Primaquine-Induced Hemolytic Anemia: Formation of Free Radicals in Rat Erythrocytes Exposed to 6-Methoxy-8-hydroxylaminoquinoline
- 1Department of Pharmacology (L.J.C.B., D.J.J., D.C.M.) and 2Division of Nephrology, Department of Medicine (A.K.G.), Medical University of South Carolina, Charleston, South Carolina; and 3Research Service (A.K.G.), Ralph H. Johnson Veteran's Administration Medical Center, Department of Veteran's Affairs, Charleston, South Carolina
- Dr. David C. McMillan, Dept. of Pharmacology Med. Univ. South Carolina 171 Ashley Ave. Charleston, SC 29425. E-mail: mcmilldc{at}musc.edu
Abstract
Primaquine is an important antimalarial drug that is often dose-limited in therapy by the onset of hemolytic anemia. We have shown recently that an N-hydroxy metabolite of primaquine, 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH), is a direct-acting hemolytic agent in rat red cells and that the hemolytic activity of this metabolite is associated with GSH oxidation and oxidative damage to both membrane lipids and skeletal proteins. To determine whether the formation of free radicals may be involved in this process, rat red cells (40% suspensions) were incubated with hemolytic concentrations of MAQ-NOH (150–750 μM) and examined by EPR spectroscopy using 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) as a spin trap. Addition of MAQ-NOH to red cell suspensions containing 10 mM EMPO gave rise to an EPR spectrum with hyperfine constants consistent with those of an EMPO-hydroxyl radical adduct standard. Of interest, formation of EMPO-OH was constant for up to 20 min and dependent on the presence of erythrocytic GSH. Although no other radical adduct signals were detected in the cells by EPR, spectrophotometric analysis revealed the presence of ferrylhemoglobin, which indicates that hydrogen peroxide is generated under these experimental conditions. The data support the hypothesis that oxygen-derived and possibly other free radicals are involved in the mechanism underlying MAQ-NOH-induced hemolytic anemia.
Footnotes
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This study was supported by grants from the National Institutes of Health to D.C.M (AI46424) and A.K.G. (DK59950) and by a National Institutes of Health Shared Equipment Grant. The studies reported in this article are part of the graduate dissertation of Laura J. C. Bolchoz.
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DOI: 10.1124/jpet.102.041459
- Abbreviations:
- MAQ-NOH
- 6-methoxy-8-hydroxylaminoquinoline
- DEM
- diethyl maleate
- EMPO
- 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide
- DTPA
- diethylenetriaminepentaacetic acid
- PBSG
- phosphate-buffered saline supplemented with d-glucose
- DMSO
- dimethyl sulfoxide
- HPLC
- high-performance liquid chromatography
- DMPO
- 5,5-dimethyl-pyrroline-N-oxide
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- Received July 9, 2002.
- Accepted September 4, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
