The objective of this study was to define the relationship between
peroxyl radical-mediated cytotoxicity and
lipid,
protein and sulfhydryl oxidation using human erythrocytes as the target mammalian cell. We found that incubation of human erythrocytes with the
peroxyl radical generator 2,2' azobis (2-amidinopropane) hydrochloride (
AAPH) resulted in a time and dose-dependent increase in
hemolysis such that at 50 mM
AAPH maximum
hemolysis was achieved at 120 min.
Hemolysis was inhibited by
hypoxia and by the addition of certain water soluble
free radical scavengers such as
5-aminosalicylic acid (5-ASA), 4-ASA, N-acetyl-5-ASA and dimethyl
thiourea.
Peroxyl radical-mediated
hemolysis did not appear to involve significant peroxidation of erythrocyte
lipids nor did they enhance
protein oxidation at times preceding
hemolysis. Peroxyl radicals did however, significantly reduce by approximately 80% the intracellular levels of GSH and inhibit by approximately 90% erythrocyte Ca(2+)-
Mg2+ ATPase activity at times preceding the hemolytic event. Our data as well as others suggest that extracellular
oxidants promote the oxidation of intracellular compounds by interacting with certain redox active membrane components. Depletion of intracellular GSH stores using
diamide did not result in
hemolysis suggesting that oxidation of GSH alone does not promote
hemolysis. Taken together, our data suggest that neither GSH oxidation, lipid peroxidation nor
protein oxidation alone can account for
peroxyl radical-mediated
hemolysis. It remains to be determined whether
free radical-mediated inactivation of Ca(2+)-
Mg2+ ATPase is an important mechanism in this process.