PURPOSE: 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.
PURPOSE: 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.
Authors: Lifang Hou; Sheng Wang; Chang Dou; Xiao Zhang; Yue Yu; Yinan Zheng; Umakanth Avula; Mirjam Hoxha; Anaité Díaz; John McCracken; Francesco Barretta; Barbara Marinelli; Pier Alberto Bertazzi; Joel Schwartz; Andrea A Baccarelli Journal: Environ Int Date: 2012-08-05 Impact factor: 9.621
Authors: John McCracken; Andrea Baccarelli; Mirjam Hoxha; Laura Dioni; Steve Melly; Brent Coull; Helen Suh; Pantel Vokonas; Joel Schwartz Journal: Environ Health Perspect Date: 2010-11 Impact factor: 9.031
Authors: Simone A Joosten; Vanessa van Ham; Claire E Nolan; Maria C Borrias; Alan G Jardine; Paul G Shiels; Cees van Kooten; Leendert C Paul Journal: Am J Pathol Date: 2003-04 Impact factor: 4.307