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J. Yin, W. Huo, J. C. Lang, E. Chaum; Vitamin C Modulation of Oxidative Stress Responses in the Human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1844.
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Our studies of oxidative stress (OS) in the retinal pigment epithelium demonstrated quantitative changes in the expression of various genes including transcription factors, chaperone proteins, and antioxidant genes. The purpose of this study was to determine whether the molecular responses to quantified levels of OS in the human RPE are modulated by antioxidant vitamin treatment.
Confluent ARPE-19 cells were cultured for three days in defined medium in the presence of vitamin C (0.1mM to 0.2mM) to stabilize gene expression. The vitamin C was removed 24 hours prior to treatment with 500uM H2O2. RNA was isolated using a no-rinse method at 1-, 4-, 8-, and 24-hours after OS and compared to no-vitamin C controls. Gene-specific expression was quantified by real-time qPCR.
We quantified the expression of the AP-1 transcription factors c-Fos, FosB and ATF3, as well as heme oxygenase-1, EGR1, and EGR2. Significant reductions in the level of transcription following OS were seen after treatment with 0.2mM vitamin C, but not after 0.1mM vitamin C. Pretreatment with 0.2mM vitamin C reduced the OS-response of c-Fos and FosB by 97% and 87%, respectively, after 1-, and 4-hours. Similarly, EGR2 transcription after OS was reduced by ~97% after 1-, and 4-hours (from 64-fold to 2-fold, and from 16-fold to <2-fold), and by ~99% after 8-hours. HO-1 transcription was reduced more than 67% after 4-, 8-, and 24-hours. A more modest response was also seen after treatment with 0.1mM vitamin C (50%) at 8-hours, which was sustained through 24-hours after OS.
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