Vanita A. Padgaonkar

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)

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. We investigated whether nPG protected cultured lens epithelial cells from H(2)O(2)-induced damage. Cells were treated with H(2)O(2) or with nPG and then H(2)O(2). H(2)O(2) inhibited growth, caused membrane blebbing, decreased lactate production, increased the level of GSSG, decreased the levels of GSH, ATP and NAD(+), and G3PDH activity, stimulated the hexose monophosphate shunt and induced single-strand breaks in DNA. nPG prevented the H(2)O(2)-induced growth inhibition, membrane blebbing, drop in NAD(+) and single-strand breaks in DNA. The mechanism by which nPG acts at the chemical level was investigated using electron paramagnetic resonance (EPR), direct spectrophotometric kinetic measurements, and cyclic voltammetry. When nPG at low concentrations (nM to microM) was mixed with a large excess of O(2)(-)*, the superoxide signal was destroyed as indicated by UV visible spectroscopy and EPR. Kinetic analysis indicated that nPG dismutated O(2)(-)* in repetitive additions of superoxide with little loss of activity. The rate constant for the overall reaction of nPG with O(2)(-)* was ca. 10(6)M(-1)s(-1). nPG had a very low specific binding constant for Fe(2+) as determined by cyclic voltammetry. The evidence indicates that nPG dismutates the superoxide ion in a catalytic manner.

Disulfide cross-linking of urea-insoluble proteins in rabbit lenses treated with hyperbaric oxygen

In vivo exposure of human patients and experimental animals to hyperbaric O2 has been shown by other investigators to lead to opacification of the lens especially in the nuclear region. In the present study, cultured rabbit lenses were treated with hyperbaric O2 in order to investigate possible formation of disulfide-cross-linked proteins in the urea-insoluble fraction of lens cortex and nucleus. When lenses were treated with 100 atmospheres of 100% O2 for 24 hr. intermolecular disulfide-linked proteins formed in both the cortical and nuclear regions. Under these conditions the level of reduced glutathione and the activity of glyceraldehyde-3 phosphate dehydrogenase (G-3PD) were depleted by greater than 95% in both regions. The lenses were hazy in appearance but not opaque. Two-dimensional diagonal electrophoresis followed by immunoblotting indicated that the majority of the cross-linked proteins were beta- and gamma-crystallins. Also involved in the cross-linking was the enzyme G-3PD but not the main intrinsic membrane protein. MIP26 kDa. Treatment of the nuclear urea-insoluble fraction of O2-treated lenses with sodium borohydride showed a nearly fourfold increase in the level of protein disulfide compared to that present in the same fraction of either fresh lenses or N2-treated controls. It was determined that an increase of approximately one disulfide group per 10(5) Da molecular weight corresponded to cross-linking of nearly 20% of the urea-insoluble protein present in the O2-treated lenses. Experiments carried out at 8 atmospheres O2 were used to determine the region of the lens in which urea-insoluble disulfide first formed after exposure to O2. After 8 hr of treatment of lenses with 8 atmospheres O2 an increase in protein disulfide was observed in the urea-insoluble proteins of the lens nucleus but not of the cortex. Under these conditions, the level of glutathione had decreased by 62% in the nucleus compared to only 13% in the cortex. Increasing the culture time to 16 hr under 8 atmospheres O2 produced a further increase in protein disulfide in the nuclear region. The formation of a small amount of cross-linked protein in the cortex and a significantly greater decrease of G-3PD activity in the lens nucleus compared to the cortex. The overall results of the study demonstrate that exposure of lenses to hyperbaric O2 leads to disulfide-cross-linking of crystallins in the urea-insoluble fraction and that the initial formation of protein disulfide as well as the initial loss of glutathione occurs first in the lens nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of H2o2-induced Effects on Cultured Bovine Trabecular Meshwork Cells

The trabecular meshwork is continuously challenged by oxidants that are both present in the aqueous humor and generated within the tissue. In this study we have investigated the antioxidant properties of cultured calf trabecular meshwork cells and evaluated the ability of the compound 4-hydroxy-2,2,6,6-tetramethypiperidine 1-oxyl (TEMPOL), a superoxide dismutase mimic, to prevent H2O2-induced cell damage. The cells were found to possess a high level of reduced glutathione, an undetectable amount of oxidized glutathione, and significant activities of glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and the hexose monophosphate shunt. The cells tolerated a 3-h exposure to a maintained, physiological level of H2O2 (0.02 mM); however, if the activity of glutathione reductase was inhibited, the same level of peroxide caused damage as indicated by cell contraction and blebbing. At a level of 0.05 mM H2O2, added to the medium as a single pulse, the shunt was stimulated eightfold and there were no significant effects on growth or morphology. However, a level of 0.1 mM H2O2 overwhelmed the antioxidant capability of the cells and produced severe effects. Treatment of the cells with TEMPOL prevented H2O2-induced inhibition of growth, formation of single-strand breaks in DNA, activation of the DNA-repair enzyme poly-ADP-ribose polymerase, and decrease in NAD, but TEMPOL was not able to prevent other changes such as the loss of GSH, decrease in glyceraldehyde-3-phosphate dehydrogenase activity, and stimulation of the shunt. Thus, certain intracellular effects of H2O2 in trabecular cells were shown to be caused directly by H2O2 whereas others were mediated through metal-catalyzed free radical reactions. The results indicate the presence of significant antioxidant activity in trabecular meshwork cells with a major contribution provided by the glutathione redox cycle.

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.