Li-Ren Lin

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)

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.

Glutaredoxin 2 knockout increases sensitivity to oxidative stress in mouse lens epithelial cells

Glutaredoxin belongs to the oxidoreductase family, with cytosolic glutaredoxin 1 (Grx1) and mitochondrial glutaredoxin 2 (Grx2) isoforms. Of the two isozymes, the function of Grx2 is not well understood. This paper describes the effects of Grx2 deletion on cellular function using primary lens epithelial cell cultures isolated from Grx2 gene knockout (KO) and wild-type (WT) mice. We found that both cell types showed similar growth patterns and morphology and comparable mitochondrial glutathione pool and complex I activity. Cells with deleted Grx2 did not show affected Grx1 or thioredoxin expression but exhibited high sensitivity to oxidative stress. Under treatment with H(2)O(2), the KO cells showed less viability, higher membrane leakage, enhanced ATP loss and complex I inactivation, and weakened ability to detoxify H(2)O(2) in comparison with the WT cells. The KO cells had higher glutathionylation in the mitochondrial proteins, particularly the 75-kDa subunit of complex I. Recombinant Grx2 deglutathionylated complex I and restored most of its activity. We conclude that Grx2 has a function that protects cells against H(2)O(2)-induced injury via its peroxidase and dethiolase activities; particularly, Grx2 prevents complex I inactivation and preserves mitochondrial function.

Crystallins and their synthesis in human lens epithelial cells in tissue culture.

Explants of epithelial cells from young human lenses of 5-12 months of age, obtained from patients who underwent surgery for retinopathy of prematurity, were cultured in Dulbeccos modified Eagles medium supplemented with 20% fetal calf serum. Without exception, every piece of the anterior capsule explant showed cell outgrowth within 48-72 h and resulted in confluent monolayer culture within 2 weeks. From these monolayer cultures, two to three passages of subcultures were obtained by routinely seeding cells in a ratio of 1:4. The doubling times for these human lens epithelium (HLE) cultures during the first 4 weeks of two passages were found to be 24-36 h. In a majority of cultures through the first three passages, more than 12 population doublings were attained. However, no lentoid bodies were formed during this period. These cells were studied for the presence of crystallins and their synthesis. Using SDS-polyacrylamide gel electrophoresis, the presence of alpha- and beta-crystallins was demonstrated in HLE cells through three passages. The amount of alpha-crystallin in the first two passages amounted to nearly 13% of the total protein, but decreased significantly in the third passage. The presence of crystallins was corroborated by antibody reaction to the specific crystallins. Indirect immunofluorescence revealed the presence of actin and vimentin in these cell cultures. The synthesis of crystallins in HLE cultures was shown by the incorporation of [35S]methionine which was time dependent. The crystallin synthesis was found to decrease in third passage when the cell growth slowed down without consistent formation of confluent monolayer. These studies have demonstrated that primary cultures of HLE cells can be successfully grown from young lenses through several passages which continue to express the characteristic crystallins of the epithelial cells.

Study of crystallin expression in human lens epithelial cells during differentiation in culture and in non-lenticular tissues.

Crystallin expression in human lens epithelial cells in culture and a number of non-lenticular tissues was studied by the technique of immunoblotting using monoclonal antibodies. The expression of alpha A, beta 5 and beta 6 crystallins per unit number of cells increased with passage number while alpha B appeared to be constant Lentoid bodies derived from cultured human lens epithelial cells not only expressed gamma-crystallin and MP26 as previously demonstrated, but also produced alpha A, alpha B, beta 5 and beta 6 crystallins. In human non-lenticular tissues including ciliary body, vitreous body, neural retina, cultured retinal pigment epithelial cells and scleral fibroblasts, alpha B-crystallin was detected, but was undetectable in cornea and iris. Alpha A was present only in the lens. These studies demonstrate that HLE cells maintain the ability to synthesize crystallins through several passages. Following differentiation, they not only synthesize gamma-crystallin and MP26 but continue to express alpha- and beta-crystallins similar to differentiated lens fiber cells in vivo. Consistent with previous observations, the expression of alpha B-crystallin does not appear to be specific for the lens.

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, 1.7 mW/cm(2) on the cornea). Eyes were irradiated in the presence of either a UV-blocking senofilcon A contact lens, a minimally UV-blocking lotrafilcon A contact lens, or no contact lens at all. Effects on the cornea and lens were evaluated at various times after exposure. RESULTS Eyes irradiated with no contact lens protection showed corneal epithelial cell loss plus lens epithelial cell swelling, vacuole formation, and DNA single-strand breaks, as well as lens anterior subcapsular opacification. The senofilcon A lens protected nearly completely against the UVB-induced effects, whereas the lotrafilcon A lens showed no protection. CONCLUSIONS The results indicate that use of a senofilcon A contact lens is beneficial in protecting ocular tissues of the rabbit against the harmful effects of UVB light, including photokeratitis and cataract.

Neuroactive steroids protect retinal pigment epithelium against oxidative stress

This study was undertaken to assess whether neuroactive steroids, 17&bgr;-estradiol and dehydroepiandrosterone-sulfate, enhance survival and protect DNA of human retinal pigment epithelial cells challenged by oxidative stress, and to investigate the role of σ1 receptors in the effects of neuroactive steroids. Retinal pigment epithelial cells were treated with various concentrations of neuroactive steroids and then exposed to hydrogen peroxide. Pretreatment with steroids resulted in significant increased viability in a dose-related manner. DNA damage induced by oxidative insult was significantly lower with steroid pretreatment. The effects of 17&bgr;-estradiol and dehydroepiandrosterone-sulfate were antagonized by pretreatment with a σ1 receptor antagonist. The results suggest that neuroactive steroids protect retinal cells from oxidative stress, and that this effect is mediated by σ1 receptors.

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.

Immunohistochemical localization of Na,K-ATPase in the in situ lens, cultured human lens epithelium and lentoid

The localization of Na,K-ATPase in the lens is quite controversial. We explored this problem through immunoelectron microscopic examination of rat and human lens. Unlike previously reported results, we have found that Na,K-ATPase is localized in the basal plasma membrane, but not in the lateral or apical plasma membrane of both rat and human lens epithelium. The lens fiber lacked immunoreaction. Localization of Na,K-ATPase was also investigated in the cultured human lens epithelium and in lentoid. Immunoreaction was detected in the apical (facing the media) plasma membrane of the lens epithelium cultured on the lens capsule, whereas the reaction was observable in both apical and basal plasma membrane of the lens epithelium cultured on the biopore membrane filters. Immunoreaction in lentoid was observed in the surface plasma membrane. These data indicate that the polarized distribution seen in the in situ lens epithelium changes when these cells are cultured, and that Na,K-ATPase in the cultured lens cells including lentoid is located in the plasma membrane which is in contact with the growth media. This change in polarity of Na,K-ATPase distribution in cultured epithelial cells may be dictated by the need to maintain ion homeostasis by extrusion of sodium ions across the cell membrane facing the media.