide and hydrogen peroxide radicals during reoxygenation. 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.