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 H2O2 is 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, H2O2 induced
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 H2O2 concentration, 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 H2O2 was needed to achieve the same results. Damage induced by H2O3 was 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 mM H2O2 (0.5 mM for rat lens) which was chosen for further studies in
cataract progression and recovery. At 1.5 mM H2O2, pig lens showed superficial opacity within 24 hr and deeper cortical opacity in 48 hr. The pre-exposed lens became less cloudy when H2O3 was removed from the medium. Incubation of the lens in 1.5 mM H2O2 for 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 H2O3 exposure 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.