Understanding the mechanisms for metabolism-linked hemolytic toxicity of primaquine against glucose 6-phosphate dehydrogenase deficient human erythrocytes: Evaluation of eryptotic pathway

Abstract

Therapeutic utility of primaquine, an 8-aminoquinoline antimalarial drug, has been limited due to its hemolytic toxicity in population with glucose 6-phosphate dehydrogenase deficiency. Recent investigations at our lab have shown that the metabolites generated through cytochrome P₄₅₀-dependent metabolic reactions are responsible for hemotoxic effects of primaquine, which could be monitored with accumulation of methemoglobin and increased oxidative stress. The molecular markers for succeeding cascade of events associated with early clearance of the erythrocytes from the circulation were evaluated for understanding the mechanism for hemolytic toxicity of primaquine. Primaquine alone though did not induce noticeable methemoglobin accumulation, but produced significant oxidative stress, which was higher in G6PD-deficient than in normal erythrocytes. Primaquine, presumably through redox active hemotoxic metabolites generated in situ in human liver microsomal metabolism-linked assay, induced a dose-dependent methemoglobin accumulation and oxidative stress, which were almost similar in normal and G6PD-deficient erythrocytes. Primaquine alone or in presence of pooled human liver microsomes neither produced significant effect on intraerythrocytic calcium levels nor affected the phosphatidyl serine asymmetry of the normal and G6PD-deficient human erythrocytes as monitored flowcytometrically with Annexin V binding assay. The studies suggest that eryptosis mechanisms are not involved in accelerated removal of erythrocytes due to hemolytic toxicity of primaquine.

Introduction

Primaquine, regarded as the drug of choice for radical cure (complete elimination of the dormant liver stages of the parasite from the body) of Plasmodium vivax malaria, has limited utility due to its narrow therapeutic index, rapid clearance, and hemolytic toxicities (Tekwani and Walker, 2006, Vale et al., 2009, Fernando et al., 2011). Primaquine is the only approved drug that kills the key parasite stages necessary for the survival of malaria. For Plasmodium falciparum (Pf) malaria, this is the mature (stage 5) gametocytes (Pfg), which transmit the infection (Bousema and Drakeley, 2011). For P. vivax (Pv) and Plasmodium ovale (Po), this is the sleeping liver stage or hypnozoite, which emerges weeks to months after the initial infection and causes relapse (Wells et al., 2010). The major drawback of primaquine is its hemotoxicity, namely methemoglobinemia and hemolysis in individuals who suffer from glucose-6-phosphate dehydrogenase (G-6-PD) deficiency (Taylor and White, 2004, Youngster et al., 2010). Due to their oxidant nature, primaquine metabolites oxidize hemoglobin and generate reactive oxygen species thus leading to depletion of protective thiols (Bloom et al., 1983, Summerfield and Tudhope, 1978). These events finally lead to a dose related hemolytic anemia. The severity of hemolysis is determined by the dose of primaquine, extent of G6PD-deficiency, and also on the patient's physiological condition (Clyde, 1981, Youngster et al., 2010).

Many studies have been done to better understand the nature of the hemolysis and reasons for primaquine sensitivity in G6PD-deficient individuals (Beutler et al., 1954, Bowman et al., 2004, Bowman et al., 2005a, Bowman et al., 2005b, Flanagan et al., 1958, Jansson et al., 1980). Initial studies demonstrated the increased susceptibility of the sensitive individuals to Heinz body formation as compared to non-sensitive individuals (Beutler et al., 1954). Further studies demonstrated the acute fall in reduced glutathione level in primaquine sensitive as compared to non-sensitive individuals (Flanagan et al., 1958). All these studies helped in concluding the importance of G6PD and the role of reduced glutathione as an antioxidant in human red blood cells (Bloom et al., 1983).

Erythrocytes are believed to be devoid of classical apoptotic pathway of cell death, due to the absence of mitochondria and nucleus, the key organelles involved in apoptosis. But recent studies have shown that the stimulation of cation channels (Ca²⁺ channels) in erythrocytes, either by oxidative stress, osmotic shock or energy depletion, can lead to cellular shrinkage, exposure of phosphatidylserine on the cell surface, and finally cause apoptosis (Lang et al., 2006, Lang et al., 2008). This process has been termed as “eryptosis”. Eryptosis has some similar characteristics to apoptosis such as cellular shrinkage, phosphatidylserine exposure, ceramide exposure and activation of cation channels in erythrocytes (Lang et al., 2008). All these changes lead to faster removal of the cells by the macrophage system, in liver and spleen. Intraerythrocytic proliferation of malaria parasite is accompanied with formation of new permeation pathways, due to the requirement of additional nutrients and disposal of waste metabolites (Föller et al., 2009). Activation of cation channels (primarily calcium and sodium), which are essential for the intracellular growth of the pathogen, P. falciparum, is believed to cause apoptosis in erythrocytes (Föller et al., 2009).

Oxidative stress causes activation of similar channels in non-infected erythrocytes, which leads to hemolysis in G6PD-deficient erythrocytes (Duranton et al., 2002). Entry of calcium leads to activation of intraerythrocytic scramblase, resulting in bidirectional movement of phospholipids and breakdown of phosphatidylserine asymmetry (Lang et al., 2008). Opening of Ca²⁺-permeable cation channels in erythrocytes have been shown to trigger apoptosis during oxidative stress (Lang et al., 2006). Phosphatidylserine exposure has been shown to enhance the removal of erythrocytes from the circulation. Eryptosis has been suggested to be an alternative pathway or as a non-immune intravascular hemolysis (Lang et al., 2008). The exposed phosphatidylserine is then identified by the macrophage system leading to engulfment and degradation of the erythrocytes (Duranton et al., 2002). Oxidized glutathione has been shown to be a strong intracellular mediator of non-selective cation channel activation during oxidative stress (Koliwad et al., 1996).

Current study is the follow-up of our recent studies on cytochrome P₄₅₀-dependent hemotoxic effects of primaquine on human erythrocytes (Ganesan et al., 2009). Multiple cytochrome P₄₅₀ isoforms were reported to contribute to generation of toxic metabolites and hemotoxicity of primaquine. These studies have been further extended to evaluate the effect of primaquine on normal and G6PD-deficient human erythrocytes. Accumulation of methemoglobin and generation of oxidative stress by primaquine, in presence of human liver microsomes, were monitored as biochemical markers for hemotoxic response (Ganesan et al., 2009). The microsomal metabolism-linked assay was employed to investigate eryptosis mechanism for hemotoxicity of primaquine on normal and G6PD-deficient human erythrocytes.