Glutathione S-Transferase Pi Isoform (GSTP1) Modulation by Sulforaphane Protects Human Retinal Pigment Epithelial Cells against Oxidative Stress

Abstract

Purpose: Glutathione S-transferase pi isoform (GSTP1) is a phase 2 intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls. We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to UV light, and GSTP1 over-expression protects them against UV light damage. Previously, we determined the dose-response effect of oxidative stress on GSTP 1 expression in the survival of human RPE cells exposed to hydrogen peroxide (H₂O₂) and cigarette smoke component hydroquinone (HQ). In the present study, we determined the effect of sulforaphane, a phase 2 enzyme inducer, on GSTP1 levels in the viability of human RPE cells exposed to oxidants H₂O₂ and HQ.

Methods: Human RPE cell culture (ARPE-19) was maintained using established protocols. ARPE-19 cells were plated at sub-confluent density in six-well plates and grown to confluence. Cells were pre-treated without or with sulforaphane (5 µM) for 24 hours before exposing to H₂O₂ (50, 100, 200 µM) or HQ (50, 100, 150 µM) for 8-12 hours. Cell survival was measured using trypan blue exclusion assay. GSTP1 expression was assessed using Western blot analysis.

Results: At baseline, ARPE-19 cell viability was not negatively affected when exposed to increasing concentrations of H₂O₂ compared to untreated control, whereas ARPE-19 cell survival decreased when exposed to increasing concentrations of HQ. However, GSTP1 expression decreased in response to both H₂O₂ and HQ. When pre-treated with sulforaphane, ARPE-19 cell survival and GSTP1 levels improved when compared to untreated control upon exposure to H₂O₂ and HQ.

Conclusions: H₂O₂ and HQ affect human RPE cell viability to different degree, but both are associated with decreased GSTP1 expression. Sulforaphane induces GSTP1 expression and increases survival of human RPE cells exposed to H₂O₂ and HQ. Enhanced GSTP1 expression may protect human RPE cells against oxidative stress.