Nitrone spin traps and a nitroxide antioxidant inhibit a common pathway of thymocyte apoptosis

Abstract

Oxidative stress has recently been suggested to be a mediator of apoptotic cell death [Buttke and Sandstrom (1994) Immunology Today 15, 7-10], although evidence that this phenomenon is a widespread component of apoptosis is lacking. When rat thymocytes were exposed to the glucocorticoid methylprednisolone (MPS), a progressive increase in intracellular peroxides and a decrease in glutathione (GSH) were observed to accompany the onset of apoptosis. Using Percoll density gradients to isolate subpopulations of thymocytes at different stages of apoptosis, the increase in peroxide content was found to be restricted to apoptotic cells, while a significant depletion of GSH and reduced protein thiol was detected in both pre-apoptotic and fully apoptotic cells. To investigate the biological significance of these redox changes, the free radical spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide (TMPO), and the related nitroxide-radical antioxidant 2,2,6,6-tetramethyl-1-piperidinyl-1-oxyl (TEMPO) were tested as inhibitors of thymocyte apoptosis. The cell shrinkage and DNA fragmentation induced by four different initiators of apoptosis were reduced by each compound. TEMPO inhibition of both etoposide- and MPS-induced thymocyte DNA fragmentation was also found to correlate with an increase in intracellular GSH, providing support for the proposal that its antioxidant properties were responsible for the observed protective activity. We conclude that some form of intracellular oxidation (here measured indirectly by changes in intracellular GSH and peroxide levels) is required during thymocyte apoptosis even when this process is initiated by an agent that does not exert a direct oxidant action.

Cisplatin: mode of cytotoxic action and molecular basis of resistance

“…These include the role of elevated GSH in either increasing DNA repair (Kelland, 1993) or increasing the inhibitory effect on apoptosis by buffering an endogenous drug-induced oxidative stress (Chiba et al, 1995;Slater et al, 1995). This is consistent with reports that cells overproducing the Bcl-2 protein have correspondingly higher intracellular GSH levels, which may contribute to the antiapoptotic functions of Bcl-2 (Hockenbery et al, 1993;Chiao et al, 1995).…”

Thymocytes selected for resistance to hydrogen peroxide show altered antioxidant enzyme profiles and resistance to dexamethasone-induced apoptosis

“…Studies aimed at establishing the chronology of apoptotic events support an early role for ROS. When apoptotic thymocyte populations are collected using Percoll gradients 13 or identified by flow cytometry, 16 small increases in ROS can be seen shortly after steroid treatment. The evidence of lipid peroxidation seen by Bustamante et al 19 also appeared shortly after drug treatment.…”

Role of reactive oxygen species in brucein D-mediated p38-mitogen-activated protein kinase and nuclear factor-κB signalling pathways in human pancreatic adenocarcinoma cells

“…Our results showed that in pancreatic cancer cells, treatment with BD caused reduction of intracellular GSH in a does-dependent manner. Glutathione is the most abundant bulk antioxidant, which prevents apoptosis and maintains viability of the cells (Slater et al, 1995). PANC-1 cells deprived of GSH on exposure to BD became more vulnerable to oxidative stress when their ability to scavenge and detoxify the various metabolically generated reactive oxygen intermediates was impaired.…”