In vitro assays for evaluating the ultraviolet B-induced damage in cultured human retinal pigment epithelial cells.
The present study demonstrates broadband UV-B-induced damage of cultured human retinal pigment epithelial cells as an effort to develop an in vitro model that can be used, along with in vivo research and other in vitro efforts, to evaluate the need for retinal UV protection in humans after cataract removal. The human retinal pigment epithelial cell line, ARPE-19, was cultured in two groups: control and treated. Treated cells were irradiated with three broadband UVB radiations at energy levels of 0.05, 0.1 and 0.2J/cm(2). After irradiation, cells were incubated for 48h while cellular viability, morphology, and phagocytotic activity were analyzed using the Alamar blue assay, confocal microscopy, and fluorescent microspheres. Confocal analysis concentrated on the study of the cell nuclei and mitochondria. The Alamar blue assay of UV-B-exposed cells showed dose and time-dependent decreases in cellular viability in comparison to control cells. Loss of cell viability was measured at the two higher energy levels (0.2 and 0.1J/cm(2)), but the cell group exposed to 0.05J/cm(2) showed no significant viability change at 1-h time point. Morphological evaluation also showed dose and time-dependent degradation of mitochondria and nucleic acids. Cells exposed with 0.05J/cm(2) UVB did not show significant degradation of mitochondria and nucleic acids during the entire culture period. Phagocytotic activity assay data for UVB-exposed cells showed dose-dependent decreases in phagocytotic activity in comparison with the control cells. The control cells have significantly greater capacities for uptake than the 0.1 and 0.2J/cm(2) UV-B-exposed cells, while the 0.05J/cm(2) UV-B-exposed cell group showed no significant difference from the control cell group. The findings suggest that UVB radiation-induced cultured RPE cell damage can be evaluated by assays that probe cellular viability, morphological change, and phagocytotic activity, and that these assay methods together provide a valuable in vitro model for ultraviolet radiation-induced retinal toxicology research.
Youn
Hyun-Yi
HY
School of Optometry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
Bantseev
Vladimir
V
Bols
Niels C
NC
Cullen
Anthony P
AP
Sivak
Jacob G
JG
D000165:Acridine Orange / Q000737:chemistry; D002470:Cell Survival; D002478:Cells, Cultured; D004249:DNA Damage; D004847:Epithelial Cells / Q000378:metabolism / Q000528:radiation effects*; D006801:Humans; D018613:Microscopy, Confocal; D010078:Oxazines / Q000737:chemistry; D010587:Phagocytosis; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000528:radiation effects*; D014466:Ultraviolet Rays; D014966:Xanthenes / Q000737:chemistry
D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D000165:Acridine Orange