H2O2 Stress Sensitivity in Cultured Primary Mouse Lens Epithelial Cells Derived From Wild Type and Thioltransferase Knockout Mice

Abstract

Abstract: : Purpose: To investigate the importance of thioltransferase (TTase) as an antioxidant enzyme in primary cultures of lens epithelial cells obtained from wild type (WT) and TTase knockout (KO) mice. Methods: Lens epithelial cells were prepared from lens capsules obtained from one–week–old WT and TTase KO mice. The lens capsules were treated with trypsin and placed in MEM medium with 10% FBS followed by centrifugation. The cell pellet was re–suspended in 1.5 ml MEM with 10% FBS and cultured at 37° C in a CO₂ incubator. Cell lysate of the 90% confluent cells was assayed for glyceraldehyde–3–phosphate dehydrogenase (G3PD), lactate dehydrogenase (LDH), and TTase activities following standard spectrophotometric methods. Both WT and KO cells were compared for their resistance to oxidative stress by exposing to H₂O₂. G3PD activity was monitored at 0, 15, 30, 60, and 90 min following 0.15 mM bolus H₂O₂ treatment. The activity of LDH leaked into the medium was determined at 0, 15, 30, 60, and 120 min in WT and KO cells treated with 1.5 mM bolus H₂O₂. Results: Untreated KO cells showed very low level of TTase enzyme activity (1 mU/mg protein), only 1/6 of the WT cells. The G3PD activity in the TTase KO cells dropped 70% at 15 min after H₂O₂ treatment as compared to a 20% drop in the WT cells. G3PD activity recovered within 90 min in the WT cells while the TTase KO cells only reached 65% of control activity. LDH leakage into the culture medium, an indicator of oxidative damage, was minimal in the WT cells (0.3% of total LDH) but a 5–fold LDH activity was found in the medium of KO cells. Conclusions: The data provide strong evidence that TTase is an important enzyme for protecting the cells against oxidative damage and cell injury.

Keywords: oxidation/oxidative or free radical damage