Differential Mechanisms of Action and Efficacy of Vitamin E Components in Antioxidant Cytoprotection of Human Retinal Pigment Epithelium

Abstract

The purpose of this study was to determine if different vitamin E components exhibit similar efficacy and mechanism of action in protecting Retinal pigment epithelium (RPE) cells from oxidative damage. We hypothesized that α-tocopherol (αT) is unique among vitamin E components in its cytoprotective mechanism of action against oxidative stress in RPE cells and that it requires protein synthesis for optimal antioxidant effect. We used cell viability assays, fluorescent chemical labeling of DNA and actin and immuno-labeling of the antioxidant proteins Nrf2 and Sod2 and of the tight junction protein, ZO-1, and confocal microscopy to determine the effects of αT and γT against oxidative stress in immortalized human RPE cells (hTERT-RPE). Using the four main vitamin E components, αT, γT, δ-tocopherol (δT) and α-tocotrienol (αTr), we ascertained that they exhibit similar, but not identical, antioxidant activity as αT when used at equimolar concentrations. In addition, we determined that the exposure time of RPE cells to α-tocopherol is critical for its ability to protect against oxidative damage. Lastly, we determined that αT, but not γT, partially requires the synthesis of new proteins within a 24-h period and prior to exposure to tBHP for optimal cytoprotection. We conclude that, unlike γT and δT, αT appears to be unique in its requirement for transport and/or signaling for it to be an effective antioxidant. As a result, more focus should be paid to which vitamin E components are used for antioxidant interventions.

Proteome changes in a human retinal pigment epithelial cell line during oxidative stress and following antioxidant treatment

“…We previously reported that αT, but not tBHP, exposure of hTERT-RPE cells induced the expression of Nrf2 but did not lead to the nuclear translocation of Nrf2 ( 10 ). Exposure to tBHP, on the other hand, had no effect on Nrf2 expression but it led to an increase in Nrf2 nuclear translocation, suggesting it had become activated.…”

“…Cells were exposed to tocopherols for 24 hours prior to treatment with the 100μM tert-butyl hydroperoxide (tBHP), the oxidant, or water as a vehicle control for 24 hours. The six paired treatment groups (24h treatment 1 - 24h treatment 2) were vehicle-control, vehicle-tBHP, αT-control, γT-control, αT-tBHP and γT-tBHP, the same as in a previous study ( 10 ) (see Supplemental Figure 1 ). A tocopherol concentration of 100μM was selected based on previous reports (~25 – 32 μM vitamin E in serum) and calculations based on the daily recommendation dose of vitamin E at 400IU.…”

“…Gamma-tocopherol (γ-Toc or γT) is highly abundant in vitamin E containing foods but is not present at high levels in the blood and tissues due to first pass metabolism. γT is a more effective antioxidant than αT and therefore may be a superior antioxidant for therapeutic purposes ( 10 ). Tocopherols have been shown to activate signaling pathways that promote cytoprotection ( 6 ).…”

Molecular Mechanisms Underlying the Therapeutic Role of Vitamin E in Age-Related Macular Degeneration

“…A common experimental model to study AMD and oxidative stress is the use of the ARPE-19 cell line, immortalized human RPE cells, and human telomerase reverse transcriptase (hTERT)-RPE. In Duncan et al, 2022 they demonstrate that α-tocopherol, γ-tocopherol, δ-tocopherol, and α-tocotrienol all exhibited similar, but not identical, antioxidant activity. In addition, exposure time is important for its protective properties against oxidative stress.…”

“…In addition, exposure time is important for its protective properties against oxidative stress. Synthesis of new proteins was also found to be partially required with α-tocopherol, but not γ-tocopherol, within a 24-h period and before exposure to tertiary butyl hydroperoxide (tBHP) for optimal cytoprotection ( Duncan et al, 2022 ). In another study, ARPE-19 cells were subjected to pre-treatment of ≥2.5 mM α-tocopherol, which significantly decreased oxidative stress-induced activator protein-1 (AP1) transcription factor expression at 14 h but was not further reduced with higher levels of α-tocopherol ( Yin et al, 2011 ).…”