The comet assay applied to cells of the eye.

The human eye is relatively unexplored as a source of cells for investigating DNA damage. There have been some clinical studies, using cells from surgically removed tissues, and altered DNA bases as well as strand breaks have been measured using the comet assay. Tissues examined include corneal epithelium and endothelium, lens capsule, iris and retinal pigment epithelium. For the purpose of biomonitoring for exposure to potential mutagens in the environment, the eye-relatively unprotected as it is compared with the skin-would be a valuable object for study; non-invasive techniques exist to collect lachrymal duct cells from tears, or cells from the ocular surface by impression cytology, and these methods should be further developed and validated.

Cell death pattern in lens epithelium of cataract patients.

PURPOSE: Apoptosis, a type of programmed cell death, is observed in various types of cataract and in cultured lens epithelium subjected to oxidative damage. We have recently described oxidative DNA base damage in epithelium in age-related cataract and cultured cells, and we here aimed to examine such epithelium for markers for proliferation, initiation of apoptosis and morphological patterns of cell damage. METHODS: Samples (n = 75) were analysed by light microscopy/electron microscopy (LM/EM); immunohistochemistry (IHC) for PCNA and Ki67 (DNA synthesis/proliferation); TUNEL assay (DNA fragmentation/apoptosis); and protein/gene expression of Caspase-3 (apoptotic effector molecule) and BAX/Bcl2 (pro-/anti-apoptotic marker) in fresh/cultured epithelium by IHC and qRT-PCR. RESULTS: In fresh samples, the majority of cells were Ki67-/PCNA+. BAX/BCL-2-ratio was approximately 1, and Caspase-3 levels were low. TUNEL stained scattered nuclei/nuclear fragments (9/6302 cells). Main morphological signs of cell damage included rupture of cell membranes and hydration of cytoplasm and nuclei. Cultivation increased levels of BAX and Bcl2 by IHC and qRT-PCR (approximately 10-fold upregulation). Caspase-3 levels remained low by IHC with similar expression in fresh and cultured samples by qRT-PCR. CONCLUSION: Genomic stress and DNA repair may explain the contrasting expression of Ki67/PCNA in fresh epithelium. Despite low levels of Caspase-3 and similar expression of BAX/Bcl-2, a low incidence of apoptosis may be detected in epithelium in age-related corticonuclear cataract. Epithelium may be transferred to culture without an increase in expression of Caspase-3, one of the central mediators of apoptosis.