Abstract
The purpose of this study was to characterize the potential of vitamins to protect the retinal pig-ment epithelium (RPE) from oxidative stress (OS). We have previously shown that OS induces the expression of AP1 transcription factors (FOSB, CFOS and ATF3), but is modulated by pretreatment with vitamin C (200 μM). We propose that OS-induced AP1 expression can be used as a biomarker of OS to test the efficacy of vitamins to limit the impact of OS in the RPE. Here we examined the efficacy of vitamin E or combined vitamin A plus vitamin C to modulate OS-induced AP1 expression in the RPE. We pretreated human ARPE-19 cells with vitamin E (0 - 7.5 μM) or with combined vitamin A (10 or 15 μM) plus vitamin C (50 or 100 μM) for 3 days prior to exposure to 500 μM H2O2 OS for 1 - 4 h. AP1 expression was assessed using qRT-PCR. Pretreatment with ≥2.5 μM vitamin E significantly decreased OS-induced AP1 expression at 1 - 4 h OS, compared to controls. Lower doses of vitamin E were ineffective at modulating OS responses. Pretreatment with 100 μM vitamin C combined with 15 μM vitamin A protected RPE cells from OS-induced AP1 expression. There is an additive and potentially protective effect of 100 μM vitamin C and 15 μM vitamin A on FOSB expression at 4 h, and a potentially protective effect of 100 μM vitamin C and 15 μM vitamin A on CFOS expression at 1 h OS. A protective effect was also seen with 15 μM vitamin A pretreatment alone on ATF3 expression. Thus, “sub-therapeutic” levels of multiple vitamins may protect RPE cells better than higher doses of a single vitamin. This OS-induced AP1 expression biomarker assay may be useful to identify complex antioxidant formulations as therapeutics for degenerative diseases that are thought to be caused by OS, like age-related macular degeneration.
Highlights
Age-related macular degeneration (AMD) is the most common cause of visual impairment in aging populations in the industrialized world and results from complex interactions between environmental, nutritional and genetic factors
We have previously shown that AP1 genes FBJ murine osteosarcoma viral oncogene homolog B (FOSB), CFOS, and ATF3 are key early molecular responders whose mRNA and protein levels are dose and time dependently activated in retinal pigment epithelium (RPE) cells in response to H2O2 oxidative stress (OS)
Because oxidative stress (OS) exposure is considered a key mechanism in AMD disease pathology and because we have shown that 500 μM H2O2 OS time-dependently increases mRNA and protein levels of AP1 transcription factors, we examined the ability of vitamin E treatment to modulate OS-induced AP1 expression in RPE cells in vitro
Summary
Age-related macular degeneration (AMD) is the most common cause of visual impairment in aging populations in the industrialized world and results from complex interactions between environmental, nutritional and genetic factors. The RPE in vivo is exposed to chronically high levels of harmful reactive oxygen and nitrogen species (ROS/RNS), which are produced from high rates of mitochondrial respiration, high retinal oxygen content, direct photic damage, digestion of oxidized outer segment membrane lipids and lipofuscin-sensitized photo-oxidation [1]-[4]. This OS can be modulated by endogenous and dietary antioxidants, if the ROS levels exceed these repair mechanisms, oxidative damage to proteins, DNA, RNA and organelles can occur. Because limiting ROS accumulation is important for maintaining normal retinal function, and because multivitamin supplementation was shown in the Age-Related Eye Disease Study to reduce the risk of moderate AMD disease progression to advanced AMD [5] [6], current research has focused on identifying dietary and nutritional antioxidants that limit OS in the retina and that may be efficacious for AMD therapy
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