Relationship of Protein-Glutathione Mixed Disulfide and Thioltransferase in H2O2-Induced Cataract in Cultured Pig Lens

Abstract

It has been previously shown in H2O2-induced cataract model in the rat lens that protein-GSH (PSSG) formation precedes protein–protein disulfide (PSSP) conjugation and lens opacity. This elevated PSSG spontaneously reduces to a normal level when H2O2is removed. To verify if thioltransferase (TTase), an enzyme that is known in other tissues to dethiolate PSSG, takes part in this recovery process, we examined the relationship of PSSG and TTase in this cataract model. To ensure enough tissue would be available for various biochemical studies, H2O2induced cataract in pig lens was established and validated with the rat lens model. The study was divided into two parts. One part was to examine the effect of H2O2concentration, ranging from 0.1 mM–10 mM, during 24 hr. Another part was to study the H2O2(1.5 mM) induced cataract progression and recovery, parallel to the long-term study in rat lenses reported previously. These lenses were compared for transparency, wet weight, GSH, PSSG levels and the activity of two redox regulating enzymes, glutathione reductase (GR) and TTase. For the most part, pig lens responded to oxidation parallel to the rat lens except that a higher concentration of H2O2was needed to achieve the same results. Damage induced by H2O2was concentration dependent. In general TTase activity and GSH level were depleted with a concomitant increase in PSSG. The D50(50% damage) for GSH in pig lens was 1.5 mMH2O2(0.5 mMfor rat lens) which was chosen for further studies in cataract progression and recovery. At 1.5 mMH2O2, pig lens showed superficial opacity within 24 hr and deeper cortical opacity in 48 hr. The pre-exposed lens became less cloudy when H2O2was removed from the medium. Incubation of the lens in 1.5 mMH2O2for one day also induced 50% GSH depletion and four fold PSSG elevations. This accumulated PSSG was dethiolated spontaneously in the absence of H2O2, similar to the findings in the rat lens and human lens models. In contrast protein-cysteine (PSSC) showed little change and did not respond to the recovery condition. TTase lost 50% activity in these lenses during 24-hr H2O2exposure but regained most of it under recovery. The study on rat lens showed similar results as before, therefore only data on the relationship of TTase activity to PSSG level during cataract development and recovery is reported here. It was found that in the H2O2(0.5 mM)-exposed rat lenses, the TTase activity was depleted but PSSG accumulation was accelerated within 8 hr. Both recovered quickly (within 8 hr) as soon as the oxidant was removed. Therefore, protein thiolation and dethiolation processes in the cultured rat or pig lenses display a mirror image with the activity pattern of TTase. Based on the close relationship between lens TTase and PSSG indicated above, it is speculated that TTase may regulate PSSG and maintain it at a low concentration in situ. This repair process may contribute to the improved transparency during recovery. Further studies are planned to substantiate this hypothesis.