Influence of soy aglycon isoflavones on bone-related traits and lens protein characteristics of ovariectomized rats and bioactivity performance of osteoprogenitor cells.

Health benefits of soy isoflavones have attracted the concern of the public and the interest of health-care professionals. In this study, two trials were conducted in characterizing bone-related traits and lens proteins as affected by supplementation of soy aglycon isoflavones (SAI). In trial 1, an in vivo study, 20 Sprague-Dawley rats were ovariectomized (OVX) and randomly distributed into OVX and OVX+SAI (135 mg of SAI/kg of feed; 8.33 mg/kg body weight; 2.5 mg/day) groups. Another group containing 10 rats with a sham operation was control (Sham). The experiment period was 3 months, and the rats were subjected to bone-related traits and lens protein characterization. In trial 2, an in vitro study, osteoprogenitor cells (UMR-106) were divided into SAI-supplemented (0.5 mg of SAI/mL of medium) and unsupplemented groups. Results of the in vivo study indicated that daily BW gains in the OVX and OVX+SAI groups were greater than that of the Sham group (p < 0.05). Bone ash and Ca contents of the Sham and OVX+SAI groups were higher than those of the OVX group (p < 0.05), while bone density, strength, and phosphorus contents among groups varied insignificantly (p > 0.05). When the lens proteins were extracted and analyzed with size-exclusion HPLC, the contents of beta- and gamma-crystallins were lowest in the OVX group and the protein solubility decrease could be recovered by dietary SAI supplementation (shown by OVX+SAI group). Based on Raman spectra of the isolated lens proteins, disulfide bonds were observed more in OVX lens than in the Sham and OVX+SAI lens. Results of in vitro study with osteoprogenitor cells revealed that cell viability, alkaline phosphatase activity, osteocalcin, and Ca contents of the SAI-supplemented group were higher than those of the unsupplemented group (p < 0.05). The likely potency to enhance bone and lens health by SAI supplementation is worth pointing out.

Differential protein expression in lens epithelial whole-mounts and lens epithelial cell cultures.

PURPOSE: Lens fibergenesis is a problem in several types of cataract and in the posterior capsular opacification following cataract surgery. To correct improper fiber differentiation or to prevent unwanted growth on the posterior capsule following cataract surgery requires a thorough understanding of normal and abnormal fiber formation. To this end, studies were initiated to characterize fiber differentiation in the bovine lens and in lens epithelial cell cultures. METHODS: Indirect immunofluorescence and immunoblot analysis were employed to study the expression of vimentin, beta-crystallin, gamma-crystallin, filensin, aquaporin 0 and the Na, K-ATPase catalytic subunit isoforms (alpha1, alpha2, alpha3) in bovine lens epithelium whole-mounts as well as lens epithelial cell cultures propagated in medium containing 10% bovine serum or in medium supplemented with bovine serum concentrations < or =4%. RESULTS: Three distinct cell types were observed in the bovine lens epithelium. The cells of the central zone were identified by a polarized distribution of two distinct Na, K-ATPase catalytic subunit isoforms, alpha1 to the apical (fiber side) and alpha3 to the basal (aqueous humor side) membranes. Lateral to the polarized central zone, was the germinative zone of cells, best characterized by perinuclear vimentin basket-like structures and the loss of polarized Na, K-ATPase catalytic subunit isoforms. Lateral to the germinative zone were the cells of the transition zone (meridinal rows) where expression of the lens specific proteins beta-crystallin, gamma-crystallin, filensin and aquaporin 0 as well as the lens fiber-, adipocyte- and brain glia-specific Na, K-ATPase catalytic subunit, alpha2 are expressed. The cultured cells propagated in medium supplemented with 10% serum bore no resemblance to any of the cells of the bovine lens epithelium whole-mounts. The cells propagated in the medium supplemented with the lower bovine serum levels resembled the differentiating fibers of the transition zone of the bovine lens epithelium whole-mounts as well as superficial cortical fibers. CONCLUSIONS: Since the low-serum lens epithelial cell cultures bear a remarkable resemblance to early differentiating fibers, they are reasonable models for the study of early fiber differentiation or prevention of differentiation. The culture conditions employed do not yield the polarized cells of the central zone. Nor has the function of these polarized cells in lens fluid, nutrient and ion homeostasis been determined.

The presence of a human UV filter within the lens represents an oxidative stress.

It has recently been demonstrated that, with age, UV filters such as 3-hydroxykynurenine glucoside, bind to proteins in the human lens. This covalent interaction leads to colouration of the normal lens, and results from the instability of the kynurenine side chain. Other primate UV filters, in addition to containing the same side chain, can also be readily oxidized. One such compound is 3-hydroxykynurenine (3OHKyn). It has been proposed that oxidation of bound and/or free UV filters, such as 3OHKyn may give rise to the lens colouration associated with age-related nuclear cataract. Therefore it has become important to understand the oxidation of 3OHKyn within the lens. In this study, intact bovine lenses (which lack UV filters) were incubated with 3OHKyn and various lens parameters monitored. The effect of exposure to hyperbaric oxygen (HBO) was also assessed, both alone, and in combination with prior 3OHKyn incubation. Glutathione (GSH), protein sulfhydryl and protein-bound sulfhydryl levels, as well as soluble protein content and gel filtration profiles, were obtained for cortical and nuclear regions after defined periods of incubation. The presence of the primate UV filter, 3OHKyn, at concentrations similar to those present in the human lens, was shown to produce considerable oxidative stress within the lens, as judged by its effect on GSH. This effect was noted under normobaric conditions, but was exacerbated by increased oxygen. Exposure of lenses to HBO caused a marked fall in GSH in cortical and nuclear regions. This effect was exaggerated in the presence of 3OHKyn. HBO treatment also lead to a fall in protein sulfhydryl content, however, this was only partial (approximately 1 mol SH per mol protein) and changed only slowly, even with extended periods of exposure to HBO, suggesting that most crystallin sulfhydryl groups may be buried. 3OHKyn did not appreciably affect this oxidation although it did cause an increase in the level of protein-bound sulfhydryl. HBO treatment produced a more than two-fold increase in protein-bound sulfhydryl content in the cortex. There was little influence of 3OHKyn alone on protein solubility, even with extended periods of incubation, however, incubation for 72 hr in the presence of HBO caused a significant increase in insoluble protein particularly in the nucleus. This insolubilization was further increased in the presence of 3OHKyn. FPLC profiles showed that the proportion of gamma and beta crystallins in the soluble fraction decreased following HBO, suggesting that these may be involved in disulfide bond formation. This study demonstrates that a readily oxidized compound, such as the primate UV filter 3OHKyn, represents an oxidative stress within the lens and that such oxidative processes can be exacerbated if the concentration of oxygen within the lens is increased. We speculate that this factor may account for the evolution of unusually high levels of glutathione reductase in human lenses.

Calcium-induced changes on crystallins in organ-cultured porcine lens.

In this study, intact porcine lenses were cultured in vitro for 7 days supplemented with commercial balanced salt solution (BSS) which is usually used as an irrigation solution during intraocular surgery, and the lenses were maintained under various culture conditions, e.g. temperature and CO2 concentration. The intact porcine lenses after 7 days culture were analyzed with optical density scanner, gel permeation chromatography on TSK HM-55 column and SDS-PAGE (polyacrylamide gel electrophoresis). It was found that lenses exhibited the least opacity when lenses were cultured with Ca(+2)-free BSS buffer, CO2-free incubator and maintained at a temperature of 25 degrees C. After the lenses were cultured with Ca(+2)-free BSS or BSS medium, the composition of crystallins in lenses was separated with TSK HM-55 column. It was indicated that the percentage of high molecular weight (HMW) protein and (alpha-crystallin increased, and gamma-crystallin decreased in lenses incubated with BSS medium compared with lenses incubated with Ca(+2)-free BSS medium. Following an increase in the concentration of calcium in the medium from 4.3 mM, 20 mM, 50 mM, 100 mM to 200 mM, the opacity of the lens was measured with a densitometer. The changed percentage of various crystallins was similar to lenses with BSS media that increased in HMW protein and alpha-crystallin, decreasing in gamma-crystallin. In the case of lens protein pattern, the crystallin washed from TSK HM-55 gel was separated with SDS-PAGE (polyacrylamide gel electrophoresis). It was indicated that some of proteins disappeared when lenses were incubated with various concentrations of calcium. The vanished pH proteins were 20.5 kDa at 50 mM calcium, 20.5 kDa and 21 kDa at 100 mM, 20.5 kDa, 21 kDa, 22 kDa and 23 kDa at 200 mM which were compared with the protein bands in the presence of 20 mM calcium in BSS medium. This study indicates that the commercial balanced salt solution (BSS) which is usually used as an irrigating solution during intraocular operations may increase the risk for lens opacity because of the calcium contained in the solution.

Anti-inflammatory effects of vitamin E on experimental lens-induced uveitis.

The anti-phlogistic effect of dietary vitamin E supplementation on the acute inflammation observed in experimental lens-induced uveitis in Brown Norway rats was studied. The effects of vitamin E were examined using histopathologic parameters as well as by measuring the levels of arachidonic acid metabolites. Histologic examination of the eyes revealed that the vitamin E-deficient animals had the most severe destruction of the retina, while those animals receiving the vitamin E-supplemented diet exhibited the best preservation of the retinal architecture. Levels of arachidonic acid metabolites, as determined by radioimmunoassay, were significantly higher in vitamin E deficient rats as compared with rats on a normal diet.

Prevention of selenite cataract by vitamin C.

Studies have been conducted to determine the efficacy of vitamin C in the prevention of cataracts induced by selenite. Administration of the latter to rat pups results in the development of advanced cataracts within 5 days. Treatment with ascorbate had a significant preventive effect. The observations indicate that selenite cataract is due to an oxidative stress to the lens. In addition, the findings are in conformity with our view that ascorbate functions as an anticataractogenic substance.

The decomposition and aggregation of rat lens protein induced by selenite in vitro and in vivo.

A single subcutaneous injection of sodium selenite at the dose of 20 mumol/kg body weight induced bilateral nuclear cataracts in suckling rats. This selenite-induced cataract incidence can be increased by pretreating animals with a glutathione synthesis inhibitor. Sodium dodecyl sulfate polyacrylamide electrophoresis of urea-soluble proteins from selenite-induced cataractous lenses showed the appearance of high molecular weight aggregates and decomposed products of lens proteins. These products were found in association with the emergence of a 45 K band. Incubation of water-soluble lens proteins with selenite in vitro produced changes similar to those demonstrated in selenite-induced cataractous lenses. Furthermore, selenite induced the gradual development of opalescence and the oxidation of sulfhydryl in the lens protein solution. Therefore, we presume that the oxidation of lens protein sulfhydryl by selenite is associated with both aggregate formation and the decomposition of lens proteins, and that these changes may provide a partial explanation for the mechanism of selenite cataract.

Lens crystallins in aqueous and vitreous humor in selenite overdose cataract.

The purpose of this research was to determine if crystallins were released from the lens into the aqueous and vitreous humor in selenite-overdose cataract. At 20 days post injection, aqueous and vitreous were obtained from lenses containing nuclear cataract or "mature" cataract (nuclear and cortical opacities). Humors were analyzed by direct ELISA and immunoblotting using antibodies against lens crystallins. Both types of selenite cataract showed increased alpha-, beta- and gamma-crystallins in aqueous and vitreous. Increases in crystallins in vitreous humor were larger than in aqueous humor, possibly because of slower turnover of vitreous. Extensive membrane damage in selenite cataract probably caused leakage of crystallins from the lens to aqueous and vitreous humors. This model may be useful for studies on the immunologic response of the body to lens crystallins released by cataract.

Selenium-induced cataract--a correlation of dry mass content and light scattering.

Selenium-induced cataracts in young rats were used to compare light scattering and dry mass concentration along the lens axis. Selenium-treated rats and control rats were examined 1, 2, 4 and 6 days after subcutaneous injection of selenium or 0.9% NaCl. The development of nuclear cataract was followed with a Scheimpflug slit-lamp camera. Light-scattering was determined by densitometry of the negatives taken by the Scheimpflug camera. Dry mass concentration was determined by quantitative microradiography of the extracted lenses. Increasing light scatter was seen from day 2 to day 6. The dry mass concentration, however, was not changed until day 6. Two distinct zones were found surrounding the center of the nucleus, one with increased and one with decreased dry mass. The rapid changes in dry mass concentration were probably caused by shifts in water distribution within the lens. Similar zones of hydration have also been found in human senile nuclear cataract. Selenium-induced cataract in the rat is discussed in relation to human nuclear cataract.

State of sulfhydryl in selenite cataract.

A dose of 20 mumol selenite/kg body weight is a potent and a very rapid inducer of cataracts in young rats. We investigated the rate at which physiological concentrations of selenite would catalyze the oxidation of glutathione in vitro and found that selenite was a strong sulfhydryl oxidant. To test if selenite had the same effect in vivo, the oxidation state of five kinds of lenticular sulfur were measured in suckling rats following a cataractous dose of selenite. The measurements included reduced glutathione (GSH), oxidized glutathione (GSSG), protein-bound glutathione ( PSSG ), reduced protein sulfhydryl ( PSH ), and oxidized protein sulfhydryl ( PSSP ). While selenite caused a 44% decrease in lens GSH by 6 days postinjection, there was no concurrent increase in either GSSG or PSSG . Likewise, there was no evidence for increased oxidation of PSH to PSSP . To determine if GSH loss were the cause of the selenite cataracts, we injected normal rats with the glutathione synthesis inhibitor buthionine sulfoximine (BSO). Lens GSH dropped more than 96% by 4 days post-BSO injection; however, no cataracts formed. Thus, selenite cataract does not appear to be caused by extensive sulfhydryl oxidation and cannot be attributed exclusively to GSH loss.

Alterations in the lenticular proteins of rats on riboflavin deficient diet.

Effect of feeding riboflavin deficient diet to rats on lens protein composition was investigated. Total proteins and profile of soluble and insoluble proteins in lenses from rats fed on a riboflavin deficient diet for seven weeks were found to be similar to that of the vitamin supplemented diet. Distribution of high molecular weight protein (above 4 X 10(6) daltons) isolated from the 9,900 g supernatant fraction was found to be significantly higher and gamma crystallin was significantly lower in riboflavin deficient group as compared to the normal lenses. However, the distribution of alpha and beta crystallins was not affected. Gel electrophoresis in sodium dodecyl sulfate of soluble lens proteins from riboflavin deficient animals had lower proportions of polypeptide species with molecular weight above 40,000 daltons while insoluble protein fraction had higher proportions of these polypeptide species as compared to control rats. These data suggest that the composition of lens proteins is altered in riboflavin deficiency.