Oxidative stress--induced single-strand breaks in chromosomal telomeres of human retinal pigment epithelial cells in vitro.
OBJECTIVE
To demonstrate that chronic hyperoxia induces single-stranded breaks in chromosomal telomeres as a measure of oxidative DNA damage in cultured RPE cells.


METHODS
RPE340 cells were cultured in 40% and 20% (control) O(2). 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% O(2) shortened at a faster rate than those grown in 20% O(2). 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% O(2), 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 damage.
Honda
S
S
Departments of Ophthalmology, University of California, Davis, USA.
Hjelmeland
L M
LM
Handa
J T
JT
D015687:Cell Hypoxia; D002478:Cells, Cultured; D016922:Cellular Senescence; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005452:Fluoresceins; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics; D051547:Heme Oxygenase-1; D006801:Humans; D008565:Membrane Proteins; D018384:Oxidative Stress; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D016615:Telomere / Q000378:metabolism*
C110400:2',7'-dichlorodihydrofluorescein diacetate; D004277:DNA, Single-Stranded; D005452:Fluoresceins; D008565:Membrane Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1