Lens epithelium: a primary target of UVB irradiation.

Cultured rabbit lenses and cultured rabbit lens epithelial cells were irradiated with UV to correlate morphological changes in the epithelium with physiological changes in the whole lens during the development of UV-induced cataract. Two UV spectral ranges were utilized; one spanned 290 to 340 nm and was designated near-UV, the other was a narrower, pure UVB region: 303 to 313 nm, designated UVB. Irradiation with either spectrum of the anterior surface of whole lenses caused opacification and a dose-dependent loss of ion homeostasis as measured by Na+ and Ca2+ concentrations in whole lenses. It was determined that cation pump activity, assessed by 86Rb uptake, continued to decline steadily during culture after UV irradiation. Whole mount preparations of the epithelial cell layer of UVB-irradiated lenses revealed morphological changes within 2 hr of irradiation and cell death after 20 hr. Following posterior irradiation of whole lenses, the epithelial cells remained viable and lenses remained transparent during 3 days of culture, presumably because UV photons did not reach the epithelium. Absorption of UV photons by posterior fiber cell membranes and proteins did not cause opacification. To learn more about the epithelial damage, cultured rabbit lens epithelial cells were irradiated, UVB treatment retarded growth over a 7-day period in cultured cells. The surviving cells at day 7 were abnormal in appearance and the potassium concentration was approximately 50% less than controls, a finding which may explain the previously reported reduction in protein synthesis by UVB irradiation. Collectively, the data suggest that UV cataract is initiated by damage to the epithelium, including a change in membrane permeability leading to loss of ion homeostasis in the lens.

Membrane damage in UV-irradiated lenses.

The purpose of this study was to investigate three possible causes of membrane damage following UV irradiation: photooxidation of membrane thiol (SH) groups, peroxidation of membrane lipids and inhibited synthesis of membrane proteins. Thiol loss was not observed. Thin-layer chromatography showed a four-fold increase in several primary lipid peroxidation products such as hydroperoxyl lipids in the epithelial membrane preparations isolated from irradiated lenses. The formation of new hydroxyl lipid bands not seen in control preparations was also observed in isolated membranes from irradiated lenses. Irradiation in the presence or absence of oxygen produced lipid peroxidation products. Aerobic irradiation produced small, but statistically significant increases in lipid hydroxyls and hydroperoxyls relative to controls. Repair of initial damage might be compromised by the observed 60% reduction in rate of protein synthesis measured in lens membranes following irradiation. Synthesis was affected by means other than depleted potassium or elevated calcium levels.

Effect of selenite on epithelium of cultured rabbit lens.

Selenite (Se) cataract in rabbit lenses was investigated in vitro to define target sites of Se that might be involved in calcium elevation and lens opacification. Experiments in which the anterior or the posterior surface of the lens was exposed to Se showed that anterior exposure led to ionic imbalances and opacification in the whole lens. Posterior exposure to Se (1 mM, 2 hr) had no effect. Se treatment (0.1 mM) of epithelial homogenates led to a 56% loss of thiol (SH) groups, and treatment of lenses cultured in Se led to a 22% loss. Experiments to assess the effects of Se on SH groups of Ca-ATPase showed that the transport enzyme was not affected by the poison. To determine whether this negative finding was due to the lack of accessibility of Se for SH sites in an ordered membrane, Ca-ATPase was also assayed in homogenate preparations treated with Se; still no inhibition of Ca-ATPase activity was observed. Therefore, an alternative explanation of calcium elevation was explored. The passive movement of labeled chloride (36Cl) was found to be twice as fast in Se-treated lenses as it was in control lenses. Measurement of the lens voltage indicated an 18-mV depolarization in Se-treated lenses, suggesting that Se increased membrane permeability. All cataractogenic changes that occurred after Se treatment were irreversible-despite intervention with external application of reduced glutathione or cysteine. This finding suggests that irreversible loss of SH groups in lens membranes is important in maintaining ion homeostasis.

Effects of selenium on ion homeostasis and transparency in cultured lenses.

Selenium toxicity was investigated in cultured rabbit lenses to provide further information about the role of Ca++ in Se cataract. At a dose of 0.1 mM for 20 hr, Se induces a 10% change in Na levels within 6 hr, a 30% increase after 20 hr, and a three-fold increase within 48 hr of subsequent culture after removal of Se. In contrast, Ca++ levels remained normal throughout the first 24 hr. Only a small, 25% decline in GSH was noted. Not until lenses begin to swell and become noticeably opaque and turbid were Ca++ levels found to be elevated. Thus, at 72 hr, 48 hr following the removal of selenium, Ca++ had increased to a concentration of 0.7 mM. Ca++ accumulation appears to be a consequence of osmotic stress rather than pump inhibition while Na accumulation is a direct consequence of Se-inhibited Na pump.

Influence of the activity of glutathione reductase on the response of cultured lens epithelial cells from young and old rabbits to hydrogen peroxide.

Our previous studies on cultured rabbit lens epithelial cells from 4-day-old rabbits showed that the glutathione redox cycle plays an important role in detoxifying H2O2, a potentially damaging oxidant present in the aqueous humor. Here we report the effect of donor age and cell density on the ability of cultured rabbit lens epithelial cells to detoxify H2O2. Lens epithelial cells (8 x 10(5] from a 4-day-old and an 8-year-old rabbit were cultured for 3 hr in minimal essential medium (MEM) or in MEM containing 0.01-0.1 mM H2O2 maintained with glucose oxidase. We determined the effect of H2O2 on the level of reduced glutathione (GSH), hexose monophosphate shunt activity, cell growth, and morphology. For growth studies, cells were exposed to the desired concentration of H2O2 for 3 hr and then cultured in MEM plus 10% rabbit serum for 7 days and counted. Young and old untreated cells contained high levels (30-40 nmol/8 x 10(5) cells) of GSH. Cells from 4-day-old rabbits tolerated 0.03 mM H2O2 with no effect on GSH and a minimal decrease in subsequent cell growth. However, in the older cells, GSH and growth were substantially diminished following treatment with 0.03 mM H2O2. Cells plated out at high density (8 x 10(5] were more tolerant of 0.03 mM H2O2 than cells plated out at low density (5 x 10(4]. Maximum shunt activity in the younger cells exposed to H2O2 was twice that of the older cells and occurred at a higher level of H2O2 (0.04 compared with 0.03 mM). Enzyme activities in untreated young and old cells were comparable for hexokinase, glucose-6-phosphate dehydrogenase, and glutathione peroxidase. However, glutathione reductase activity was 50% lower in the cells from the 8-year-old rabbit. The toxicity of H2O2 to cultured lens epithelial cells was directly related to donor age and inversely related to cell density. The damage in the older lens epithelial cells at 0.03 mM H2O2 was apparently due, in part, to a diminished response of the glutathione redox cycle to oxidative challenge.

Calcium-induced opacification is dependent upon lens pH.

The intracellular pH of a normal lens is 6.8 in the cortex and remains unchanged during culture in media buffered at pH 7.2. Incubation of rabbit lenses in calcium enriched media, either at 24 degrees C or 37 degrees C, results in lens opacification provided that the lens pH remains slightly acidic. Opacities are prevented in cultured lenses with an alkaline interior (pH 7.1-7.3) despite the accumulation of calcium (1.3 mM). The mechanism by which an intracellular pH shift from 6.8 to 7.1 prevents opacification in the presence of excess calcium is not known, but does not appear to depend upon the total level of bound calcium. This study provides the first data that opacification caused by calcium is associated with lens pH.

Peroxide-induced effects on lens cation transport following inhibition of glutathione reductase activity in vitro.

Previous studies from this laboratory have shown that the normal lens can tolerate exposure to 0.05 mM H2O2 without apparent damage and that this is due in part to an active glutathione redox cycle. The present studies were designed to investigate the role of glutathione reductase in protecting cation transport systems in the lens against potentially damaging effects of peroxide. Pre-treatment of rabbit lenses with 0.5 mM 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a relatively specific inhibitor of glutathione reductase, brought about a 71% inhibition of the enzyme in the capsule-epithelia of the lenses. Subsequent exposure of the lenses for 3 hr to a constant level of 0.05 mM H2O2 in culture medium produced significant accumulation of oxidized glutathione (GSSG) in the lens epithelium and severe effects on the electrolyte balance in the lens, on the activity of Na, K-ATPase and on the accumulation and efflux of 86Rb. The effects included a 35% decrease in activity of Na, K-ATPase, a 10 mM increase in the concentration of Na+ and an 8 mM decrease in K+. BCNU-H2O2 treatment also resulted in loss of transparency of the lenses in the form of vacuoles present in the anterior, subcapsular region, encircling the entire periphery of the organ near the germinative zone of the epithelium. Treatment with either BCNU or 0.05 mM H2O2 alone had only minimal effects on accumulation of GSSG in the epithelium, on lens transparency and on the parameters of cation transport which were investigated. When lenses were treated with 0.05 mM H2O2 alone and then placed in normal medium to measure the accumulation of 86Rb it was found that the cation pump was stimulated 20% above the normal level of activity. Levels of H2O2 higher than 0.05 mM without BCNU pre-treatment produced significant inhibition of Na, K-ATPase and the effects of 0.3 mM H2O2 on cation transport and GSSG accumulation were comparable to those of BCNU-0.05 mM H2O2. While inhibition of the activities of glutathione reductase and Na, K-ATPase in the lenses was found to be irreversible, a partial recovery of the Na+ level and nearly complete recovery of the K+ level were observed when treated lenses were cultured in normal medium for an additional 6 hr. In addition, the rate of efflux of 86Rb which was significantly faster from the BCNU-H2O2-treated lenses compared with the controls, was found to return to the control value during the recovery period.(ABSTRACT TRUNCATED AT 400 WORDS)

Detoxification of H2O2 by cultured rabbit lens epithelial cells: participation of the glutathione redox cycle.

Although it has been shown that cultured rabbit lenses can adequately defend against the 0.03-0.05 mM level of H2O2 normally found in aqueous humor, the contribution of the epithelium in this process has not been well defined. In the present study, the peroxide-detoxifying ability of the epithelium is evaluated in cultured rabbit lens cells established from 4-6-day-old rabbits and compared to that of skin fibroblasts from rabbits of the same age. When cells were cultured in medium containing H2O2, the concentration of peroxide rapidly decreased; however, various concentrations could be maintained for 3-hr periods by using glucose oxidase to enzymically generate H2O2. At an extracellular level of 0.03 mM H2O2, the rate of detoxification of peroxide by epithelial cells was 2 mumol H2O2 (8 x 10(5) cells)-1 3 hr-1, twice as fast as that for fibroblasts. Epithelial cells contained a high level of reduced glutathione (GSH) equal to 36 nmol (8 x 10(5) cells)-1, twice that present in the fibroblasts. The concentration of GSH in 8 x 10(5) epithelial cells, a number of cells normally present in one intact rabbit lens epithelium, remained constant during 3 hr of exposure to H2O2 levels as high as 0.03 mM, even though the amount of H2O2 taken up under these conditions was sufficient to oxidize completely the cellular GSH every 2 min. In contrast, the GSH content of fibroblasts declined at levels of peroxide above 0.01 mM. Participation of the glutathione redox cycle in the H2O2-detoxification process was demonstrated from studies of hexose monophosphate shunt (HMPS) activity as measured by oxidation of [1-14C]-labeled glucose. The oxidation of [1-14C]-glucose in epithelial cells was stimulated 13 times that of controls during exposure to 0.04-0.05 mM H2O2, while the corresponding increase in oxidation of [6-14C]-labeled glucose was only 1.6 times. In contrast, maximum shunt activity in fibroblasts occurred at 0.03-0.04 mM H2O2 and was six times the control value. The growth potential of the cells following a 3-hr exposure to H2O2 was also used as a measure of oxidant toxicity in both cell types. Concentrations of H2O2 up to 0.03 mM had no effect on the growth of 8 x 10(5) epithelial cells but did diminish the growth of the same number of fibroblasts. Cell density was found to be an important parameter in the ability of the cells to tolerate H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)

A direct correlation between the levels of ascorbic acid and H2O2 in aqueous humor.

There is evidence that H2O2 present in aqueous humor arises from ascorbic acid which is also present in this fluid, but the extent to which peroxide is derived from ascorbic acid is not known. We have measured the concentrations of H2O2 and ascorbic acid normally present in the aqueous humor of various species and also under conditions in which the level of ascorbic acid in the fluid was experimentally altered. In aqueous humor of rabbit and guinea pig the concentration of ascorbic acid was 10 times higher than that present in aqueous of rat and frog. Similarly, the concentration of H2O2 was four to 10 times higher in rabbit and guinea pig aqueous compared to that in rat and frog. Consistent with the higher concentration of ascorbic acid in posterior compared to anterior aqueous humor in the rabbit, the concentration of H2O2 was also significantly higher in the posterior aqueous. When ascorbic acid in rabbit aqueous humor was elevated by intraperitoneal administration of the compound, there was a significant increase in the level of H2O2 in both anterior and posterior aqueous humor. Moreover, when the level of ascorbic acid was lowered experimentally by placing guinea pigs on an ascorbic acid deficient diet, a 10-fold decrease in the level of both ascorbic acid and H2O2 was observed in the aqueous humor. Upon returning the animals to a normal diet, the concentrations of both compounds returned to control values. The direct correlation between the concentrations of ascorbic acid and H2O2 in aqueous humor suggests that ascorbic acid is the primary source of H2O2 in this fluid.

The effect of inhibition of glutathione reductase on the detoxification of H2O2 by rabbit lens.

Mechanisms by which the lens protects against H2O2 are believed to include the metabolism of glutathione (GSH). In the present study, rabbit lenses were exposed to constant concentrations of H2O2 (0.01 to 0.1 mM) that were maintained in culture media with the use of a peristaltic pump. The rate at which H2O2 entered the lens was proportional to its concentration in the medium and reached 2.6 mumol H2O2/lens/3 hr at 0.1 mM H2O2. Up to 0.06 mM H2O2, a concentration that approximates that present in normal rabbit aqueous humor, the activity of the hexose monophosphate shunt (HMPS) increased linearly with no significant decrease in the concentration of lens GSH. However, at 0.1 mM H2O2, there was indication of oxidative damage to the lens as shown by a sharp decrease in HMPS activity and a coincident drop in the concentration of GSH. Pretreatment of lenses with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), blocked the normal threefold stimulation of HMPS activity occurring in the presence of 0.06 mM H2O2 and resulted in accumulation of oxidized GSH. This result demonstrated the inability of H2O2 to react directly with NADPH in the lens. BCNU was shown not to affect the potential of the HMPS to respond to compounds other than H2O2 since it did not alter methylene blue-stimulation of HMPS activity. The study supports the hypothesis that detoxification of H2O2 in the aqueous humor is linked to the metabolism of GSH in the lens and demonstrates that lenses with impaired GR activity are more susceptible to oxidative damage by peroxide.

The role of glutathione metabolism in the detoxification of H2O2 in rabbit lens.

Aqueous humor is known to contain a significant level of H2O2, but the mechanisms by which ocular tissues protect against oxidative damage are not well understood. With the use of C-1-, C-2-, and C-6-labeled glucose, the contribution of glutathione (GSH) metabolism and the hexose monophosphate shunt (HMS) to the detoxification of peroxide in the lens has been evaluated. It was observed that H2O2 in the culture medium disappeared rapidly (0.5 mumol H2O2/lens/hr) upon incubation of a rabbit lens at 37 degrees C. At 0 degrees to 3 degrees C, however, the rate of disappearance of H2O2 was only one fifth of that observed at the higher temperature. In the absence of a lens or after pretreatment of the lens with methyl mercuric hydroxide, the rate of disappearance of peroxide from the medium was reduced to nearly zero. When a nearly constant level of H2O2 (0.05 to 0.07 mM) was maintained in the medium by means of a peristaltic pump, the amount of CO2 liberated by the HMS at 37 degrees C was found to be three times that liberated from lenses cultured in the absence of peroxide. No change was noted in the level of GSH in the H2O2-treated lenses at 37 degrees C. A significant decrease in GSH was observed, however, at 0 degrees to 3 degrees C, suggesting nonenzymatic oxidation of the tripeptide at the lower temperature. The results indicate that GSH metabolism and the HMS pathway contribute significantly to the detoxification of H2O2 in the lens.