Thioredoxin reductase activity may be more important than GSH level in protecting human lens epithelial cells against UVA light

Abstract

This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2 , 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm(-2) of UVA radiation (338-400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage.

Fig. 1

Effect of various treatments on the concentration of GSH in cultured human lens epithelial cells. Cells were exposed to either UVA light alone (7 mW/cm², 365 nm peak wavelength, 1 h, 3% or 20% O₂, 37°C, PBS + 5mM glucose), BSO alone (0.5 mM, overnight), CDNB alone (0.02 mM, 10 min), BSO (overnight) + UVA (1 h, 20% O₂) or CDNB (10 min) + UVA (1 h, 20% O₂). GSH levels were measured immediately after each treatment. Control cells were cultured normally. Each result is expressed as the mean +/− SD. The number of experiments is in parentheses. α: p<0.001 to control; β: p<0.01 to control; γ: p<0.001 to UVA alone.

Fig. 2

Effect of UVA light on the activities of various enzymes in cultured human lens epithelial cells. Cells were exposed to UVA light (20% O₂; see Fig. 1 for details) for 1 hr, and enzyme activity was determined immediately. Control cells were cultured normally. Each result is expressed as the mean +/− SD. The number of experiments is in parentheses. α: p<0.05 to control; β: p<0.001 to control.

Fig. 3

Photomicrographs of cultured human lens epithelial cells. Cells (5×10⁵) were either (a) cultured normally for one hour or (b) exposed to UVA light (20% O₂; see Fig. 1 for details) for one hour. Note the lack of change in the appearance of the UVA-treated cells immediately following the 1 h exposure. Each photograph is representative of five experiments.

Fig. 4

Effect of various treatments, plus 24 hr of normal culture, on the number of cultured human lens epithelial cells. Conditions are as described in Fig. 1. Control cells were cultured normally, and the average number of control cells at the end of each experiment was 915,375 +/− 245,000 . Each result is expressed as the mean +/− SD. The number of experiments is in parentheses. α: p<0.01 to control; β: p<0.05 to CDNB alone; γ: p<0.05 to BSO + UVA (3% O₂); δ: p<0.001 to control; ε: p<0.01 to CDNB alone; ζ: p<0.001 to BSO + UVA (20% O₂).

Fig. 5

Effect of CDNB (0.02 mM) plus UVA light, followed by 24 hr of normal culture, on the morphology of cultured human lens epithelial cells. Cells were exposed to either CDNB alone (10 min) or CDNB (10 min) + UVA light (1 h) at either 3% or 20% O₂, cultured normally for 24 hr, and photographed. Conditions for UVA light exposure are shown in Fig. 1. (a) Control cells cultured normally; (b) CDNB alone (10 min) + 24 hr normal culture; (c) CDNB (10min) + UVA light (1 hr, 3% O₂) + 24 h normal culture; (d) CDNB (10 min) + UVA light (1 hr, 20% O₂) + 24 h normal culture. Note the lack of change in appearance of cells treated with CDNB alone, 24 h later (b). However, cells treated with CDNB + UVA light at 3% O₂ (c) showed a change in morphology after 24 h, compared to control cells (a), including enlarged cells (arrows) and spaces (arrowheads) that are indicative of cell death. Cells treated with CDNB + UVA light at 20% O₂ (d) showed threadlike structures (arrowheads), dead cells (arrows) and an increased number of spaces, compared to cells treated with CDNB + UVA light at 3% O₂ (c). Each photograph is representative of 6-9 experiments.

Fig. 6

Effect of BSO plus UVA light followed by 24 h of normal culture, on the morphology of cultured human lens epithelial cells. Cells were exposed to either BSO alone (overnight) or BSO (overnight) + UVA light (1 h) at either 3% or 20% O₂, cultured normally for 24 h, and photographed. Conditions for UVA light exposure are shown in Fig. 1. (a) Control cells cultured normally; (b) BSO alone (overnight) + 24 h normal culture; (c) BSO + UVA light (1 hr, 3% O₂) + 24 h normal culture; (d) BSO + UVA light (1 hr, 20% O₂) + 24 h normal culture. Note the lack of change in appearance after 24 h of culture for cells treated with BSO alone (b), BSO + UVA light at 3% O₂ (c), and BSO + UVA light at 20% O₂ (d). Each photograph is representative of 4-6 experiments.

Fig. 7

Effect of CDNB (0.02 mM) plus UVA light on the growth of cultured human lens epithelial cells. Cells were exposed to either CDNB alone (10 min) or CDNB (10 min) + UVA light (1 h, 3% O₂), cultured normally for 7 days, and counted on days 1, 3 and 7 with a Coulter counter. The data for day 1 are the same as those shown in Fig. 4. Conditions of the UVA light exposure are shown in Fig. 1. Control cells cultured normally (open circles); CDNB alone (10 min) (closed triangles); CDNB (10 min) + UVA light (open triangles) Each result is expressed as the mean +/− SD. The number of experiments is in parentheses. α: p<0.01 to control; β: p<0.001 to control; γ: p<0.001 to CDNB alone; δ: p=0.01 to CDNB alone.

Fig. 8

Effect of CDNB (0.02 mM) plus UVA light, followed by 7 days of normal culture, on the morphology of cultured human lens epithelial cells. Cells were exposed to either CDNB alone (10 min) or CDNB (10 min) + UVA light (1 h, 3% O₂), cultured normally for 7 days, and photographed. Conditions for UVA light exposure are shown in Fig. 1. (a) Control cells cultured normally for 7 days; (b) CDNB alone (10 min) + 7 days normal culture; (c) CDNB + UVA light (1 hr, 3% O₂) + 7 days normal culture. Note the multilayering of cells (arrows) in (b) and the enlarged cells (arrows) and threadlike structures (arrowheads) in (c). Each photograph is representative of four experiments.

Fig. 9

Quantification of antioxidant enzyme mRNA expression in challenged human lens epithelial cells using real-time PCR. Conditions for UVA light exposure are shown in Fig. 1. Fold-upregulation was calculated as the fold difference in the amount of mRNA for control and experimental samples, both normalized to β-actin (see Methods). (a) CDNB (10 min) alone plus 8 h normal culture. (b) UVA light alone (1 h) at 3% and 20% O₂ plus 8 h normal culture. (c) CDNB (10 min) + UVA light (1 h, 3% and 20% O₂) plus 8 h normal culture. Each result is expressed as the mean +/− S.D. for 2-3 experiments (average of triplicate samples for each). α: p<0.01 to 3% O₂; β: p<0.05 to 3% O₂.