Reactive oxygen species and DNA damage in 2-bromo-(glutathion-S-yl) hydroquinone-mediated cytotoxicity.
Exposure of renal proximal tubular epithelial cells (LLC-PK1) to the nephrotoxicants 2-bromo-6-(glutathion-S-yl)hydroquinone, 2-bromo-3-(glutathion-S-yl)-hydroquinone, and 2-bromo-(diglutathion-S-yl)hydroquinone caused DNA fragmentation and cytotoxicity. Viability measured by lysosomal neutral red accumulation was the most sensitive parameter of cytotoxicity, and preceded toxicity determined by either the mitochondrial MTT assay or by measuring intracellular lactate dehydrogenase activity. DNA fragmentation was detected as early as 15 min after exposure to 2-bromo-6-(glutathion-S-yl)hydroquinone (100 microM), 2-bromo-3-(glutathion-S-yl)hydroquinone (200 microM), and 2-bromo-(diglutathion-S-yl)hydroquinone (400 microM) and prior to other indices of toxicity. The ability 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-(glutathion-S-yl)hydroquinone from the incubation medium. Moreover, inhibition of poly(ADP-ribose)polymerase with 3-amino-benzamide (10 mM), following exposure of LLC-PK1 cells to 0.5 mM 2-bromo-6-(glutathion-S-yl)hydroquinone or 2-bromo-(diglutathion-S-yl)hydroquinone, decreased cytotoxicity, indicating that DNA repair processes, activated in response to DNA damage, exacerbate toxicity. Treatment with the endonuclease inhibitor, aurintricarboxylic acid did not decrease cytotoxicity. A decrease in the cytotoxicity caused by 2-bromo-6-(glutathion-S-yl)hydroquinone and 2-bromo-(diglutathion-S-yl)hydroquinone was observed when cells were incubated with catalase or pretreated with deferoxamine (10 mM). The data suggest a mechanism whereby the conjugates generate hydrogen peroxide, and the subsequent iron-catalyzed generation of hydroxyl radicals causes DNA fragmentation and cytotoxicity.
Mertens
J J
JJ
Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin 78712, USA.
Gibson
N W
NW
Lau
S S
SS
Monks
T J
TJ
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000633:toxicity; D006873:Hydroquinones / Q000633:toxicity*; D007501:Iron / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D018374:LLC-PK1 Cells; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013552:Swine
C090344:2-bromo-3-(glutathion-S-yl)hydroquinone; D004277:DNA, Single-Stranded; D006873:Hydroquinones; D017382:Reactive Oxygen Species; C066791:2-bromo-(diglutathion-S-yl)hydroquinone; D011064:Poly Adenosine Diphosphate Ribose; D007501:Iron; D004720:Endonucleases; D005978:Glutathione