Purpose: In view of the important antioxidant roles of thioltransferase (TTase), thioredoxin (Trx), and thioredoxin reductase (TR) in the lens, the present study was conducted to investigate the induction of these cytosolic enzymes in response to H(2)O(2) stress in cultured lenses.
Methods: Porcine lenses were cultured, exposed to H(2)O(2) for various lengths of time between 0 and 24 hours, and photographed to detect morphologic changes. The lenses were then harvested; dissected into epithelial layer, cortex, and nucleus; and homogenized for the determination of the glutathione (GSH) level. Pooled epithelial layers were used to examine TTase, Trx, and TR protein or mRNA levels.
Results: Treatment of lenses with H(2)O(2) caused distinct morphologic changes. Lower concentrations of H(2)O(2) (0.2 mM) caused the lens to be hazy after 6 hours and to worsen progressively between 12 and 24 hours. Higher levels of H(2)O(2) (0.5 mM) induced similar morphologic changes, but sooner (within 1 hour) and more severe. Both H(2)O(2)-treated groups showed a dramatic and gradual GSH depletion during the 24-hour incubation, but the GSH level at 50% or above appeared to be essential in maintaining lens clarity. However, TTase, Trx, and TR activities, protein expressions, and mRNA transcriptions in the epithelial layers of these lenses were increased, but each enzyme had a distinct pattern. Under mild H(2)O(2) stress, a slow and transient activation of TTase, Trx, and TR was observed. However, under stronger H(2)O(2) stress, all three enzymes showed a very rapid increase and then a steady decline in activity. Western blot and RT-PCR analyses revealed that this increase in activity in all three enzymes was due to the induction of protein and mRNA expression. In the control group (no oxidative stress) all three enzyme activities and their respective expressions remained constant throughout the experimental period.
Conclusions: The data show that TTase, Trx, and TR activity and expression are induced in lens cells under oxidative stress, probably to protect and maintain the health of the lens.