Pro-oxidant Properties and Cytotoxicity of AZT-Monophosphate and AZT

Abstract

The effects of zidovudine (AZT) and AZT-monophosphate (AZT-MP) on lipid peroxidation and oxidative cell injury were studied. When microsomal membranes from rat livers were peroxidized by a superoxide-driven, Fe-catalyzed oxy-radical system (ORS), both AZT-MP and, to a lesser extent AZT, but not thymidine, concentration dependently (2-100 microM) enhanced lipid peroxidation (TBARS formation) up to 51% above control. Significance (p < 0.05) was achieved by 6.7 microM AZT-MP. When cultured bovine aortic endothelial cells were incubated with the ORS for 60 min, total glutathione (GSH) decreased by 40% and 24-h cell survival, determined by the tetrazolium salt MTT assay, decreased by 38%. Using this cell system, AZT-MP (7-100 microM) promoted cell death further; at 20 microM 50% (p < 0.01), cell death was induced. In comparison, AZT was less effective. Concurrently, AZT-MP significantly promoted ORS-mediated loss of GSH. These cytotoxic effects were further exacerbated by low extracellular magnesium. Interestingly, when the endothelial cells were exposed to an iron-independent peroxynitrite generating system (SIN-1), the AZT-MP effects were absent. We propose that these pro-oxidant properties of AZT-MP are iron dependent. Because AZT-MP is a major phosphorylated metabolite, the data suggest that potential pro-oxidative activities may be associated with AZT use when catalytic iron is present.

Absence of a Universal Mechanism of Mitochondrial Toxicity by Nucleoside Analogs

“…In general, for the respective doses and time frames, the HepG2 data presented here are in agreement with previous studies using this cell line (7,39,50) and confirm the observation that AZT is cytotoxic without depleting mtDNA. Oxidative damage and ROS have been observed to increase with AZT treatment (14,15,46,53), and both AZT and AZTmonophosphate have been shown to be pro-oxidant in vitro (31). In this study, we demonstrate that AZT exposure increases intramitochondrial superoxide production in HepG2 cells.…”

“…However, this shared locus of toxicity belies the apparent complexity of NRTI toxicity. Suggested mechanisms of toxicity include, but are not limited to, the inhibition of DNA Pol-␥ (23,24,32), inhibition of endogenous nucleotide kinases (30,35,49), direct inhibition of oxidative phosphorylation (4,5,29,40), reactive oxygen species (ROS) generation (31,44,46), and mtDNA and nuclear DNA mutagenesis (1,10,45), yet mtDNA abundance is commonly employed as the hallmark of NRTI toxicity. The results of this investigation demonstrate that inhibition of DNA Pol-␥ is but one of many possible cytotoxic mechanisms of the different NRTIs to alter the metabolic status of the cell and that the essences of mtDNA depletion in the mechanism of cytotoxicity differ among the different cell lines.…”

“…At the center of these differences lies the mitochondrion, which has long been suspected to be the inadvertent target of NRTI toxicity. While there are some studies that provide mechanisms aside from mitochondria (31,46,54), based on the observed pathologies, the mitochondria remain the primary focus. Irrespective of this common focus, NRTIs display surprisingly variable toxicities, and it is becoming increasingly difficult to accommodate these differences within a single mechanism (i.e., the "DNA Pol-␥ hypothesis" [23,24]).…”

The nerve-heart connection in the pro-oxidant response to Mg-deficiency

“…AZT alone or moderate MgD alone only caused mild cardiac lesions; however, combined AZT and MgD led to significant elevation in small-size ventricular lesions and promoted prominent atrial inflammatory lesions, fibrosis and scarring. In association, the loss of RBC glutathione was further aggravated by AZT treatment [91]. Interestingly, we have shown that low extracellular Mg (0.2 mM) can potentiate AZT-phosphate-mediated oxidative injury in cultured endothelial cells [91], as well as the pro-oxidant effect of AZT-phosphate in phosphate buffer [92].…”

“…Myocardial necrosis induced by isoproterenol (β-adrenergic agonist) was dramatically augmented by dietary Mg-deficiency in hamsters [90]. More recently, using a mouse model [91], we studied the cardiac histopathologic consequences of AZT (zidovudine) administration with concurrent Mg-deficiency. AZT alone or moderate MgD alone only caused mild cardiac lesions; however, combined AZT and MgD led to significant elevation in small-size ventricular lesions and promoted prominent atrial inflammatory lesions, fibrosis and scarring.…”