Purpose: To study the effects of hydrogen peroxide exposure on mitochondrial DNA (mtDNA) in cultured human corneal epithelial cells. In addition, we compared the integrity of mtDNA found in epithelial cells isolated from keratoconus (KC) and normal (NL) corneas.
Methods: Telomerase immortalized human corneal epithelial cell line (hTCEpi) were cultured at pH 7.0 or pH 5.0 with or without 200 microM hydrogen peroxide (H2O2). Immunohistochemistry with a marker for oxidative damage, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), was performed on KC and NL corneas (n = 10). Epithelial cells were isolated from KC corneas (n = 5) and NL corneas (n = 7). Total DNA was extracted, and the mtDNA was analyzed by long extension polymerase chain reaction (LX-PCR). The ratios of mtDNA to nuclear DNA were measured by PCR. The mtDNA control regions were PCR amplified and sequenced.
Results: In the epithelial cell cultures, the full-length LX-PCR mtDNA decreased 54% and 44% in the H2O2 + pH7 cultures and H2O2 + pH5 cultures, respectively. 8-OH-dG was present in all layers of KC epithelial cells but only in superficial layers of NL epithelial cells. The isolated KC and NL epithelial cells had comparable levels of full-length LX-PCR mtDNA (16.2 kb) and smaller sized mtDNA bands (4.3 +/- 0.99 vs 4.0 +/- 0.83 bands per individual, respectively). There were no significant differences in the control region nucleotide sequences in KC and NL epithelia.
Conclusions: Hydrogen peroxide can significantly degrade LX-PCR mtDNA in vitro. Although the KC epithelium showed a higher degree of oxidative damage, the levels of mtDNA damage in NL and KC epithelial cells were similar to each other.