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

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.

The role of metallothionein IIa in defending lens epithelial cells against cadmium and TBHP induced oxidative stress.

PURPOSE: Heavy metals and other forms of oxidative stress have been implicated as key factors in the formation of age-related cataract in humans. Metallothioneins are a group of proteins known to play important roles in defending cells against the cytotoxic effects of heavy metals. However, little is known about their involvement in defending against other forms of oxidative stress. Here, we examined the ability of metallothionein IIa (MTIIa) to protect human lens epithelial cells against cadmium and tertiary butyl hydroperoxide (TBHP)-induced oxidative stress. METHODS: MTIIa over-expressing human lens epithelial cells (SRA01/04) were created by retroviral mediated gene transfer. Normal and MTIIa over-expressing cells were exposed to various concentrations of cadmium and TBHP and subsequently monitored for cell death, changes in cell phenotype and differences in growth rate. In addition, expression levels of three other important antioxidant genes, heme oxygenase-1, thioredoxin reductase-1, and manganese superoxide dismutase were monitored by real-time RT-PCR following exposure to TBHP. RESULTS: Analysis of the over expressing cell lines revealed an approximate 3-4 fold increase in MTIIa expression relative to control cells, resulting in as much as 20% protection against cadmium-induced oxidative stress (p<0.001). The MTIIa over expressing cells were also significantly more resistant to TBHP treatment while control cells exhibited significant shrinking and rounding-up following 3-6 h TBHP treatment, no changes were observed in TBHP-treated over expressing cells. When control cells were treated for 3 h or overnight with TBHP, 40-45% cell death occurred by day three. However, no cell death was observed at this time for the treated MTIIa over-expressing cell line. In addition, TBHP induced the expression of MTIIa, heme oxygenase-1, thioredoxin reductase-1, and MnSOD in both normal and MTIIa over-expressed cell lines. Interestingly the latter three genes were induced at 2-3 fold higher levels in TBHP-treated MTIIa over-expressing cells, compared to treated control cells (p=0.001, p=0.02, and p=0.01, respectively). CONCLUSIONS: These data indicate that over-expression of MTIIa in human lens epithelial cells results in protection against cadmium and TBHP-induced oxidative stress. In addition, the results suggest that MTIIa, and/or its ability to chelate metals, may play a role in regulating expression of other important antioxidant genes in response to oxidative stress.

Thioredoxin reductase may be essential for the normal growth of hyperbaric oxygen-treated human lens epithelial cells.

We have shown previously with in vivo and in vitro animal models that the lens epithelium, in contrast to the nucleus, is remarkably resistant to hyperoxia. The main purpose of this study was to investigate the mRNA response of cultured human lens epithelial cells (LECs) to challenge by a high level of hyperbaric oxygen. Cells were treated for 3 hr with 50 atm of 99% O2, and then cultured normally for various times up to 11 days. Although the cells appeared normal immediately after the O2-treatment, they failed to grow and suffered 50% cell loss, as well as significant mitochondrial damage, during normal post-culture. Growth of the cells resumed after 3 days and by day 11, the number of O2-treated cells was the same as the controls. Remarkably, the 3 hr O2-treatment produced no immediate effects on either the cellular level of GSH, or on the activities of a number of antioxidant enzymes including glyceraldehyde-3-phosphate dehydrogenase, which is generally regarded as being highly sensitive to oxidation. In contrast, the activity of thioredoxin reductase (TrxR) was severely affected by the O2, decreasing by 51% after the 3 hr exposure. O2-induced death of the cells appeared to be caused by loss of ATP since a 31% decrease in ATP level occurred immediately after the O2-treatment, in spite of a 46% increase in lactate production. Analysis with real-time PCR showed a maximum 3-6-fold increase in mRNA levels 9 hr after the 3 hr O2-exposure for the enzymes heme oxygenase-1 (HO-1), MnSOD and TrxR1 (the cytoplasmic form of TrxR). These results were confirmed with the use of one-step RT-PCR and Northern blotting. Initial upregulation of message for HO-1 occurred a few hours before any upregulation of MnSOD could be detected, suggesting that release of free iron from the degradation of heme by HO-1 may have played a role in the upregulation of the dismutase. No significant changes in mRNA levels were observed for the antioxidant enzymes catalase, CuZnSOD, glutathione reductase and glutathione peroxidase, or for the antioxidant protein thioredoxin. Recovery of TrxR activity over a 4-day period appeared to parallel the return of the cells to a normal rate of growth. The results indicate that damaging effects of hyperoxia on cultured LECs occur primarily in the mitochondria, rather than in the cytoplasm. Cells avoid O2-induced cell death, and return to a normal rate of proliferation by upregulating mRNA levels for HO-1, MnSOD and TrxR1. It appears that full activity of TrxR1, an enzyme required for the production of deoxyribonucletides for DNA synthesis, is essential for the normal growth of O2-challenged LECs.

UVA light in vivo reaches the nucleus of the guinea pig lens and produces deleterious, oxidative effects.

The possible role of ultraviolet light in the formation of cataract is not well understood. In this study, guinea pigs were exposed to a chronic, low level of UVA light (0.5 mWcm(-2), 340-410 nm wavelength, peak at 365 nm) for 4-5 months. It is known that the lens of the guinea pig possesses unusually high levels of the UVA chromophore NADPH. In a preliminary analysis, it was found that isolated guinea pig corneas transmitted 70-90% of 340-400 nm light, and that UVA radiation was able to penetrate deep into the nucleus of the guinea pig lens, where it was absorbed. Exposure of guinea pigs to UVA in vivo produced a 60% inactivation of lens epithelial catalase; however, analysis by transmission electron microscopy (TEM) showed no apparent morphological effects on either the lens epithelium or the cortex. A number of UVA-induced effects were found in the nucleus of the guinea pig lens, but were observed either not at all or to a lesser extent in the cortex. The effects included an increase in light scattering (two-fold; slit-lamp examination), distention of intercellular spaces (TEM), an increase in lipid peroxidation (30-35%; infrared spectroscopy), a decrease in GSH level (30%), an increase in protein-thiol mixed disulfide levels (80%), loss of water-soluble protein (20%), an increase in the amount of protein disulfide (two-fold; two-dimensional diagonal electrophoresis), degradation of MIP26 (15%) and loss of cytoskeletal proteins including actin, alpha- and beta- tubulin, vimentin and alpha-actinin (60-100%). The results indicate that a 4-5 month exposure of guinea pigs to a biologically relevant level of UVA light produces deleterious effects on the central region of the lenses of the animals. UVA radiation, coupled presumably with the photoreactive UVA chromophore NADPH and trace amounts of O(2) present in the lens nucleus, produced significant levels of oxidized products in the nuclear region over a five month period. The data demonstrate the potentially harmful nature of UVA light with respect to the lens, and highlight the importance of investigating a possible role for this type of radiation in the formation of human cataract.