Variation in Cellular Glutathione Peroxidase Activity in Lens Epithelial Cells, Transgenics and Knockouts Does Not Significantly Change the Response to H₂O₂Stress

Abstract

This investigation examines the contribution of glutathione peroxidase (GSHPx-1) in degrading H₂O₂in lens preparations. Rabbit (N/N1003A) and normal and GSHPx-1 transfected mouse (αTN4-1) lens epithelial cell lines and normal and GSHPx-1 transgenic and knockout mouse lenses were utilized. GSHPx-1 activity in the cell lines was increased from two-fold to about four-fold, in the lenses from transgenics more than four-fold and the lenses from knockouts had less than 3% of normal GSHPx-1 activity. The transgenic and knockout mice as well as their lenses appeared normal for up to 3 to 4 months, the longest period of observation. The preparations were subjected to oxidative stress by placing them either in a medium containing 120 or 300μmH₂O₂or utilizing photochemical stress where the H₂O₂levels normally rise to about 100μmover a few hours in the presence of a normal lens. With all preparations, it was found that either markedly increasing or eliminating GSHPx-1 activity had only a small effect on the system's ability to metabolize H₂O₂. 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase (GSSG Red) and 3-aminotriazole (3-AT), an inhibitor of catalase, also had little effect. However, the addition of both inhibitors caused a marked decrease in H₂O₂degradation. Examination of the distribution of GSHPx-1 in the lens indicated that the activity per milligram of protein was evenly distributed between the epithelium and the remainder of the lens in the normal lens and was about 1.7-fold greater in the epithelium of transgenic lenses than in the remainder of the lens. Surprisingly, the distribution of GSSG Red was quite different with eight- to ten-fold more activity in the epithelium. Catalase was also found to be concentrated in the epithelium. With H₂O₂exposure, a rapid loss of non-protein thiol (NP-thiol) was found in cell cultures and in the epithelia of cultured lenses. However, the remainder of the lens showed little change in NP-thiol. The variation of GSHPx-1 activity did not influence the NP-thiol changes which occurred more rapidly and to a greater extent in the presence of BCNU. The addition of BCNU also caused a decrease in total lens NP-thiol. Examination of thymidine incorporation and choline transport, indicators of nuclear and membrane function, also reflects the H₂O₂degradation data, showing little difference in the degree to which H₂O₂effects these parameters in lenses from normal and transgenic animals. Catalase activity is four- to six-fold greater than GSHPX-1 activity in the αTN4-1 cell lines, about three-fold lower in the rabbit cell line and, remarkably, about 18-fold lower than the peroxidase in the normal mouse lens. In spite of such observations, the consistent overall conclusion is that GSHPx-1 and catalase function together but when GSHPx-1 is knocked out or GSSG Red is inhibited, catalase is able to protect the system from H₂O₂stress. Indeed, the young mouse does not appear to require GSH Px-1 for normal function.