We reported that 50% of cisplatin-induced apoptosis in primary cultures of rabbit renal proximal tubule cells (RPTC) proceeded via caspase-independent mechanisms. This study determined whether caspase-independent apoptosis, using multiple and diverse endpoints, could be produced by toxicants other than cisplatin and in cell models other than RPTC. Cisplatin, staurosporine, vincristine, and A23187 induced RPTC apoptosis after 24 h as indicated by 2-to 2.5-fold increases in annexin V and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining, and 2-to 10-fold increases in cell shrinkage. All toxicants induced 8-to 50-fold increases in caspase-3 activities, which were completely inhibited by the pan caspase inhibitor ZVAD-fmk. However, ZVAD-fmk only decreased cisplatin-and staurosporine-induced annexin V staining and cell shrinkage 30 to 50%, staurosporine-induced TUNEL staining 30%, and did not affect vincristine-or A23187-induced RPTC apoptosis. All toxicants tested induced apoptotic RPTC nuclear morphology. However, similar to its effect on annexin V and TUNEL staining, ZVAD-fmk only partially inhibited toxicant-induced apoptotic nuclear morphology. Cisplatin and staurosporine also induced annexin V staining in the human epithelial cancer cell lines Caki-1 (kidney carcinoma), A549 (lung carcinoma), A172 (glioblastoma), and murine lymphocytic leukemia L1210 cells. Pretreatment with ZVAD-fmk inhibited cisplatin-induced annexin V staining in Caki-1, A172, and A549 cells but had no affect in L1210 cells. Pretreatment with ZVAD-fmk did not decrease staurosporineinduced annexin V staining in Caki-1, A549, L1210, and A172 cells. These results suggest that a significant fraction of apoptosis induced by diverse toxicants in renal epithelial cells and in four different cancer cell lines is caspase-independent.

Previous studies have shown that 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH), an N-hydroxy metabolite of the antimalarial drug, primaquine, is a direct-acting hemolytic agent in rats. To investigate the mechanism underlying this hemolytic activity, the effects of hemotoxic concentrations of MAQ-NOH on rat erythrocyte sulfhydryl status, membrane lipids, skeletal proteins, and morphology have been examined. Treatment of rat erythrocytes with a TC 50 concentration of MAQ-NOH (350 M) caused only a modest and transient depletion of reduced glutathione (GSH) (ϳ30%), which was matched by modest increases in the levels of glutathione disulfide and glutathione-protein mixed disulfides. Lipid peroxidation, as measured by thiobarbituric acidreactive substances and F 2 -isoprostane formation, was induced in a concentration-dependent manner by MAQ-NOH. However, the formation of disulfide-linked hemoglobin adducts on membrane skeletal proteins and changes in erythrocyte morphology were not observed. These data suggest that hemolytic activity results from peroxidative damage to the lipid of the red cell membrane and is not dependent on skeletal protein thiol oxidation. However, when red cell GSH was depleted (Ͼ90%) by titration with diethyl maleate, hemolytic activity of MAQ-NOH was markedly enhanced. Of interest, exacerbation of hemotoxicity was not matched by increases in lipid peroxidation, but by the appearance of hemoglobin-skeletal protein adducts. Collectively, the data are consistent with the concept that MAQ-NOH may operate by more than one mechanism; one that involves lipid peroxidation in the presence of normal amounts of erythrocytic GSH, and one that involves protein oxidation in red cells with low levels of GSH, such as are seen in individuals with glucose-6-phosphate dehydrogenase deficiency.

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...