Mitochondrial 'Movement' and Lens Optics following Oxidative Stress from UV-B Irradiation: Cultured Bovine Lenses and Human Retinal Pigment Epithelial Cells (ARPE-19) as Examples

摘要

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 photorecep-tor 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 (A) 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° 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 μm/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 that may lead to activation of caspase-mediated apop-totic pathways.