Ultraviolet B-induced mitochondrial dysfunction is associated with decreased cell detachment of corneal epithelial cells in vitro.
OBJECTIVE
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.
Shimmura
S
S
Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
Tsubota
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology / Q000528:radiation effects; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology / Q000528:radiation effects; D002470:Cell Survival / Q000502:physiology / Q000528:radiation effects; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000502:physiology / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004734:Energy Metabolism / Q000528:radiation effects; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology / Q000528:radiation effects; D005456:Fluorescent Dyes; D006801:Humans; D008928:Mitochondria / Q000502:physiology / Q000528:radiation effects*; D010101:Oxygen Consumption / Q000528:radiation effects; D011419:Propidium; D020112:Rhodamine 123; D012235:Rhodamines; D014466:Ultraviolet Rays
D005456:Fluorescent Dyes; D012235:Rhodamines; D020112:Rhodamine 123; D011419:Propidium; D000255:Adenosine Triphosphate