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N.J. Kleiman, A. Spector; Tertiary Butyl Hydroperoxide (tBHP) Induced DNA Damage, DNA Repair and Cell Death in Human Lens Epithelial (HLE) Cell Cultures . Invest. Ophthalmol. Vis. Sci. 2003;44(13):307.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The DNA damaging and cell killing effects of tBHP were investigated in HLE cell cultures. A variety of anti-oxidants, metal ion chelators, superoxide mimics, radical scavengers and other agents were employed to investigate the mechanisms whereby tBHP leads to cytotoxicity and DNA damage. Methods: HLE-B3 cells were subjected to tBHP stress. Cytotoxicity was measured by MTT assay and LDH leakage and DNA single strand breaks (SSB) analyzed by alkaline elution. Results: A single dose of 0.8 ml 80 µM tBHP to 40,000 cells was lethal. However, cytotoxicity was greatly reduced by incubation with a deferoxamine-Mn (DF-Mn) complex or by Tempol, SOD mimics, suggesting superoxide involvement. The metal chelators deferoxamine (DF) or orthophenanthroline (OP) had a limited ability to protect cells. The hydroxyl radical scavengers KI, mannitol, glycerol or DMSO and the nitroxide radical chelator PTIO were completely ineffective as was an inhibitor of nitric oxide synthase, L-NAME and the membrane permeable anti-oxidant DPPD. Incubation with either superoxide dismutase or catalase did not prevent cell death. In contrast, tBHP induced SSB were prevented by KI and relatively unaffected by Tempol and DF-Mn. OP and DF were partially protective. The SOD inhibitor diethyldithiocarbamate greatly increased the degree of SSB. Following a 30 min exposure to 400 µM tBHP, SSB repair was completed within one hour. Surprisingly, NAD levels remained relatively stable during insults of as long as 3 hours. 3-Aminobenzamide, an inhibitor of polyADP-ribosyl polymerase (PARP), led to an apparent increase in SSB during 30 min exposure to tBHP. Continued PARP inhibition during subsequent repair periods however, did not increase the apparent frequency of SSB, suggesting that PARP is important only in the early stages of DNA repair and/or that other DNA repair enzymes may compensate for the reduction in PARP activity. In contrast to H2O2-induced DNA damage, metal chelators were less effective in preventing tBHP-induced SSB and higher concentrations of tBHP were required for equivalent levels of strand breakage. Conclusions: The mechanism(s) whereby tBHP exerts cytotoxicity appears to involve superoxide or superoxide-like radicals but does not involve formation of hydroxyl radicals. Initial cytotoxic effects are independent of DNA damage. In contrast to cytotoxicity, DNA damage appears to involve formation of hydroxyl radical. It is hypothesized that the primary cytotoxic effect of tBHP involves superoxide mediated membrane damage while the DNA damaging effects reflect Haber-Weiss chemistry at the nuclear membrane.
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