title: Variation in cellular glutathione peroxidase activity in lens epithelial cells, transgenics and knockouts does not significantly change the response to H2O2 stress.
abstract: This investigation examines the contribution of glutathione peroxidase (GSHPx-1) in degrading H2O2 in lens preparations. Rabbit (N/N1003A) and 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 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 300 microM H2O2 or utilizing photochemical stress where the H2O2 levels normally rise to about 100 microM over 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 H2O2 degradation. 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 H2O2 exposure, 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 H2O2 degradation data, showing little difference in the degree to which H2O2 effects these parameters in lenses from normal and transgenic animals. Catalase activity is four- 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 out or GSSG Red is inhibited, catalase is able to protect the system from H2O2 stress. Indeed, the young mouse does not appear to require GSH Px-1 for normal function.
authors: Department of Ophthalmology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA.
authors: A
authors: A
authors: Spector
authors: Y
authors: Y
authors: Yang
authors: Y S
authors: YS
authors: Ho
authors: J L
authors: JL
authors: Magnenat
authors: R R
authors: RR
authors: Wang
authors: W
authors: W
authors: Ma
authors: W C
authors: WC
authors: Li
mesh_terms: D000818:Animals; D002374:Catalase / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism*; D005980:Glutathione Reductase / Q000502:physiology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007908:Lens, Crystalline / Q000201:enzymology*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D018384:Oxidative Stress / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide; D002374:Catalase; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase