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).

Shotgun proteomic analysis of S-thiolation sites of guinea pig lens nuclear crystallins following oxidative stress in vivo.

Purpose: To compare levels of S-glutathiolation and S-cysteinylation occurring at more than 60 cysteine residues of 12 different guinea pig lens water-soluble nuclear crystallins following treatment of the animals with hyperbaric oxygen (HBO).

Methods: Guinea pigs (initially 18 months old) were treated 30X (3X per week for 10 weeks) with HBO (2.5 atm 100% O(2) for 2.

A Class I UV-blocking (senofilcon A) soft contact lens prevents UVA-induced yellow fluorescence and NADH loss in the rabbit lens nucleus in vivo.

It is known that fluorescence, much of it caused by UVA light excitation, increases in the aging human lens, resulting in loss of sharp vision. This study used an in vivo animal model to investigate UVA-excited fluorescence in the rabbit lens, which contains a high level of the UVA chromophore NADH, existing both free and bound to λ-crystallin. Also, the ability of a Class I (senofilcon A) soft contact lens to protect against UVA-induced effects on the rabbit lens was tested.

A class I (Senofilcon A) soft contact lens prevents UVB-induced ocular effects, including cataract, in the rabbit in vivo.

Purpose: UVB radiation from sunlight is known to be a risk factor for human cataract. The purpose in this study was to investigate the ability of a class I UV-blocking soft contact lens to protect against UVB-induced effects on the ocular tissues of the rabbit in vivo.

Methods: Eyes of rabbits were exposed to UVB light for 30 minutes (270-360 nm, peak at 310 nm, 1.

Expression and purification of his-tagged recombinant mouse zeta-crystallin.

Zeta-crystallin is an NADPH-binding protein consisting of four identical 35kD subunits. The protein possesses quinone oxidoreductase activity, and is present in large amounts in the lenses of camelids, certain hystricomorphic rodents, and the Japanese tree frog, and in lower catalytic amounts in certain tissues of various species. In this study, recombinant methods were used to produce substantial quantities of his-tagged recombinant mouse zeta-crystallin, which was then purified to homogeneity.

Enzyme-induced posterior vitreous detachment in the rat produces increased lens nuclear pO2 levels.

It has been proposed that disruption of normal vitreous humor may permit O(2) to travel more easily from the retina to the center of the lens where it may cause nuclear cataract (Barbazetto, I.A., Liang, J.

Measurement of lens protein aggregation in vivo using dynamic light scattering in a guinea pig/UVA model for nuclear cataract.

The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model.

Microplasmin-induced posterior vitreous detachment affects vitreous oxygen levels.

Purpose: To determine if enzymatic induction of a posterior vitreous detachment (PVD) and/or vitreous liquefaction affects O2 concentration in the vitreous cavity in animals with vascularized and avascular retinal circulations.

Methods: Either microplasmin or hyaluronidase was injected intravitreally into guinea pigs (avascular retinal circulation), brown Norway rats (vascularized retinal circulation without fovea), or cats (vascularized retinal circulation with fovea) with the contralateral eye used as a control. One to 2 weeks post injection, vitreal oxygen concentration was measured using a highly sensitive, platinum-based fluorophore O2 sensor.

Raman spectroscopic evidence for nuclear disulfide in isolated lenses of hyperbaric oxygen-treated guinea pigs.

Our laboratory treats guinea pigs with hyperbaric oxygen (HBO) as a model for investigating the formation of nuclear cataract. Previous analyses of lens supernatants using this model have shown an increase in disulfide (-SS-) and loss of sulfhydryl (-SH) in the lens nucleus of O(2)-treated animals. In this paper, we have used the non-invasive technique of Raman spectroscopy to confirm these findings in intact, freshly-excised lenses.

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.

Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract: dynamic light-scattering and HPLC analysis.

Purpose: The role of oxygen in the formation of lens high-molecular-weight (HMW) protein aggregates during the development of human nuclear cataract is not well understood. The purpose of this study was to investigate lens crystallin aggregate formation in hyperbaric oxygen (HBO)-treated guinea pigs by using in vivo and in vitro

Methods: methods. Guinea pigs were treated three times weekly for 7 months with HBO, and lens crystallin aggregation was investigated in vivo with the use of dynamic light-scattering (DLS) and in vitro by HPLC analysis of water-insoluble (WI) proteins.

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.

Effect of hyperbaric oxygen on guinea pig lens optical quality and on the refractive state of the eye.

The main objective of this study was to investigate the effect of in vivo hyperbaric oxygen (HBO) treatment of albino guinea pigs on ocular refractive state and optical properties of the lens in vitro, as well as on the integrity of the mitochondria of the lens. The animals were treated 30-35 times (2.5-3 months) or 70 times (6 months) with HBO.

Propyl gallate is a superoxide dismutase mimic and protects cultured lens epithelial cells from H2O2 insult.

n-Propyl gallate (nPG) is a food preservative that is generally regarded as safe by the US FDA. It suppresses oxidation in biological systems. The mechanism by which nPG acts in biological systems is uncertain.

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.

The effects of hyperbaric oxygen on the crystallins of cultured rabbit lenses: a possible catalytic role for copper.

Oxidative effects on lens proteins have been linked with the formation of human age-related cataract, particularly nuclear cataract. This study investigated the effects of hyperbaric oxygen (HBO)-induced oxidative stress on nuclear and cortical alpha-, beta- and gamma-crystallins of cultured rabbit lenses, using high performance liquid chromatography (HPLC). The lenses were treated with 50 atm of either 100% N(2)(control) or 100% O(2)(experimental) for 3, 6, 16 and 48 hr.

Impact of aging and hyperbaric oxygen in vivo on guinea pig lens lipids and nuclear light scatter.

Purpose: To measure lipid compositional and structural changes in lenses as a result of hyperbaric oxygen (HBO) treatment in vivo. HBO treatment in vivo has been shown to produce increased lens nuclear light scattering.

Methods: Guinea pigs, approximately 650 days old at death, were given 30 and 50 HBO treatments over 10- and 17-week periods, respectively, and the lenses were sectioned into equatorial, cortical, and nuclear regions.

Thiol disulfide exchange modulates the activity of aldose reductase in intact bovine lens as a response to oxidative stress.

The reversibility of S-thiolation of aldose reductase was shown in intact bovine lens subjected to oxidative stress. The glutathione modified aldose reductase generated in the lens as a consequence of hyperbaric oxygen treatment was recovered in its reduced form following culturing in normobaric air conditions. Nucleus and cortex were differently affected by both oxidative treatment and normobaric air recovery.

Hyperbaric oxygen in vivo accelerates the loss of cytoskeletal proteins and MIP26 in guinea pig lens nucleus.

Previous studies have shown that treatment of guinea pigs with hyperbaric oxygen (HBO) produces certain changes in the lens nuclei of the animals which are typical of those occurring during aging. These include an increase in nuclear light scattering (NLS), elevation in levels of oxidized thiols, loss of water-soluble protein and damage to nuclear membranes. The present study investigated the effect of HBO-treatment in vivo on lens cytoskeletal proteins and MIP26 which are also known to undergo alteration with age.

Protection from oxidative insult in glutathione depleted lens epithelial cells.

It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine, compounds that limit the availability of Fe+2 and prevent the generation of hydroxyl radicals, protect cultured rabbit lens epithelial cells from H2O2-induced damage. In view of the importance of glutathione as an antioxidant and the decrease in GSH that is known to accompany most forms of cataract, we investigated whether these compounds protected cultured lens epithelial cells from H2O2 when the cells were artificially depleted of glutathione. Treatment of lens epithelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound that irreversibly binds to glutathione, or buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis, reduced the glutathione content to an average of 15-20% of the control values without a concomitant increase in oxidized glutathione.

Heme oxygenase synthesis is induced in cultured lens epithelium by hyperbaric oxygen or puromycin.

We showed previously that treatment of cultured rabbit lens epithelial cells (LECs) with hyperbaric oxygen (HBO) produced DNA strand-breaks, caused reversible inhibition of protein synthesis and induced the synthesis of a 32 kD protein. In the present work, we employed immunostaining procedures to identify the 32 kD protein as heme oxygenase-1 (HO-1). Increased synthesis of the enzyme was observed as early as 12 hr after HBO-treatment, reached a maximum at 18 hr and was not detectable at 36 hr.

A protective role for glutathione-dependent reduction of dehydroascorbic acid in lens epithelium.

Purpose: In view of the antioxidant role of ascorbic acid and the glutathione redox cycle in the lens, the authors have studied the relationship of the cycle to reduction of the oxidized product of ascorbic acid, dehydroascorbic acid (DHA), in lens epithelium.

Methods: Cultured dog lens epithelial cells and intact rabbit lenses were exposed to various concentrations of DHA in experiments performed at 20 degrees C to minimize hydrolysis of the compound (t1/2 of 5 minutes at 37 degrees C). Levels of glutathione (GSH) and oxidized glutathione (GSSG) were measured in lens cells and whole lens epithelial by electrochemical detection.

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.

Occurrence of glutathione-modified aldose reductase in oxidatively stressed bovine lens.

The optimization of an affinity chromatography method on Matrex Orange resin allowed the separation of glutathione modified and native aldose reductase in crude extracts of bovine lens. The analysis of hyperbaric oxygen treated lenses revealed the formation in the intact cultured lens of an enzyme form displaying affinity column binding properties, specific activity, sensitivity to inhibition and susceptibility to activation by thiol reducing agents, all comparable to glutathione modified aldose reductase. The extent of the enzyme modification increased with the time of the oxidative treatment and was maximal in the lens nucleus.