A study of ultraviolet radiation effects on porcine crystalline lens and development of a new assay methodology for UV cataractogenesis investigation
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Date
2000
Authors
Oriowo, Olanrewaju Matthew
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Publisher
University of Waterloo
Abstract
Four aspects of ultraviolet radiation (UVR) induced cataractogenesis were studied. Firstly, the suitability of procine lens for organ culture study and the feasibility of using two fluorescent dyes (alamarBlue TM) and 5-caroxyfluorescein diacetate-acetoxymethyl ester [CFDA-AM]) for in vitro UVR-lens study were examined. Secondly, because there were no available data for whole lens in vitra UVR threshold investigation, an experiment was conducted to determine the in vitro action spectrum for UVR-lens photodamage with a secondary purpose of assessing the recovery pattern in the exposed lenses. The third set of experiments investigated the efficacy of the two assay dyes (alamarBlueTM and CRDA-AM) to quantitatively determine the broadband UVB and UVA in vitro thresholds for impairing lens cellular function. Simulating environmental conditions, the fourth objective was to investigate the synergy of sub-solar low level UVB and UVA exposures in compromising lens integrity.
In all experiments, aseptically dissected porcine lenses were preincubated in organ culture for approximately one week. Addressing the first objective, the two dyes were assayed together. The lenses were immersed in the assay medium for fluorescence measurements at predetermined intervals over a period of 8 weeks. The assay results were validated with a scanning laser system that measured the optical quality of the same set of lenses. It was shown that porcine lenses can be cultured for at least 6 weeks without any compromise in cellular and optical integrity and that the two fluorescent indicator dyes are not toxic to the cultured lenses.
The in vitro action spectrum for UVR wavelengths from 270 nm to 370 nm was determined by subjecting cultured porcine lenses to varying UV radiant exposure levels at defined wavelengths. The action spectrum was derived with probit analysis of the data using an ascending/descending staircase method for dose determination at each waveband. In terms of repair, the data indicate shorter latency periods and slower recovery for higher UV energy levels, while the reverse is the case for lower UV energy levels. However, this observation is wavelength dependent. The damage and repair data demonstrate that at twice the threshold levels, permanent damage occurs for both UVB and UVA wavelengths.
The results from exposures involving UVB alone, UVA alone, and UVA and UVB in combination, show that exposure to low levels of UVB and moderately high levels of UVA can cause metabolic stress in the crystalline lens by inhibiting the lens epithelial cells' mitochondrial function and disrupting membrane integrity. The synergism study shows that subthreshold levels of UVB and UVA exposures acting together can cause decreased lens cellular metabolism and increased permeabilisation of lens epithelial and fibre cell membranes, triggering glutathione (GSH) depletion in the lens. These UV phototoxic events would render the lens epithelium incapable of regulating ions and water homeostasis, thereby causing lens swelling and opacity. Evaluation of the morphological changes showed that UVB irradiation cause "spoke like" equatorial opacity while UVA irradiation caused reversible anterior subcapsular vacuoles at almost 100 times the UVB exposure level.
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