NITRONE SPIN TRAPS AND A NITROXIDE ANTIOXIDANT INHIBIT A COMMON PATHWAY OF THYMOCYTE APOPTOSIS

Oxidative stress has recently been suggested to be a mediator of apopt otic 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 glucoco rticoid methylprednisolone (MPS), a progressive increase in intracellu lar peroxides and a decrease in glutathione (GSH) were observed to acc ompany the onset of apoptosis. Using Percoll density gradients to isol ate subpopulations of thymocytes at different stages of apoptosis, the increase in peroxide content was found to bt 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 inves tigate the biological significance of these redox changes, the free ra dical 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-radi cal antioxidant 2,2,6,6-tetramethyl-1-piperidinyl-1-oxyl (TEMPO) were tested as inhibitors of thymocyte apoptosis. The cell shrinkage and DN A 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 t hat its antioxidant properties were responsible for the observed prote ctive 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.