Peroxyl radical-mediated hemolysis: role of lipid, protein and sulfhydryl oxidation.
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.
Sandhu
I S
IS
Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport 71130-3932.
Ware
K
K
Grisham
M B
MB
D000636:Aminosalicylic Acids / Q000494:pharmacology; D001798:Blood Proteins / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D004912:Erythrocytes / Q000378:metabolism*; D005609:Free Radicals; D005978:Glutathione / Q000097:blood; D006461:Hemolysis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D015227:Lipid Peroxidation / Q000502:physiology; D008055:Lipids / Q000097:blood*; D010084:Oxidation-Reduction; D010545:Peroxides; D013438:Sulfhydryl Compounds / Q000097:blood*
D000636:Aminosalicylic Acids; D001798:Blood Proteins; D005609:Free Radicals; D008055:Lipids; D010545:Peroxides; D013438:Sulfhydryl Compounds; C049375:perhydroxyl radical; D005978:Glutathione