Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue

Abstract

Purpose: To examine levels of oxidative DNA base damage and expression of selected genes and proteins related to DNA damage repair in human limbal epithelium engineered ex vivo.

Methods: Cells were expanded from limbal tissue on cell culture-treated inserts in medium containing fetal bovine serum, recombinant growth factors, hormones and cholera toxin (COM) and in medium with human serum as the single growth-promoting additive (HS). Cells were analysed after two, three and four weeks in culture for DNA strand breaks and oxidized purine bases (Comet assay using the enzyme formamidopyrimidine DNA glycosylase, Fpg) and for expression of DNA repair enzymes APE1, OGG1 and Polβ by in situ hybridization (ISH) and by immunohistochemistry (IHC).

Results: Levels of strand breaks were substantial while levels of net Fpg-sensitive sites (8-oxoguanine and ring-opened FaPy bases) were relatively low in cells engineered in COM and in HS. Both types of medium were found to support expression of base excision repair (BER) enzymes APE1, OGG1 and Polβ at the gene level. At the protein level, expression of APE1 and OGG1 was noticeable in both conditions while expression of Polβ was low.

Conclusion: Our findings indicate low levels of oxidative stress and/or efficient DNA purine base damage repair in human limbal epithelium engineered in a medium with human serum as the single growth-promoting additive as well as in traditional medium with xenobiotics.

Keywords: DNA damage; cornea; epithelium; limbus; oxidative damage; tissue engineering.