Date of Award

2005

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell and Molecular Pharmacology

College

College of Graduate Studies

First Advisor

David McMillan

Second Advisor

David Jollow

Third Advisor

Kevin J. Schey

Fourth Advisor

Craig Beeson

Fifth Advisor

Brad Schulte

Abstract

Primaquine is an important antimalarial agent because of its activity against exoerythrocytic forms of Plasmodium sp. Methemoglobinemia and hemolytic anemia, however, are dose-limiting side effects of primaquine therapy. These hemotoxic effects are believed to be mediated by metabolites, though the identity of the toxic specie(s) and the mechanism underlying hemotoxicity have remained unclear. Previous studies showed that an N-hydroxylated metabolite of primaquine, 6-methoxy-8- hydroxylaminoquinoline, was capable of mediating primaquine-induced hemotoxicity. The present studies were undertaken to investigate the hemolytic mechanism of 5-hydroxyprimaquine (5-HPQ), a phenolic metabolite that has been detected in experimental animals. 5-HPQ was synthesized, isolated by flash chromatography and characterized by NMR spectroscopy and mass spectrometry. In vitro exposure of 51Cr-Iabeled erythrocytes to 5-HPQ induced a concentration-dependent decrease in erythrocyte survival (TC50 ~40 µM) when the exposed cells were returned to the circulation of isologous rats. 5-HPQ also induced methemoglobin formation and depletion of glutathione (GSH) when incubated with suspensions of rat erythrocytes. Furthermore, when red cell GSH was depleted (>95%) by titration with diethyl maleate to mimic GSH instability in human glucose-6-phosphate dehydrogenase deficiency, a 5-fold enhancement of hemolytic activity was observed. These data indicate that 5-HPQ also has the requisite properties to contribute to the hemotoxicity of primaquine. To investigate the fate of erythrocytes in vivo after in vitro exposure to 5-HPQ, rat 51Cr-Iabeled erythrocytes were incubated with hemolytic concentrations of 5-HPQ and then re-administered intravenously to rats. The time-course of loss of radioactivity from blood and uptake into the spleen and liver was measured. In rats given 5-HPQ-treated erythrocytes, an increased rate of removal of radioactivity from the circulation was observed as compared to the vehicle control. The loss of blood radioactivity was accompanied by a corresponding increase in radioactivity appearing in the spleen but not in the liver. When rats were pretreated with clodronate-Ioaded liposomes to deplete splenic macrophages, there was a decreased rate of removal of radioactivity from the circulation and a markedly diminished uptake into the spleen. A role for phagocytic removal of 5-HPQ-treated red cells was confirmed in vitro using the J774A.1 macrophage cell line. Furthermore, depletion of red cell GSH with diethyl maleate significantly enhanced in vitro phagocytosis of 5-HPQ-treated red cells. These data indicate that splenic macrophages are responsible for removing 5-HPQ-treated red cells and support a role for depletion of GSH as a key event in the process leading to macrophage recognition and phagocytosis of 5-HPQ-damaged erythrocytes. 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 (DCFDA). 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. These 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. Cummulatively, the data presented in this dissertation describe a mechanism by which exposure to 5-hydroxyprimaquine leads to the hemolytic removal of red cells; i.e. the generation of intracellular oxidative stress causes protein oxidation and hemoglobin binding to the membrane and subsequent phagocytosis by macrophages. A relationship between drug-induced hemolytic anemia and mechanisms of red cell senescence is discussed, as are possible implications for the antimalarial therapeutic effect of primaquine. The relative contribution of N-hydroxy vs. phenolic metabolites to the overall hemotoxicity of primaquine remains to be assessed.

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