THIOLTRANSFERASE IS PRESENT IN THE LENS EPITHELIAL-CELLS AS A HIGHLY OXIDATIVE STRESS-RESISTANT ENZYME

The redox homeostasis is controlled by several enzyme systems. Sulfhyd ryl groups in lens proteins are Very sensitive to oxidative stress and can easily conjugate with nonprotein thiols (S-thiolation) to form pr otein-thiol mixed disulfides. We have observed an elevation of protein S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC) in cataractous lenses from humans and from animal models subjected to oxidative stre ss. We also observed that these protein-thiol mixed disulfides could b e spontaneously dissociated and lowered to basal levels if the lens wh ich was pre-exposed to H2O2 was subsequently cultured in H2O2-free med ium. This suggests that the lens has a system to repair oxidative dama ge through dethiolation thereby restoring its redox homeostasis. In ot her tissues, an enzyme, thioltransferase (TTase), has been shown to be responsible for thiol/disulfide regulation. We recently demonstrated the presence of this enzyme in the lens and in cultured lens epithelia l cells. Here, we investigated the response of TTase to H2O2 stress an d its possible repair function in cultured lens epithelial cells. Rabb it lens epithelial cell line N/N 1003A was raised to confluence, tryps inized and plated at 0.8 million cells per 60 mm culture dish. The cel ls were incubated overnight in Eagle's minimum essential medium (MEM) with 1% rabbit serum and then in serum-free MEM for 30 min before a bo lus of 0.5 mM H2O2 was added. At intervals of 5, 15, 30 min and up to 3 hr, the cells were harvested and used for enzyme assays for TTase, g lutathione reductase (GR), glutathione peroxidase (GPx) and glyceralde hyde-3-phosphate dehydrogenase (G-3PD), Free GSH, total SH and PSSG an d PSSC were also determined. Hydrogen peroxide in the medium was measu red at each time point. Cells incubated without H2O2 were used as cont rols, The results showed that the H2O2 concentration was reduced to 50 % within 30 min and was undetectable at 2 hr. Cellular GSH dropped to 40% within 5 min and stayed at this level before it began to increase at 90 min and completely recovered by 2 hr. The total SH groups were s imilar to free GSH, PSSG and PSSC increased 6.5 and 2 times respective ly before 30 min and then decreased when GSH started to recover. G-3PD was most sensitive to H2O2 and lost 95% activity within 5 min. The ac tivity was regained quickly when H2O2 diminished in the medium. A simi lar but less severe pattern was observed in both GPx (60% loss at 60 m in) and GR (30% loss at 90 min). In contrast, TTase activity remained constant during the entire 3 hr. Only when a higher dose of H2O2 (0.8- 1.0 mM) was used, did TTase activity show a brief loss (<30% at 60 min ) and a swift recovery. Cells exposed to H2O2 exhibited a normal morph ology with no evidence of DNA fragmentation. The lens epithelial cells showed a remarkable ability to repair the early damages induced by H2 O2. The unusual oxidative stress-resistant property displayed by TTase , coupled with its known function suggest that it plays an important r ole in the repair of oxidative damage. (C) 1998 Academic Press Limited .