Potential Protection by Antioxidants of the Action of Tobacco Smoke on the Metabolism of Cultured Bovine Lenses

Abstract

Purpose: Smoke from cigarette smoking (CS) has been proposed to be a major environmental risk factor for a variety of human diseases and was implicated also in cataract, an eye lens pacification, which is a major cause of blindness. We have undertaken a study to investigate the effect of smoke on the physiological integrity and metabolism of organ cultured lenses. Lenses in organ culture are metabolically active and have functional defense systems, thus they provide an appropriate model for studying the effects of smoke. Also the possible protective action of N-acetylL-cysteine (NAC) which is a precursor of glutathione and the iron chelators Deferoxamine (DFO), was estimated as potential protective agents against cataract. Methods: Bovine lenses were incubated in organ culture conditions at 35°Cfor 6 days. Treated lenses were 4 day in the culturally environment daily sated with a cigarette smoke under various doses of pressure-250,500 psi. Two of the experimental groups were treated with NAC (1 mM) and DFO (2.5 µg/ml) as antioxidants. An automated scanning laser system was used for daily testing of both treated and control lenses. At the end of the culture period, lenses were analyzed by inverted microscopy. We have used preparations of the forward monolayer epithelium bovine lenses from all experiments. For this purpose the capsule opened and crystalline lens fibers were cleaned. On the object-plate them there was only a capsule and a cellular monolayer epithelium. Changes of morphology of cells and the contents of the nucleic acids was estimated using the Einarson - DNA-RNA staining method. Reactive Oxygen Species (ROS) were estimated with 5- (and 6-) chloromethyl-2’,7’-dichloro-dihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA, C6827) to measure the level of cellular oxidation in the cells of lens epithelium. The levels of ROS were measured by monitoring the fluorescent intensity relative to that of control cultures under fluorescent microscopy. Nucleic acids were stained with Propidium Iodide. Results: Exposure of cigarette smoke in cultured medium under negative pressure, optical quality of lenses and the structural changes were demonstrated by decreased light transmission, increase in focal length variability and a decrease in morphological integrity such as hyperplasia and hypertrophy of epithelial cells. The group exposed to NAC and to DFO, demonstrated reduced optical changes representing smaller lens injury. The lenses show almost no volume changes. However, the baseline fluorescence of controls varied between experiments. A dose-dependent increase in ROS generation in cultures was also evident. Conclusions: We have shown that increasing the amount CS exposure, for a relatively short time, causes a sharp increase in the damage to the lens. Smoking is an independent risk factor that has dose-response effects. It causes morphological and functional changes to the lens. Based on the independent effects of NAC and DFO, we propose to possibly use them as means of prevention and/or treatment of cataract in heavily smoking people.