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S.W. Cousins, D. Espinosa Heidman, M.E. Marin Castaño; Smoking-Related Oxidants and RPE Injury In Vitro and In Vivo . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1619.
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Purpose: Epidemiological studies have established a strong correlation between cigarette smoke and ARMD. Numerous potentially cytotoxic substances of cigarette smoke circulate in the blood of smokers and may induce oxidant injury in RPE. We postulate that a specific tar-associated oxidant in cigarette smoke called hydroquinone (HQ), might stimulate RPE injury by the induction of sublethal bleb formation and dysregulation of matrix molecules, causing sub-RPE deposits. HQ metabolism in the mitochondria generates superoxide anion, which in turn, damages mitochondrial membranes and leaks into the cytoplasm participating in protein or other oxidation reactions. We sought to evaluate the impact of HQ on regulation of cell membrane blebbing and molecules important for extracellular matrix turnover molecules in RPE, and to determine if chronic oral feeding of HQ causes increased severity of subRPE deposits in a mouse model. Methods: Confluent cultures of the human RPE-19 cell line were incubated in phenol red free medium containing 0.1% fetal bovine serum with or without hydroquinone (from 300 to 0.1 mM). In a parallel experiment, the cells were incubated with or without HQ at 10 mM for various times. Cells count and viability assay was performed. The supernatants were collected to assess MMP-2 activity by zymography and collagen type IV accumulation by ELISA. 16 month old C57BL/6 female mice received high fat diet with or without HQ 0.8% for 4.5 months. After the first month of diet the mice were exposed to argon blue-green light. The eyes were removed for TEM of the retina and choroid after treatment. Direct measurement of median thickness of BrM and quantitative assessment of the choriocapillaries was done. Results: In vitro, high dose HQ (300 to 200 mM) killed a significant fraction of RPE cells (~ 60% of control). Low dose HQ (10 mM) was nonlethal to RPE but induced significant blebbing and down regulated MMP-2 activity within 2 hrs of exposure. In contrast, HQ increased collagen type IV. Transient exposure to oxidant was associated with diminished MMP-2 activity and with increased collagen type IV accumulation during the acute injury, but quickly recovered to normal levels 18 hrs after removal. In vivo, chronic oral exposure to HQ to aged mice induced moderately thick sub-RPE deposits corresponding to BLD, with thickening of the basement membrane. Conclusions: Acute oxidant injury transiently upregulated blebbing but decreased ECM turnover. In vivo HQ induced moderately thick sub-RPE deposits. These data suggest that cigarette smoke-derived oxidants may contribute to ARMD pathogenesis. CR: N.
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