We developed a cerebellar granule cell model of peroxynitrite toxicity and showed that certain sulfhydryl-containing compounds (e.g., penicillamine) present as concurrent treatments could inhibit this toxicity. In the present study, 21-aminosteroid and pyrrolopyrimidine lazaroids were tested for cytoprotection in this peroxynitrite toxicity model. In addition, we tested for added protection using a peroxynitrite scavenger concurrent treatment combined with a lazaroid post-treatment. The toxicity assay utilized cells that were previously exposed to 100 microM L-buthionine (S,R)-sulfoximine (BSO), an inhibitor of gamma-glutamyl-cysteine synthetase, for 24 h. This sublethal concentration of BSO shifted the peroxynitrite (1-1000 microM) toxicity curve to the left by more than one-half of a log unit. The half-maximal toxicity concentration (TC50) of peroxynitrite in cells treated with BSO was 50 microM. The 21-aminosteroids, U-74006F and U-74500A, and the pyrrolopyrimidines, U-91736B and U-101033E, were tested as post-treatments. U-74006F and U-74500A had EC50 values of approximately 100 microM (concentrations which blocked 50% of the toxicity). U-91736B and U-101033E had EC50 values of 1 microM and showed 100% protection at 3-10 microM. Treatment with either 100 microM U-74006F or 1 microM U-101033E resulted in a right-hand shift (protection) in the peroxynitrite toxicity curve. Further, combination treatment of lazaroids with 1 mM penicillamine resulted in additive protection compared to either treatment alone.