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Abstract
PURPOSE: To evaluate the effects of ultraviolet B light (UV-B) on mitochondrial inner membrane function, cell viability, and migration of cultured human corneal epithelial cells. METHODS: After UV-B exposure in SV-40 transfected human corneal epithelial cells (T-HCEC), mitochondrial function was assessed by digital microfluorography using the mitochondrial marker, rhodamine 123 (Rh 123). The oxygen consumption rate of T-HCEC suspensions (10(7) cells/ml) was measured by an O2 meter, and adenosine triphosphate contents were measured by luciferase-driven chemiluminescence. Cell viability and migration was observed by propidium iodide (PI) staining and migration assays. RESULTS: UV-B exposure caused an immediate drop in O2 consumption by T-HCEC suspensions, whereas exposure of a monolayer culture of T-HCEC to UV-B at radiant exposures of 50 mJ/cm2 caused a reversible decrease in Rh 123 fluorescence (22.4%) and a significant decrease in adenosine triphosphate contents (1.52 +/- 0.05 nmol/10(6) cells) compared to control (2.93 +/- 0.12 nm/10(6) cells) after 10 minutes. The effects on Rh 123 fluorescence were irreversible at 100 mJ/cm2, which approximately corresponded with the threshold dose at which cells positive to PI staining (PI+) appeared. UV-B doses of 50 mJ/cm2 caused detachment of T-HCEC, primarily PI-, whereas higher doses (100 mJ/cm2) resulted in PI+ cells that did not detach from the dish. These PI+ cells hindered the migration of surrounding viable cells; detachment of PI- cells allowed cells to migrate and to cover a trough created by a 500 microns wide beam of UV-B. CONCLUSIONS: Threshold levels of UV-B (100 mJ/cm2) are associated with irreversible mitochondrial dysfunction and with the loss of the ability for cultured corneal epithelial cells to detach in vitro.