Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation: Is the hydroxyl radical really the most damaging radical species?

Isolated adult rat cardiac myocytes were subjected to anoxia and substrate deprivation for 15, 30, 60, 90, and 120 minutes and reoxygenation for 120 seconds. The supernatant and cell extract were analyzed for hydroxyl radicals (·OH) with high-performance liquid chromatography using salicylate as a trapping agent. The production of intracellular H₂O₂ as a possible precursor of ·OH was also documented using the fluorescent probe dichlorofluorescein diacetate. The release of the cytosolic enzyme lactate dehydrogenase (LDH) and malondialdehyde (MDA) formation were used as cell injury markers. Trypan blue and horseradish peroxidase stains were used as markers for altered membrane permeability. Maximum formation of ·OH was observed in myocytes subjected to 15 minutes of anoxia/reoxygenation (2.83±0.27 nmol/mg protein), at which time no injury was observed at light and ultramicroscopic levels. On the other hand, there was no correlation between the amount of ·OH production and different parameters of cell injury in myocytes subjected to anoxia/reoxygenation longer than 15 minutes. Myocytes developed extensive blebbing, loss of cell membrane permeability, and ultrastructural damage. The enzyme leakage was minimal at 15 minutes (0.094±0.021 units/mg protein) and increased fivefold after 120 minutes (0.428±0.069 units/mg protein). Similarly, MDA increased from 0.78±0.14 nmol/mg protein at 15 minutes to 1.65±0.35 nmol/mg protein at 120 minutes. Incubation with 1 mM deferoxamine reduced the ·OH production at all anoxic intervals, most significantly at 15 minutes, but did not decrease LDH and MDA release or provide ultrastructural preservation. However, preincubation with 2.5 μM diphenylphenylenediamine markedly reduced both LDH and MDA release and offered prominent ultrastructural protection. These results suggest that 1) myocytes were able to generate ·OH endogenously; 2) maximum ·OH was produced at 15 minutes after anoxic reoxygenation without compromising cell viability; 3) prolongation of the anoxic period exacerbated cell damage without parallel increase in ·OH generation; 4) there was no significant production of ·OH after 15 minutes of anoxia/reoxygenation with or without treatment of deferoxamine, suggesting that prolonged anoxia/reoxygenation does not induce additional ·OH formation and thus mediate cell injury; and 5) it is likely that the damage to myocytes in this system was still mediated by free radicals other than ·OH, as indicated by the protection by diphenylphenylenediamine against the cellular injury.