REACTIVE OXYGEN SPECIES AND DNA-DAMAGE IN 2-BROMO-(GLUTATHIONE-S-YL) HYDROQUINONE-MEDIATED CYTOTOXICITY

Exposure of renal proximal tubular epithelial cells (LLC-PK1) to the n ephrotoxicants 2-bromo-6-(glutathion-S-yl)hydroquinone, 2-bromo-3-(glu tathion-S-yl)-hydroquinone, and 2-bromo-(diglutathion-S-yl)hydroquinon e caused DNA fragmentation and cytotoxicity, Viability measured by lys osomal neutral red accumulation was the most sensitive parameter of cy totoxicity, and preceded toxicity determined by either the mitochondri al MTT assay or by measuring intracellular lactate dehydrogenase activ ity, DNA fragmentation was detected as early as 15 min after exposure to 2-bromo-6-(glutathion-S-yl)hydroquinone (100 mu M), 2-bromo-3-(glut athion-S-yl)hydroquinone (200 mu M), and 2-bromo-(diglutathion-S-yl) h ydroquinone (400 mu M) and prior to other indices of toxicity, The abi lity of the cells to repair DNA damage was evident by the decrease in the extent of single strand breaks following removal of 2-bromo-3-(glu tathion-S-yl)hydroquinone from the incubation medium, Moreover, inhibi tion of poly(ADP-ribose)polymerase with 3-amino-benzamide (10 nM), fol lowing exposure of LLC-PK1 cells to 0.5 mM 2-bromo-6-(glutathion-S-yl) hydroquinone or 2-bromo-(diglutathion-S-yl)hydroquinone, decreased cyt otoxicity, indicating that DNA repair processes, activated in response to DNA damage, exacerbate toxicity, Treatment with the endonuclease i nhibitor, aurintricarboxylic acid did not decrease cytotoxicity, A dec rease in the cytotoxicity caused by 2-bromo-6-(glutathion-S-yl)hydroqu inone and S-bromo(diglutathion-S-yl)hydroquinone was observed when cel ls were incubated with catalase or pretreated with deferoxamine (10 mM ), The data suggest a mechanism whereby the conjugates generate hydrog en peroxide, and the subsequent iron-catalyzed generation of hydroxyl radicals causes DNA fragmentation and cytotoxicity. (C) 1995 Academic Press, Inc.