Efficacy of lipid soluble, membrane-protective agents against hydrogen peroxide cytotoxicity in cardiac myocytes.

We examined the efficacy of a group of drugs that stabilize the cell membrane and can potentially prevent cytotoxicity in cultured fetal chick cardiac myocytes exposed to hydrogen peroxide (H2O2). The effects of various membrane-protective agents were determined by analysis of the kinetics of lactic dehydrogenase (LDH) release. The kinetic parameters calculated from the data include a rate constant for release of LDH (kb) and the fraction of total LDH that is released from the cells (CIIMax). The CIIMaxs derived from a range of H2O2 concentrations reveal that the mean toxic concentration of H2O2 is 1.1 mM and that the pattern of toxicity is consistent with the damage being directly proportional to the concentration of the free radicals generated from the H2O2. Maximum nontoxic concentrations of three amphiphilic membrane protective agents had no effect upon cytotoxicity from H2O2. The slightly polar lipophilic agent, Trolox C, a vitamin E derivative, was also without protective effect at a maximum nontoxic concentration. The highly lipophilic agent, probucol, had a small protective effect at 50 microM, the maximum concentration we succeeded in solubilizing in the culture medium. However, the lipophilic 21-aminosteroid U74500, delivered to the cells in an emulsion, markedly reduced cytotoxicity from H2O2. The CII Max was significantly reduced and the protection was concentration dependent over a range of concentrations from 50-400 nmol/ml. Furthermore, the inhibition by U74500 was fully consistent with a mechanism of scavenging of free radicals formed during lipid peroxidation. In support of this hypothesis, a dose of 400 nmoles/ml completely prevented an increase in lipid peroxides due to H2O2 exposure, whereas there was a sixfold increase during exposure to H2O2 in untreated myocytes. Thus, a lipid soluble 21-aminosteroid prevented lipid peroxidation and reduced cardiac myocyte injury during exposure to H2O2, probably by scavenging of free radicals formed during lipid peroxidation in the cell membrane, whereas amphiphilic agents, which probably altered the physicochemical structure of the cell membrane but did not scavenge free radicals, were not protective.