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M. C. Kenney, S. R. Atilano, M. Chwa, N. Udar, J. V. Jester, P. Coskun, D. Wallace; Mitochondrial DNA Susceptibility to Hydrogen Peroxide Induced Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2710.
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© ARVO (1962-2015); The Authors (2016-present)
To understand if corneal mitochondrial DNA (mtDNA) can be damaged by oxidative reactions as a possible determinant in keratoconus (KC). We analyzed mtDNA isolated from epithelial cells of human normal (NL) and KC corneas. We also examined human telomerase immortalized corneal epithelial cells (hTCEpi) treated with an oxidizing agent, hydrogen peroxide (H2O2).
Total DNA was extracted from NL corneal epithelium (n=7, mean 54 yrs, range 35 to 70), KC corneal epithelium (n=5, mean 40 yrs, range 26 to 59) and the hTCEpi cultures which had been incubated at pH 7.0 or pH 5.0 with or without 200µM H2O2. Long Extension-Polymerase Chain Reaction (LX-PCR) was performed on all samples. The mtDNA to nuclear DNA (nDNA) ratio was measured by quantifying the PCR products of ND2 and 18s genes. The mtDNA control region (CR, 16024 - 576 bp) of the hTCEpi samples was sequenced.
The NL and KC epithelial cells had a major LX-PCR product of ~16.2kb, the expected size for mtDNA, and also smaller sized mtDNA bands, 4.28 ± 0.99 vs 4.0 ± 0.83 bands per individual, respectively. The mtDNA to nDNA ratios were similar for both NL and KC epithelium. With LX-PCR, the untreated hTCEpi cultures had the mtDNA 16.2kb band and smaller sized bands. After incubation at pH 5.0, the LX-PCR mtDNA bands were decreased by 32%. After H2O2-treatment, the LX-PCR mtDNA 16.2 kb band was not observed and 82% of the smaller sized bands degraded. Sequencing of the mtDNA CR showed multiple sites of change in the H2O2-treated hTCEpi cultures.
Intact KC corneas have higher levels of mtDNA damage compared to NL corneas (Atilano et al., 2005 IOVS 46:1256-1263). Our findings show minimal mtDNA damage in the isolated KC corneal epithelial cells, implying that the accumulated mtDNA damage found in intact KC corneas may be attributable to the stroma and endothelium, cell types with slower turnover rates. In addition, in vitro hTCEpi studies show that H2O2 challenges can cause significant mtDNA damage, including mtDNA CR changes, and provide a correlation between oxidative stress and mtDNA dysfunction.
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