The Activation of Thioltransferase in the Cultured Pig Lenses Under Oxidative Stress

Abstract

Abstract: : Purpose: Thioltranferase (TTase) is an oxidoreductase enzyme, which regulates the thiol/disulfide homeostasis in the cells and rescues the oxidatively damaged proteins/enzymes by dethiolation. In this study, we investigated how this enzyme is regulated in the pig lens when stressed in the presence of hydrogen peroxide (H₂O₂). Methods : Fresh pig lenses were cultured in TC 199 medium with one group containing 0.2 mM H₂O₂, the second group containing 0.5 mM H₂O₂ and the last group without H₂O₂ as control. Appropriate amount of glucose oxidase was added to the medium to maintain a constant level of H₂O₂. The lenses were first recorded for morphology by photography and then harvested at 1, 2, 4, 6, 9, 12, 18 and 24 hrs. Each lens was dissected into epithelial layer, cortex and nucleus, and homogenized for determination of glutathione (GSH) levels and the specific activity of TTase. Results : TTase activity was found mostly in the epithelial layer, which was 3-5 x more than the TTase equally distributed in the cortex and nucleus. Cortical opacity appeared at 2 hrs in the high H₂O₂ group and at 6 hrs in the low H₂O₂ group. The severity of cataract progression was proportional to the H₂O₂ used in the medium. The epithelial layer in the low H₂O₂ group showed a slow activation of TTase, witch began at 60 min with 3x over control (1.1 mU/mg) and peaked at 12 hrs to 3.3-fold before it gradually declined. The high H₂O₂ group, however showed a dramatic transient increase in TTase activity within the first 6-hr (peaked at 4 hr to 6-fold). TTase activity in the control group remained constant throughout the experiment. GSH levels in all groups showed a gradual depletion during the first 6 hrs before reaching to a reversed plateau, in which the control group lost 35% while low H₂O₂ group lost 70% and high H₂O₂ group lost 80%. Conclusion : GSH was consumed and oxidized under the experimental conditions but the cellular TTase was resistant to oxidative stress. The cells appear to combat the stress by initially activating TTase in an attempt to rescue and maintain the health of the lens.

Keywords: cataract • enzymes/enzyme inhibitors • oxidation/oxidative or free radical damage