The effect and recovery of long-term H2O2 exposure on lens morphology and biochemistry

Oxidative stress has long been speculated to play an important role in cataractogenesis. In the H2O2-induced cataract model, rat lens showed extensive biochemical damage but very mild morphological changes after being exposed to H2O2 (0.5 mM) for 24 hr in culture. This damage included reduced glutathione (GSH) depletion, protein-GSH mixed disulfide (PSSG) elevation but not protein-protein disulfide (PSSP) formation. In order to understand the role of protein-thiol mixed disulfide formation in relation to the sequence of events during cataract induction, we conducted a long term H2O2 exposure study for up to 96 hr to monitor the dynamic changes in GSH and PSSG levels, the formation of PSSP aggregate, protein solubility, and the progression in lens opacity. Rat lenses were cultured in 0.5 mM H2O2 and harvested at intervals of 24, 48, 72 and 96 hr for the examination of morphological and biochemical changes. Contralateral lenses cultured in H2O2-free media were used as controls. It was found that the lenses had only patchy opacity at the equator after 24 hr, but became hydrated suddenly at 48 hr (31% heavier than the control), with an opacity which involved the entire outer cortical region. By 72 hr incubation, the nucleus was opacified. Lens GSH progressively decreased with time of H2O2 exposure, 40% was lost by 24 hr and over 95% by 48 hr. There was a concomitant elevation of PSSG, 16-fold over the controls by 24 hr and 45-fold by 48 hr followed by a decline to 34-fold after 72 hr. In addition, the level of protein-cysteine mixed disulfide (PSSC) was elevated after 48 hr incubation in H2O2. At this time point, PSSP aggregates began to appear both in water soluble (WS) and urea soluble (US) fractions along with a drastic reduction in protein solubility. Western blot analysis of the protein fractions identified beta and gamma, but not alpha-crystallin in the disulfide-containing aggregates. The lens clarity and biochemical changes partially recovered if the oxidant was removed within 24 hr, indicating a potential therapeutic role for antioxidants. The complete normalization of PSSG level under this recovery condition signifies that cells may have a natural defense system for controlling PSSG elevation.