DNA Oxidation and Expression of Repair Enzymes in Organ- Cultured Human Limbal Epithelium

DNA oxidation damage and its repair are essential for maintaining genomic integrity in the human limbal epithelium, which harbors corneal epithelial stem cells. This study investigated the distribution of the DNA base oxidation 8-oxoguanine (8-oxoG) and the base excision repair (BER) enzymes 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease 1 (APE1) in non-cultured and eye-bank organ-cultured human limbal epithelia. Immunohistochemistry was used to assess the localization and staining intensity of 8-oxoG, OGG1, and APE1, evaluated semi-quantitatively and by image analysis. In situ hybridization was performed to detect the distribution of OGG1 and APE1 gene expression in organ-cultured tissue. In non-cultured limbal epithelia, nuclear 8-oxoG staining was more frequently observed in superficial epithelial layers, whereas nuclear OGG1 and APE1 staining predominated in basal layers. In organ-cultured epithelia, a higher proportion of superficial nuclei exhibited 8-oxoG staining, while the basal predominance of OGG1 was reduced and that of APE1 was preserved. Transcripts of OGG1 and APE1 were detected in basal- as well as in suprabasal layers of organ-cultured epithelia. These findings demonstrate the presence of DNA base oxidation and BER-related enzymes in basal and suprabasal human limbal epithelial cells during storage of corneal tissue under commonly used eye-bank organ-cultured conditions prior to transplantation.