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Shigeru Honda, Leonard M. Hjelmeland, James T. Handa; Oxidative Stress–Induced Single-Strand Breaks in Chromosomal Telomeres of Human Retinal Pigment Epithelial Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2001;42(9):2139-2144.
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purpose. To demonstrate that chronic hyperoxia induces single-stranded breaks in
chromosomal telomeres as a measure of oxidative DNA damage in cultured
methods. RPE340 cells were cultured in 40% and 20% (control) O2.
DNA damage was assessed by mean terminal restriction fragment (TRF)
length, and the S1 nuclease assay was used to determine the frequency
of single-strand breaks in telomeric DNA. The degree of oxidative
stress in cells was estimated by flow cytometric analysis of reactive
oxygen intermediate (ROI)-induced
2′,7′-dichlorodihydrofluorescein diacetate fluorescence and Northern
blot analysis of heme oxygenase-1 (HO-1) mRNA induction.
results. The mean TRF length of cells grown in 40% O2 shortened at
a faster rate than those grown in 20% O2. The S1 nuclease
assay showed that the accelerated mean TRF length shortening was due to
an increased accumulation of single-stranded breaks in telomeric DNA.
The degree of ROI production and HO-1 mRNA induction was greater in
cells treated with 40% than 20% O2, an effect that was
also larger in old than young passaged cells.
conclusions. RPE340 cells in vitro grown in chronic hyperoxia exhibited evidence of
DNA damage with accelerated telomeric shortening via an increased
accumulation of single-strand breaks in telomeric DNA. These changes
could provide insight into aging of RPE cells by oxidative DNA
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