Frank J. Giblin

Nuclear light scattering, disulfide formation and membrane damage in lenses of older guinea pigs treated with hyperbaric oxygen

Nuclear cataract, a major cause of loss of lens transparency in the aging human, has long been thought to be associated with oxidative damage, particularly at the site of the nuclear plasma membrane. However, few animal models have been available to study the mechanism of the opacity. Hyperbaric oxygen (HBO) has been shown to produce increased nuclear light scattering (NLS) and nuclear cataract in lenses of mice and human patients. In the present study, older guinea pigs (Initially 17-18 months of age) were treated with 2.5 atmospheres of 100% O2 for 2-2.5-hr periods, three times per week, for up to 100 times. Examination by slit-lamp biomicroscopy showed that exposure to HBO led to increased NLS in the lenses of the animals after as few as 19 treatments, compared to lenses of age-matched untreated and hyperbaric air-treated controls. The degree of NLS and enlargement of the lens nucleus continued to increase until 65 O2-treatments, and then remained constant until the end of the study. Exposure to O2 for 2.5 instead of 2 hr accelerated the increase in NLS; however, distinct nuclear cataract was not observed in the animals during the period of investigation. A number of morphological changes in the experimental lens nuclei, as analysed by transmission electron microscopy, were similar to those recently reported for human immature nuclear cataracts (Costello, Oliver and Cobo, 1992). O2-induced damage to membranes probably acted as scattering centers and caused the observed increased NLS. A general state of oxidative stress existed in the lens nucleus of the O2-treated animals, prior to the first appearance of increased NLS, as evidenced by increased levels of protein-thiol mixed disulfides and protein disulfide. The levels of mixed disulfides in the experimental nucleus were remarkably high, nearly equal to the normal level of nuclear GSH. The level of GSH in the normal guinea pig lens decreased with age in the nucleus but not in the cortex; at 30 months of age the nuclear level of GSH was only 4% of the cortical value. HBO-induced changes in the lens nucleus included loss of soluble protein, increase in urea-insoluble protein and slight decreases in levels of GSH and ascorbate; however, there was no accumulation of oxidized glutathione. Intermolecular protein disulfide in the experimental nucleus consisted mainly of gamma-crystallin, but crosslinked alpha-, beta- and zeta-crystallins were also present.(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 relative roles of the glutathione redox cycle and catalase in the detoxification of H2O2 by cultured rabbit lens epithelial cells

The relative roles of the glutathione redox cycle and catalase in the detoxification of H2O2 were investigated in cultured rabbit lens epithelial cells. Exposure of cells to H2O2 was carried out following inhibition of either of the two antioxidant systems. Two different procedures were used to expose the cells to extracellular H2O2, one in which a low, steady state level of 0.025 mM H2O2 was maintained in the culture medium with the use of glucose oxidase and the other in which H2O2 was added to the medium as a single pulse at levels ranging from 0.03 to 0.5 mM. When lens cells were treated with a low, steady state level of H2O2, the glutathione redox cycle was the primary means of defense against oxidative damage. Cells with fully active catalase but with inhibited glutathione reductase were not able to resist the cytotoxic effects of a 0.025 mM level of extracellular H2O2. Under these conditions the cells were nearly completely depleted of reduced glutathione within 15 min. The cellular damage observed after 1.5 hr of culture included loss of cell-to-cell contact, rounding up of the cells and formation of numerous blebs. In contrast, cells with completely inhibited catalase but with an unimpaired glutathione redox cycle suffered few damaging effects from a 3-hr exposure to 0.025 mM H2O2. When lens cells were pulsed with a single challenge of 0.5 mM H2O2, both the glutathione redox cycle and catalase were found to be essential for survival of the cells. While control cells were able to withstand the pulse of H2O2, cells with impaired activities of either the glutathione redox cycle or catalase were killed. Control cells treated with 0.5 mM H2O2 may have been protected from damage by the fact that the cellular level of GSH never dropped below 35% of normal. The cause of cell death following inhibition of catalase appeared to be related to an inability of the cells to remove peroxide from the culture medium, at a rapid rate, following the H2O2-pulse. Although cells with impaired glutathione reductase activity removed H2O2 from the medium at a rate comparable to that of control cells (due to uninhibited catalase activity), they did not survive the challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyridine nucleotides in ocular tissues as determined by the cycling assay

The modified cycling assay of Nisselbaum and Green (1969) has been demonstrated to be a useful method for the determination of pyridine nucleotides in ocular tissues. With this method it is possible to assay conveniently and accurately amounts of coenzyme as low as 10 −11 mol which permits analysis of pyridine nucleotides in the epithelium of a single rabbit lens. Using this assay, the concentrations of coenzymes were determined in lens, retina and corneal epithelium of various species. The ratio of NAD + to NADH in each of the tissues analyzed was substantially greater than 1·0, varying from 1·9 in rabbit corneal epithelium to 5·5 in bovine corneal epithelium. The highest ratio of NADPH to NADP + (1·3) was found in rat retina and bovine corneal epithelium. A study of the distribution of pyridine nucleotides in rabbit lens showed that the combined levels of NAD + plus NADH were nearly equal in cortex and nucleus. However, a distinct difference between the ratios of NAD + to NADH present in cortex (3·3) and nucleus (1·3) reflected the lower metabolic activity in the nuclear region of the lens. Low concentrations of NADP + and NADPH were found in nucleus and were most likely a result of a low concentration of ATP, possibly insufficient for the phosphorylation of NAD + . A major finding was that lens epithelium contains unusually high concentrations of phosphorylated pyridine nucleotides at levels 25 times higher than those in cortex or whole lens. The abundance of NADP + and NADPH may be related to the high level of glutathione (GSH) and high activity of glutathione reductase present in epithelium. Higher ratios of both NADPH and GSH to protein-SH groups exist in epithelium compared to the rest of the lens. This may ensure that membrane sulfhydryls in the epithelium are maintained in the reduced state in an environment that is more highly oxidative than the inner regions of the lens.

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)

Stimulation of the hexose monophosphate shunt in rabbit lens in response to the oxidation of glutathione

The stimulation of hexose monophosphate shunt (HMS) activity in rabbit lens has been investigated during exposure to t-butyl hydroperoxide (TBHP), an oxidant for glutathione (GHS). Exposure of lenses to 1·0 m m -TBHP produced maximum stimulation of HMS activity as measured by the oxidation of 14C-labeled glucose, labeled in either the C-1, C-2 or C-6 position. A significant increase in the oxidation of both C-1 and C-2 labeled glucose was observed. Removal of TBHP from the medium resulted in a rapid decrease in the rate of oxidation. The maximum rate of liberation of CO2 from the HMS pathway was estimated to be 0·75 μmol/g wet wt/hr, which was 7·7 times the control value. The generation of NADPH at maximum stimulation was estimated to be 1·5 μmol/g wet wt/hr which corresponded to a complete turnover of NADP+ to NADPH in the lens every 48 sec. This rate of generation of NADPH was sufficient to result in a reduction of oxidized glutathione (GSSG) at the rate of 26% of total glutathione in the lens per hour. Exposure of lenses to 0·10 m m -TBHP stimulated shunt activity by three times the control. However, under these conditions only minimal accumulation of GSSG was detected in the lens and no effect was observed on the uptake or efflux of 86Rb. Higher levels of TBHP resulted in a significant accumulation of GSSG in the lens and impaired cation transport. The distribution of the activity of the HMS pathway was determined in homogenates of various regions of rabbit lens. It was found that 65% of the total activity was present in the equatorial cortex, which made up 45% of the total weight. The results indicate that a direct link exists between the rate of formation of GSSG in the lens and the stimulation of HMS activity. In addition, the findings exemplify the ability of the lens to protect itself against oxidative damage through an interrelationship of the shunt, glutathione peroxidase, GSH, NADPH and glutathione reductase.

Glutathione oxidation in retina: Effects on biochemical and electrical activities

This study investigates the possible role of glutathione (GSH) in defending the retina against oxidative damage. Freshly excised rat retina was found to contain 1.2 mumol/g wet wt GSH and an undetectable level of oxidized glutathione (GSSG). Whole retinas were either incubated or superfused with various concentrations of the GSH-oxidant diamide in order to study the effects of oxidation of GSH on the activity of the hexose monophosphate shunt (HMS) and on the receptor potential of the retina. It was found that exposure of the retina to diamide produced a stimulation of HMS activity up to 26-times that of the control. Significant changes in GSH content and receptor potential were observed at concentrations of diamide that produced more than a 5.4-fold stimulation of HMS activity. The diamide-induced electrical alterations included an increase in latency and peak time of the receptor potential, a delay in the onset of the off response and an increase in the time required for the potential to return to the baseline. It was found that nearly 80% of GHS could be regenerated and that most of the electrical effects of diamide could be reversed by superfusion with normal medium. The results indicate that the retina possesses an active system for maintaining GSH in the reduced state and that this may be essential for the normal function of this tissue.

The effect of X-irradiation on cation transport in rabbit lens

The effect of X-irradiation on the active transport and diffusion of 86 Rb was investigated at various times following exposure to a single dose of 2000 rad. In addition the levels of ascorbic acid, glutathione (GSH) and superoxide dismutase (SOD) in lens and ascorbic acid and H 2 O 2 in aqueous humor were studied as a function of time after irradiation. A decrease in active transport of Rb and an increase in permeability was first observed after 4 weeks following X-ray; after 7 weeks, the active transport decreased by 80% and permeability increased by 44 times the control value. Despite the dramatic changes in cation transport and permeability, the hydration of the lens remained unaffected until the development of maturre cataracts, which appeared 8–9 weeks following exposure to X-ray. In contrast to the delayed effect of X-ray on hydration and cation transport, there was a gradual decrease in the level of GSH. The values of ascorbate in aqueous humor were significantly lower in the X-rayed eyes than the contralateral controls at all time periods after irradiation, while a lower concentration of H 2 O 2 in the aqueous of X-rayed eyes was first observed 4 weeks after exposure. The activity of SOD in the lens was unaffected at any time after X-irradiation. The decrease in the level of GSH in capsule and epithelium was less pronounced than in the decapsulated or whole lens The possible role of GSH in the epithelium in the regulation of cation transport and hydration is discussed.

High molecular weight protein aggregates in x-ray-induced cataract.

Abstract The nature and the possible mechanism of formation of high molecular weight (HMW) protein aggregates were studied in X-ray-induced cataract in rabbit. The HMW protein fraction (molecular weight >4×10 6 daltons) which constituted approximately 18% of the total soluble protein in both the cataractous cortex and nucleus was isolated by gel filtration chromatography. The concentration of sulfhydryl (-SH) groups per milligram of protein in the HMW fraction was three times higher than that of normal α-crystallin. In addition, 50% of the total concentration of sulfhydryl groups contained in the HMW protein was found to be present in the oxidized state; the increased oxidation of protein -SH groups in the X-irradiated lens being observed only during the final stage of cataract which was marked by complete opacity. Treatment of HMW protein with dithioerythritol and subsequent refractionation yielded two peaks; peak I which eluted as α-crystallin and the second fraction (peak II) which eluted in the position of low molecular weight β-crystallin. Peak I accounted for approximately 40% of the deaggregated protein and peak II 60%. There was little protein eluted in the position of γ-crystallin. Thus, it appears that the HMW protein of X-ray-induced cataract is comprised of both α- and β-crystallins joined by intermolecular disulfide bonds. SDS (sodium dodecyl sulfate) electrophoresis of HMW protein and peaks I and II showed that the major bands of both peaks I and II were represented in the bands exhibited by the HMW protein. The electrophoretic mobilities of the major bands of peaks I and II were similar to those of α-crystallin and β-crystallin, respectively. The amino acid composition of peak I was found to be comparable to α-crystallin while the composition of peak II was similar to β-crystallin with the major exception that the hydrolysates of proteins in both peaks I and II were nearly lacking in tyrosine.

Peroxide-Induced Damage in Lenses of Transgenic Mice with Deficient and Elevated Levels of Glutathione Peroxidase

Transgenic mice with elevated glutathione peroxidase (GSHPx) activity and gene knockout animals with a deficiency of the enzyme were used to investigate the role of GSHPx in defending the lens against H2O2-induced damage. The effects of peroxide on cultured lenses were determined by using light and transmission electron microscopy to evaluate morphological changes occurring in the epithelium and superficial cortex of the central and equatorial regions of the lens. DNA single-strand breaks in the epithelium were also examined. Following a 30-min exposure to 25 microM H2O2, lenses from normal animals showed distinct changes in the morphology of both the epithelium and superficial cortex. The damage to these cells was extensive in lenses of gene knockout mice in which activity of GSHPx was undetectable. In marked contrast, lenses of transgenic mice, which had 5-fold higher activities of GSHPx, were able to resist the cytotoxic effects. Similar to damage to cell morphology, the extent of DNA strand breaks was significantly lower (40% of control) in H2O2-exposed lenses as compared to normal lenses while DNA damage in gene knockout lenses was 5 times greater than that of GSHPx-rich transgenic lenses. The present studies extend our previous findings on the role of the glutathione redox cycle in the detoxification of peroxide and demonstrate that an increase in GSHPx activity protects the lens against peroxide-induced changes in cell morphology and DNA strand breaks.