The mechanism of puromycin aminonucleoside (PAN)-induced nephrosis has not yet been well defined. In the present study, we examined the protective effect of active oxygen scavengers on the PAN-induced injury of cultured rat glomerular epithelial cells (GECs) and the generation of active oxygen species in PAN-treated GECs. When exposed to PAN (greater than or equal to 25 micrograms/ml), cellular damage occurred in a time- and dose-dependent manner as evaluated by both the LDH release and MTT colorimetric assays. Concomitant addition of either the hydrogen peroxide (H2O2) scavenger, catalase, or the iron chelating agent, deferoxamine, to the culture medium caused a striking reduction of cellular injury. This suggested a role for H2O2 and for hydroxyl radicals (OH.) generated via the iron-catalyzed breakdown of H2O2 in PAN nephrosis. Using the scopoletin fluorescence assay, the release of H2O2 into the culture medium by GECs exposed to PAN (greater than or equal to 50 micrograms/ml) was shown to increase dose-dependently (greater than or equal to 57 +/- 11 pmol/4.4 x 10(6) cells per h, P less than 0.01) as compared with control cells (14 +/- 2 pmol/4.4 x 10(6) cells per h). These results strongly suggested that active oxygen species, especially H2O2 and OH., might play an important role in PAN-induced GEC injury in vitro as well as in vivo.