Hydrogen Peroxide-Mediated Cell Injury: Investigating the Mechanism of Antioxidative Action by 17beta-Estradiol in Human Lens Epithelial Cells

Abstract

Abstract: : Purpose: Estrogens are potent antioxidants and act to prevent H₂O₂-mediated depletion of intracellular ATP in human lens epithelial cells (HLE-B3) cultured in 20% fetal bovine serum (FBS)-supplemented medium (Wang et al., Invest Ophthalmol Vis Sci 2002; 43:a849). Previous studies have shown that H₂O₂ insult causes downregulation of several mitochondrial transcripts (MT) from proteins involved with cellular respiration in the human epithelial cell line, SRA 01-04 (Carper et al., Invest Ophthalmol Vis Sci 1999; 40:400-406). The glycolytic pathway enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also a primary target for inactivation by H₂O₂ (Hyslop et al., J Biol Chem 1988; 263:1665-1675). Both pathways are involved with ATP production and this study examined whether 17ß-estradiol (17ß-E₂) provides antioxidative cytoprotection against H₂O₂-mediated damage to the MT of cellular respiration and GAPDH activity. Methods: HLE-B3 cells (60-80% confluence) were maintained in medium (MEM) containing 20% FBS. Prior to exposure to H₂O₂, cells were rinsed with 0.5% FBS/MEM and subsequently incubated with either 0.5% FBS/MEM containing ethanol (ETOH) vehicle or 17ß-E₂ (1µM) overnight. Approximately 18 h later, cells were rinsed with serum-free MEM and treated with either 17ß-E₂ alone, 17ß-E₂ and H₂O₂ (50-400µM) or ETOH vehicle and H₂O₂ for up to 8 h. Total RNA was isolated using TRIzol Reagent and analyzed by RT-PCR and Northern blot analysis. GAPDH activity was determined under similar experimental conditions. Results: Reduction in MT levels were observed by RT-PCR analysis for nicotinamide adenine dinucleotide dehydrogenase (NADH) subunits 4 and 5 and cytochrome c with H₂O₂ (200-400µM) treatment. Northern blot analysis further confirmed a decrease in mRNA levels at both 50 and 100µM H₂O₂. Loss of cytochrome c mRNA level was most apparent by 5 h, while NADH subunits 4 and 5 showed a marked decrease as early as 1 h. Pretreatment and simultaneous addition of 17ß-E₂ with H₂O₂ did not prevent the observed reduction in MT. H₂O₂ insult (100µM) significantly lowered GAPDH activity, but as with the MT, 17ß-E₂ failed to normalize GAPDH activity. Conclusions: The combination of serum deprivation of HLE-B3 cells and H₂O₂ insult may be so harsh a treatment as to render the antioxidative properties of 17ß-E₂ ineffective. Alternatively, the antioxidative capacity of 17ß-E₂ may be more directly related to the protection of the mitochondrial ATPase-synthase complex.

Keywords: oxidation/oxidative or free radical damage • antioxidants • cataract