title: Primaquine-induced hemolytic anemia: role of membrane lipid peroxidation and cytoskeletal protein alterations in the hemotoxicity of 5-hydroxyprimaquine.
abstract: Primaquine-induced hemolytic anemia is a toxic side effect that is due to premature splenic sequestration of intact erythrocytes. Previous studies have suggested that a phenolic metabolite, 5-hydroxyprimaquine (5-HPQ), mediates primaquine hemotoxicity by generating reactive oxygen species (ROS) within erythrocytes that overwhelm antioxidant defenses. However, the nature of the oxidative stress is not understood, and the molecular targets, whether protein and/or lipid, are unknown. To investigate the mechanism underlying the hemolytic activity of 5-HPQ, we have examined the effect of hemolytic concentrations of 5-HPQ on ROS formation within rat erythrocytes using the cellular ROS probe, 2',7'-dichlorodihydrofluoresein diacetate. In addition, we examined the effect of 5-HPQ on membrane lipids and cytoskeletal proteins. The data indicate that 5-HPQ causes a prolonged, concentration-dependent generation of ROS within erythrocytes. Interestingly, 5-HPQ-generated ROS was not associated with the onset of lipid peroxidation or an alteration in phosphatidylserine asymmetry. Instead, 5-HPQ induced oxidative injury to the erythrocyte cytoskeleton, as evidenced by changes in the normal electrophoretic pattern of membrane ghost proteins. Immunoblotting with an anti-hemoglobin antibody revealed that these changes were due primarily to the formation of disulfide-linked hemoglobin-skeletal protein adducts. The data suggest that cytoskeletal protein damage, rather than membrane lipid peroxidation or loss of phosphatidylserine asymmetry, underlies the process of removal of erythrocytes exposed to 5-HPQ.
authors.affiliation: Dept. of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA.
authors.forename: Zachary S
authors.initials: ZS
authors.lastname: Bowman
authors.forename: Jason D
authors.initials: JD
authors.lastname: Morrow
authors.forename: David J
authors.initials: DJ
authors.lastname: Jollow
authors.forename: David C
authors.initials: DC
authors.lastname: McMillan
mesh_terms: D000743:Anemia, Hemolytic / Q000139:chemically induced*; D000818:Animals; D000962:Antimalarials / Q000633:toxicity*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D008051:Lipid Bilayers; D015227:Lipid Peroxidation / Q000187:drug effects*; D008297:Male; D010718:Phosphatidylserines / Q000097:blood; D011319:Primaquine / Q000031:analogs & derivatives* / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000097:blood; D013154:Spleen / Q000378:metabolism
chemical_list: D000962:Antimalarials; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D007476:Ionophores; D008051:Lipid Bilayers; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; C056497:5-hydroxyprimaquine; D011319:Primaquine; D005033:Ethylmaleimide; D002118:Calcium; D004229:Dithiothreitol