May 2004
Volume 45, Issue 13
ARVO Annual Meeting Abstract  |   May 2004
Tertiary Butyl Hydroperoxide (tbhp) Conditioned Human Retinal Pigment Epithelial (rpe) Cells Resist Oxidative Stress Induced Cell Death And Dna Damage
Author Affiliations & Notes
  • W. Ma
    Ophthalmology, Columbia University, New York, NY
  • N.J. Kleiman
    Ophthalmology, Columbia University, New York, NY
  • Footnotes
    Commercial Relationships  W. Ma, None; N.J. Kleiman, None.
  • Footnotes
    Support  RPB
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 747. doi:
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      W. Ma, N.J. Kleiman; Tertiary Butyl Hydroperoxide (tbhp) Conditioned Human Retinal Pigment Epithelial (rpe) Cells Resist Oxidative Stress Induced Cell Death And Dna Damage . Invest. Ophthalmol. Vis. Sci. 2004;45(13):747.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: To understand the mechanism whereby RPE cells respond to oxidative stress and how such cells might be manipulated to resist the damaging effects of peroxide exposure. Methods: A variety of anti–oxidants, metal ion chelators, superoxide mimics, radical scavengers and other agents were employed to investigate the mechanism whereby tBHP leads to cytotoxicity and DNA damage in human ARPE14 cell cultures. Cytotoxicity was measured by MTT assay, LDH leakage and Trypan blue exclusion. Cell damage was measured by quantitating GSH levels, choline transport and thymidine and rubidium uptake. DNA single strand break formation (SSB) was analyzed by alkaline elution. Resistant cell cultures were created by gradually increasing tBHP levels in the medium over several months. Results: tBHP resistant cells thrived when exposed to daily concentrations of 130 µM tBHP. Even after oxidative stress was removed from the culture medium for three weeks, the cells retained their resistance to subsequent challenge with tBHP. Conditioned cells were resistant to the tBHP induced reduction in choline transport, thymidine uptake and intracellular GSH levels observed in unconditioned control cells. In contrast to conditioned cells, normal cells were killed within 12 hrs when exposed to tBHP levels as low as 70 µM. Cytotoxicity was greatly reduced in unconditoned cells by incubation with SOD mimics, suggesting superoxide involvement. Metal chelators and hydroxyl radical scavengers were ineffective in preventing cell death. In contrast to cytotoxicity, tBHP induced SSB were prevented by KI, a hydroxyl radical scavenger. When compared to H2O2–induced DNA damage, higher concentrations of tBHP were required for equivalent levels of strand breakage. Conclusions:A stable cell line resistant to the cell killing and DNA damaging effects of tBHP has been created. The mechanism(s) whereby tBHP exerts cytotoxicity appears to involve superoxide or superoxide–like radicals but may not involve formation of hydroxyl radicals. Initial cytotoxic effects are independent of DNA damage and, 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. Work to identify particular genes and/or regulatory pathways that are crucial to the maintenance of resistance is ongoing.

Keywords: oxidation/oxidative or free radical damage • age–related macular degeneration 

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