VARIATION IN CELLULAR GLUTATHIONE-PEROXIDASE ACTIVITY IN LENS EPITHELIAL-CELLS, TRANSGENICS AND KNOCKOUTS DOES NOT SIGNIFICANTLY CHANGE THERESPONSE TO H2O2 STRESS

This investigation examines the contribution of glutathione peroxidase (GSHPx-1) in degrading H2O2 in lens preparations. Rabbit (N/N1003A) a nd normal and GSHPx-1 transfected mouse (alpha TN4-1) 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 GSH Px-1 activity. The transgenic and knockout mice as well as their lense s appeared normal for up to 3 to 4 months, the longest period of obser vation. The preparations were subjected to oxidative stress by placing them either in a medium containing 120 or 300 mu M H2O2 or utilizing photochemical stress where the H2O2 levels normally rise to about 100 mu M over a few hours in the presence of a normal lens. With all prepa rations, it was found that either markedly increasing or eliminating G SHPx-1 activity had only a small effect on the system's ability to met abolize H2O2. 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibito r of GSSG reductase (GSSG Red) and S-aminotriazole (3-AT). an inhibito r of catalase, also had little effect. However, the addition of both i nhibitors caused a marked decrease in H2O2 degradation. Examination of the distribution of GSHPx-1 in the lens indicated that the activity p er milligram of protein was evenly distributed between the epithelium and the remainder of the lens in the normal lens and was about 17-fold greater in the epithelium of transgenic lenses than in the remainder of the lens. Surprisingly, the distribution of GSSG Red was quite diff erent with eight- to ten-fold more activity in the epithelium. Catalas e was also found to be concentrated in the epithelium. With H2O2 expos ure, a rapid loss of non-protein thiol (NP-thiol) was found in cell cu ltures 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 a nd membrane function, also reflects the H2O2 degradation data, showing little difference in the degree to which H2O2 effects these parameter s in lenses from normal and transgenic animals. Catalase activity is f our- to six-fold greater than GSHPX-1 activity in the alpha TN4-1 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 ou t or GSSG Red is inhibited, catalase is able to protect the system fro m H2O2 stress. Indeed, the young mouse does not appear to require GSH Px-1 for normal function. (C) 1996 Academic Press Limited