Janet P. McCready

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.

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)

Mechanisms involved in cataract development following near-ultraviolet radiation of cultured lenses

Cultured rabbit lenses were irradiated with UV (311 nm peak; 295-340 nm) for 30 to 60 min. The entire spectrum lies in the near-UV, the major component is UVB, with a minor portion (25%) of UVA, and is henceforth referred to as near-UV(B). Posterior irradiation caused no cataract and no significant ionic imbalances compared to anterior irradiation, which caused opacification and marked changes in sodium and calcium concentrations. Anterior irradiation also resulted in reduced Na/K-ATPase activity in the epithelium. ATPase activity was not immediately inhibited; rather, only after culture was enzyme activity reduced. The concentration of reduced glutathione (GSH) decreased rapidly in the epithelium and more slowly in the underlying lens fibers. Loss of GSH was more rapid and extensive when irradiation occurred in the presence of oxygen. Irradiation under anaerobic conditions resulted in opacification but was considerably less extensive than when irradiation of lenses occurred in the presence of 7% oxygen. Near-UV(B) damage following anaerobic irradiation and 20 hrs of culture resulted in an increase in sodium levels and loss of GSH; calcium levels were not significantly elevated. Since irradiation of tryptophan solutions produced small amounts of hydrogen peroxide, the possibility of hydrogen peroxide-mediated damage was investigated but no role could be substantiated. Peroxide detoxification by the epithelium of near-UV(B) cataracts was observed, as measured by its ability to eliminate hydrogen peroxide added as a bolus.

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)

MEMBRANE DAMAGE IN UV‐IRRADIATED LENSES

Abstract 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.

COMPARATIVE EFFECT OF UVA AND UVB ON CULTURED RABBIT LENS

Abstract Effects on lens physiology of UVB and UVA used separately and sequentially were investigated using 4 week old rabbit lenses in organ culture. Narrowband UVB at 0.3 J/cm2= joules/lens (1 h exposure) has little effect on sodium and calcium concentrations in the lens interior or transparency of lenses subsequently cultured for 20 h after a 1 h exposure. With an incident energy of 3 J/cm2 of broadband UVB (295–330 nm), lenses become opaque and slightly swollen with significant ion imbalances during culture over a 1 day period. In contrast, lenses exposed to approximately 6–24 J/cm2 of UVA (330–400 nm) remain transparent after 1 day of culture. Extended culture up to 4 days reveals no signs of opacification. Ion homeostasis and normal lens hydration are also maintained in UVA‐irradiated lenses. The presence of 95% oxygen during UVA irradiation is also without effect. Broadband UVA irradiation is damaging, however, if lenses are first exposed to subthreshold doses of narrowband UVB (307 ± 5 nm) irradiation, viz. 0.3 J/cm2. Thus, sequential UVB/UVA irradiation at subthreshold doses causes impaired active cation transport and accumulation of sodium and calcium accompanying lens opacification.

Regional distribution of calcium in alloxan diabetic rabbit lens

A diabetic rabbit model was developed for investigation of cataractogenesis and other changes in the anterior segment. Rabbits were fasted, injected with 0.7 mg/kg alloxan, fed 1% glucose solution for 24 hrs and returned to a normal diet. Animals showing and maintaining blood glucose of greater than 300 mg% within two days were used in this study. Concomitant with increase in blood glucose was a rise in aqueous humor glucose and osmolality, together with a decrease in ascorbate concentration. Vacuoles and small discrete opacities developed, and in some cases, at longer time periods complete opacity of anterior or posterior aspects was found. Total calcium content of the whole lens increased up to 2-fold, especially after 60 days, and was correlated with a decrease in lens transmittance of a He/Ne laser beam and also with high osmolality of the aqueous humor. Free calcium was six-fold higher in opaque areas than clear areas, and was 100-fold higher in vacuoles. It is suggested that, in addition to the recognized role in sugar cataractogenesis of osmotic stress due to sorbitol accumulation in the lens, changes of intracellular calcium in localized areas of the lens and stresses imposed by changes in aqueous humor osmolality may also be important.

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.

Effect of thiol reagents on Ca-ATPase in rabbit lens epithelium

The inhibitory effects of sulfhydryl reagents on Ca-ATPase activity in the rabbit lens epithelium were assessed. Test compounds used in this study were selected on their ability to cause a calcium increase in cultured lenses. Under conditions in which lenses were cultured in the presence of the test compound, epithelial Ca-ATPase was inhibited markedly by diamide, t-BHP, IAA and slightly by selenite. The findings demonstrated that hydrogen peroxide caused little inhibition of Ca-ATPase in the lens epithelium, both when the intact lens was cultured in the presence of the oxidant or when the epithelial homogenate contained peroxide during the assay of enzyme activity. The study suggests that if a thiol-modifying compound can reach Ca-ATPase or its critical SH groups, inhibition is likely.