Ultraviolet B-induced mitochondrial dysfunction is associated with decreased cell detachment of corneal epithelial cells in vitro

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 (10⁷ cells/ml) was measured by an O₂ meter, and adenosine triphosphate contents were measured by luciferase 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/10⁶ cells) compared to control (2.93 ± 0.12 nm/10⁶ cells) after 10 minutes. The effects of 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 μm 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.