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.

The influence of selected ␤-receptor blockers on iron overload and oxidative stress in endothelial cells (ECs) was assessed. Confluent bovine ECs were loaded with iron dextran (15 M) for 24 h and then exposed to dihydroxyfumarate (DHF), a source of reactive oxygen species, for up to 2 h. Intracellular oxidant formation, monitored by fluorescence of 2Ј,7Ј-dichlorofluorescin (DCF; 30 M), increased and peaked at 30 min; total glutathione decreased by 52 Ϯ 5% (p Ͻ 0.01) at 60 min. When the ECs were pretreated 30 min before iron loading with 1.25 to 10 M D-propranolol, glutathione losses were attenuated 15 to 80%, with EC 50 ϭ 3.1 M. D-Propranolol partially inhibited the DCF intensity increase, but atenolol up to 10 M was ineffective. At 2 h, caspase 3 activity was elevated 3.2 Ϯ 0.3-fold (p Ͻ 0.01) in the iron-loaded and DHF-treated ECs, and cell survival, determined 24 h later, decreased 47 Ϯ 6% (p Ͻ 0.01). Ten micromoles of D-propranolol suppressed the caspase 3 activation by 63% (p Ͻ 0.05) and preserved cell survival back to 88% of control (p Ͻ 0.01). In separate experiments, 24-h iron loading resulted in a 3.6 Ϯ 0.8-fold increase in total EC iron determined by atomic absorption spectroscopy; D-propranolol at 5 M reduced this increase to 1.5 Ϯ 0.4-fold (p Ͻ 0.01) of controls. Microscopic observation by Perls' staining revealed that the excessive iron accumulated in vesicular endosomal/lysosomal structures, which were substantially diminished by D-propranolol. We previously showed that propranolol could readily concentrate into the lysosomes and raise the intralysosomal pH; it is suggested that the lysosomotropic properties of D-propranolol retarded the EC iron accumulation and thereby conferred the protective effects against iron load-mediated cytotoxicity.

No abstract