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Mitochondria provide energy generated by oxidative phosphorylation and at the same time play a central role in apoptosis and aging. As a byproduct of respiration, the electron transport chain is known to be the major intracellular site for the generation of reactive oxygen species (ROS). Exposure to solar and occupational ultraviolet (UV) radiation, and thus production of ROS and subsequent cell death, has been implicated in a large spectrum of skin and ocular pathologies, including cataract. Retinal pigment epithelial cell apoptosis generates photoreceptor dysfunction and ultimately visual impairment. The purpose of this article was to characterize in vitro changes following oxidative stress with UV-B radiation in (a) ocular lens optics and cellular function in terms of mitochondrial dynamics of bovine lens epithelium and superficial cortical fiber cells and (b) human retinal pigment epithelial (ARPE-19) cells. Cultured bovine lenses and confluent cultures of ARPE-19 cells were irradiated with broadband UV-B radiation at energy levels of 0.5 and 1.0 J/cm(2). Lens optical function (spherical aberration) was monitored daily up to 14 days using an automated laser scanning system that was developed at the University of Waterloo. This system consists of a single collimated scanning helium-neon laser source that projects a thin (0.05 mm) laser beam onto a plain mirror mounted at 45 degrees on a carriage assembly. This mirror reflects the laser beam directly up through the scanner table surface and through the lens under examination. A digital camera captures the actual position and slope of the laser beam at each step. When all steps have been made, the captured data for each step position is used to calculate the back vertex distance for each position and the difference in that measurement between beams. To investigate mitochondrial movement, the mitochondria-specific fluorescent dye Rhodamine 123 was used. Time series were acquired with a Zeiss 510 (configuration Meta 18) confocal laser scanning microscope equipped with an inverted Axiovert 200 M microscope and 40-x water-immersion C-Apochromat objective (NA 1.2). The optical analysis showed energy level-dependent increases in back vertex distance variability (loss of sharp focus) from 0.39 +/- 0.04 mm (control, n = 11) to 1.63 +/- 0.33 mm (1.0 J/cm(2), n = 10) and 0.63 +/- 0.13 mm (0.5 J/cm(2), n = 9). Confocal laser scanning microscopy analysis of both bovine lenses and ARPE-19 cells showed that following treatment at 0.5 J/cm(2) the mitochondria stopped moving immediately whereas at 1.0 J/cm(2) not only did the mitochondria stop moving, but fragmentation and swelling was seen. Untreated control tissue exhibited up to 15 microm/min of movement of the mitochondria. This could represent normal morphological change, presumably allowing energy transmission across the cell from regions of low to regions of high ATP demand. Lack of mitochondrial movement, fragmentation, and swelling of mitochondria may represent early morphological changes following oxidative stress tha...

This work involves the evaluation of UV blocking efficiency of commercially available intraocular lens (IOL) materials using a retinal cell culture and a biological in vitro model that was developed in a previous study, as an effort to examine the sensitivity of this in vitro approach for evaluating toxicity of UV radiation on the retinal pigment epithelial cells. The human retinal pigment epithelial (RPE) cell line, ARPE-19, was cultured, and cells were irradiated with broadband UVB radiations at energy levels of 0.2 and 0.4 J/cm. Some treated cells were not shielded from the radiation while others were shielded using two thicknesses (0.9 and 1.5 mm) of IOL material. After irradiation, cellular viability, mitochondrial distribution, nuclei morphology, and phagocytotic activity were analyzed using the Alamar blue assay, Rhodamine 123 staining, the Hoechst assay, and a phagocytotic activity assay. The results demonstrate that UVB radiation can cause significant decreases in RPE cell viability as well as in phagocytotic activity. Also, the results show that UVB radiation can induce the degradation of DNA and mitochondria in cultured RPE cells. However, the two different thickness IOL material sheets (0.9 and 1.5 mm) showed very effective UV blocking ability, allowing no cellular damage at all. Thus, the finding suggest that these four assays together can be used as a sensitive, and meaningful in vitro biomarker method for evaluating toxicity of UV radiation on RPE cells, and also for examining IOL effectiveness.