Primaquine-Induced Hemolytic Anemia: Formation of Free Radicals in Rat Erythrocytes Exposed to 6-Methoxy-8-hydroxylaminoquinoline

Abstract

Primaquine is an important antimalarial drug that is often doselimited 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 directacting 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 M) 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.Hemolytic anemia and methemoglobinemia are well recognized dose-limiting side effects in the therapeutic use of arylamine drugs, such as primaquine and dapsone (Beutler, 1969). Because these compounds are not hemotoxic when incubated with red cells in vitro, it has long been appreciated that metabolites are responsible for the onset of the hemolytic response. In the cases of aniline (Harrison and Jollow, 1986), dapsone (Grossman and Jollow, 1988), and phenacetin (Jensen and Jollow, 1991), we have shown that the hemolytic metabolites are their N-hydroxy derivatives. Primaquine metabolism, on the other hand, is relatively more complex, and the metabolites that mediate the hemotoxic responses have not been identified. A variety of known and putative phenolic metabolites of primaquine are redox-active and therefore have the potential to mediate primaquine hemotoxicity (Strother et al., 1984;Baird et al., 1986;Fletcher et al., 1988;Agarwal et al., 1991); however, direct evidence for their hemolytic activity is lacking. We have recently explored an alternative hypothesis-that primaquine hemotoxicity is mediated by an N-hydroxylated metabolite, 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH).Although the mechanism underlying the damage and removal of red cells by hemolytic N-hydroxylamines remains unclear, oxidative stress has long been considered to play a prominent role in the process (for review, see Beutler, 1971). This concept is based on the well known association of hemotoxicity with oxidation of erythrocytic GSH (to glutathione disulfide and glutathione protein-mixed disulfides), with methemoglobin f...

Antiparasitic Activities and Toxicities of Individual Enantiomers of the 8-Aminoquinoline 8-[(4-Amino-1-Methylbutyl)Amino]-6-Methoxy-4-Methyl-5-[3,4-Dichlorophenoxy]Quinoline Succinate

“…The metabolites that are responsible for activity and toxicity appear to be minor and highly labile. Several probable metabolites of primaquine have been proposed, but none of them have been fully characterized in humans or animal models (3,12,13,19,27).…”

Primaquine revisited six decades after its discovery

“…This compound was responsible for causing hemolysis in vivo as well as for inducing the formation of MetHb when incubated with suspensions of rat erythrocytes [154]. One year later, the same authors demonstrated that, under hemolytic conditions, MAQ-NOH generates three oxygen-active species in rat erythrocytes: hydroxyl radical, hydrogen peroxide and ferryl heme, responsible for hemolytic anemia [155].…”

“…6-Methoxy-8-aminoquinoline (MAQ, 18) is thoroughly referenced in works devoted to PQ metabolism [148,[151][152][153][154][155]. MAQ is not very active in inducing MetHb formation, thus shows lower toxicity indices as compared to other PQ metabolites [148,152,153].…”

Methemoglobin—It's not just blue: A concise review

“…The hydroxylamine is also produced in the microsomes, and would act completely analogous to the aniline-like drugs. The 6-methoxy-8-hydroxylaminoquinoline derivative of primaquine also induces methemoglobinemia, glutathione depletion, and hemolytic anemia [82]. But besides the hydroxylamine the phenolytic derivative 5-hydroxyprimaquine is produced and induces methemoglobin formation and depletion of red cell glutathione.…”