1.The effective concentration and exposure time of mitomycin-C for the inhibition of lens epithelial cell proliferation in rabbit eyes.
Yong Sung YOU ; Seong Jung LIM ; Hak Sung CHUNG ; Young Gee LEE ; Chan Yun KIM ; Young Jae HONG
Yonsei Medical Journal 2000;41(2):185-189
The proliferation of residual lens epithelial cells following cataract surgery is assumed to be a major cause of posterior capsular opacification. To assess the efficacy of mitomycin-C in preventing posterior capsular opacification, we determined the effective concentration and exposure time of mitomycin-C in inhibiting rabbit lens epithelial cell proliferation. The fourth-passaged rabbit lens epithelial cells were maintained for one day and then exposed to mitomycin-C for 1, 2, 3, and 5 minutes, respectively. There were 9 different plating concentrations of mitomycin-C with two-fold serial dilution. The maintenance of the phenotypic properties of lens epithelial cells was confirmed by continuous transcription of lambda-crystalline mRNA determined by reverse transcription-polymerase chain reaction and the polymorphism of the restriction fragment. Cell proliferation was assayed with 3H-thymidine incorporation into DNA. The fourth-passaged cells maintained the expression of lambda-crystalline mRNA, suggesting that they are phenotypically authentic lens epithelial cells. The effective concentrations and exposure time of mitomycin-C were 0.1 mg/ml for 1 minute and 2 minutes, and 0.025 mg/ml for 2 minutes. By these results, we postulated that mitomycin-C at relatively short incubation times could be clinically used for prevention of posterior capsular opacification after cataract surgery.
Animal
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Antibiotics, Antineoplastic/pharmacology*
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Cell Division/drug effects
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Cells, Cultured
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DNA/biosynthesis
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Dose-Response Relationship, Drug
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Epithelial Cells/drug effects
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Epithelial Cells/cytology
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Lens, Crystalline/drug effects*
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Lens, Crystalline/cytology
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Mitomycin/pharmacology*
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Rabbits
;
Time Factors
2.Effects of ecdysterone on the expression of NF-kappaB p65 in H2O2 induced oxidative damage of human lens epithelial cells.
Chun-Yan FENG ; Xiu-Rong HUANG ; Ming-Xin QI
Chinese Journal of Integrated Traditional and Western Medicine 2012;32(1):76-79
OBJECTIVETo study the effects of ecdysterone (ECR) on the expression of nuclear factor (NF)-kappaB in H2O2 induced oxidative damage of human lens epithelial cells (HLECs).
METHODSThe cultured HLECs were divided into 5 groups, i.e., the control group, the H2O2 group, the beta-estradiol (E2) group, the ECR group, and the pyrrolidine dithiocarbamate group (PDTC) group. The expression rate of NF-kappaB p65 in the HLECs were detected by flow cytometer (FCM).
RESULTSThe expression of NF-kappaB p65 occurred in normal HLECs (9. 53%). The expression rate of NF-kappaB p65 in the H2O2 group obviously increased (39.87%, P < 0.01). The expression rate of NF-kappaB p65 in the PDTC group obviously decreased (5.90%, P < 0.01). The expression rates of NF-kappaB p65 in the ECR group (13.99%) and the E2 group (25.18%) ranged between the control group and the H2O2 group, but still lower than that of the H2O2 group (P < 0.01).
CONCLUSIONSThe activation of NF-kappaB in the HLECs could be induced by H2O2 ECR with the estrogenic activity could effectively inhibit the activation of NF-kappaB.
Cells, Cultured ; Ecdysterone ; pharmacology ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Hydrogen Peroxide ; adverse effects ; Lens, Crystalline ; cytology ; Oxidative Stress ; drug effects ; Transcription Factor RelA ; metabolism
3.Safety of intravitreally injected ciprofloxacin in phakic rabbit eyes.
Sang Ha KIM ; Jae Hong KIM ; Kyung Shin CHO ; Jung Sik KWAK
Korean Journal of Ophthalmology 1995;9(1):12-18
This study was designed to determine the maximal safe drug concentration of intravitreal ciprofloxacin in phakic rabbit eyes. Twenty-two eyes of New Zealand pigmented rabbits received midvitreal ciprofloxacin of 100, 200, 400, 600 or 800 microgram in BSS Plus, or BSS Plus only. Retinal toxicity was dose-dependent as determined with electroretinography, light microscopy, and transmission electron microscopy. At a dose of greater than 400 microgram, disorganization of the outer segments was a main pathological finding in transmission electron microscopy. We evaluated retinal function by measuring the electroretinograms for a graded series of flash intensities and by fitting electroretinogram b-wave amplitudes to the Naka-Rushton equation. At a dose of greater than 600 microgram, Rmax was significantly decreased and log K was significantly increased. N-value tended to decrease. A decrease of b-wave amplitudes caused by retinal toxicity could be detected very sensitively with lower luminance stimuli. Determination of retinal toxicity with lower luminance electroretinography revealed a significant decrease of b-wave amplitudes at a dose of greater than 400 microgram. We concluded that a safe dose of intravitreal ciprofloxacin in phakic rabbit eyes was 200 microgram in phakic eyes.
Animals
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Ciprofloxacin/administration & dosage/*toxicity
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Dose-Response Relationship, Drug
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Electroretinography/drug effects
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Injections
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Lens, Crystalline
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Photic Stimulation
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Rabbits
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Retina/*drug effects/pathology/physiopathology
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Rod Cell Outer Segment/drug effects/pathology
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Vitreous Body
4.Mitochondrial proteomic analysis of isopsoralen protection against oxidative damage in human lens epithelial cells.
Chun-Yan FENG ; Xiu-Rong HUANG ; Ming-Xin QI ; Song-Wen TANG ; Yan-Hong HU ; Sheng CHEN ; Fa-Jie KE
Chinese journal of integrative medicine 2012;18(7):529-533
OBJECTIVETo investigate the protective effects of the natural medicinal monomer isopsoralen (ISR) with estrogenic activity against oxidative damage in human lens epithelial cells B3 (HLE-B3) caused by hydrogen peroxide (H(2)O(2)) and to pursue the possible mitochondrial proteomic regularity of the protective effects.
METHODSHLE-B3 cells were treated with H(2)O(2) (300 μ mol/L), β-estradiol (E(2): 10(-8) mol/L) and H(2)O(2), ISR (10(-5) mol/L) and H(2)O(2), or left untreated. Altered expressions of all mitochondrial proteins were analyzed by protein array and surfaceenhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). The mass/charge (m/z) ratios of each peak were tested by the Kruskal-Wallis rank sum test, and the protein peak value of the m/z ratio for each treatment by pair comparison was analyzed with the Nemenyi test.
RESULTSH(2)O(2) up-regulated the expressions of two protein spots (with m/z of 6532 and 6809). E(2) mitigated the oxidative damage, and the expression of one protein spot (m/z 6532) was down-regulated. In contrast, ISR down-regulated both of protein spots (m/z 6532 and 6809).
CONCLUSIONSISR could effectively inhibit H(2)O(2)-induced oxidative damage in HLE-B3 cells. The protein spot at m/z of 6532 might be the target spot of ISR against oxidative damage induced by H(2)O(2).
Cell Line ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Estradiol ; pharmacology ; Furocoumarins ; pharmacology ; Humans ; Hydrogen Peroxide ; toxicity ; Lens, Crystalline ; pathology ; Mitochondria ; metabolism ; Oxidation-Reduction ; drug effects ; Oxidative Stress ; drug effects ; Protective Agents ; pharmacology ; Proteome ; metabolism ; Proteomics ; methods
5.Effect of cinobufagin on the expressions of bcl-2 mRNA and bax mRNA and the proliferation of lens epithelial cells.
Chinese Journal of Integrated Traditional and Western Medicine 2009;29(10):915-917
OBJECTIVETo investigate the effect of cinobufagin on the proliferation of lens epithelial cells (LECs) and the bcl-2 and bax mRNA expressions of rabbits.
METHODSCultured LECs were treated for 72 h with cinobufagin of different concentrations, the end titer was 0.1 mg/L for low, 0.2 mg/L for moderate and 0.3 mg/L for high concentration, respectively. The inhibitory rate of cinobufagin on LECs' proliferation was detected using MTT method; the mRNA expressions of bcl-2 and bax genes in LECs were examined using reverse transcription-polymerase chain reaction (RT-PCR).
RESULTSProliferation of LECs was inhibited significantly by cinobufagin in a concentration dependent manner, the inhibitory rate was 24.65%, 30.13% and 36.98% respectively for low, moderate and high concentration. With the drug concentration increasing, the mRNA expression levels of the pro-apoptotic bax were increased, whereas those of the anti-apoptotic bcl-2 decreased.
CONCLUSIONCinobufagin can remarkably inhibit the proliferation and induce the apoptosis of LECs in vitro, it might be taken as one of the drugs for the prevention and treatment of after-cataract.
Animals ; Apoptosis ; drug effects ; Bufanolides ; pharmacology ; Cell Proliferation ; drug effects ; Epithelial Cells ; drug effects ; metabolism ; Lens, Crystalline ; cytology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; genetics ; Rabbits ; bcl-2-Associated X Protein ; metabolism
6.Protective Effect of Catechin on Apoptosis of the Lens Epithelium in Rats with N-methyl-N-nitrosourea-induced Cataracts.
Sung Min LEE ; Il Gyu KO ; Sung Eun KIM ; Dong Hee KIM ; Byung Nam KANG
Korean Journal of Ophthalmology 2010;24(2):101-107
PURPOSE: To investigate the effect of catechin on apoptotic cell death in the lens epithelium of rats with cataract. METHODS: Cataract was induced by intraperitoneal injection of 100 mg/kg N-methyl-N-nitrosourea (MNU) to ten day-old Sprague-Dawley rats. The neonatal rats were randomly divided into five groups (n=15 in each group): a control group, and four cataract-induction groups, treated with either 0, 50, 100, 200 mg/kg catechin. We performed slit-lamp biomicroscopic analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, Western-blot for Bcl-2 and Bax, and immunohistochemistry for caspase-3. RESULTS: Apoptotic cell death in lens epithelial cells that increased following cataract formation in rats was suppressed by cathechin. CONCLUSIONS: Catechin inhibited cataract-induced apoptotic cell death in the lens epithelium and may prove useful for the prevention of cataract progression.
Analysis of Variance
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Animals
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Animals, Newborn
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Apoptosis/*drug effects
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Blotting, Western
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Caspase 3/metabolism
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Cataract/chemically induced/*drug therapy
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Catechin/*pharmacology
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Immunoenzyme Techniques
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In Situ Nick-End Labeling
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Lens, Crystalline/*drug effects
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Random Allocation
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Rats
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Rats, Sprague-Dawley
7.Accumulation of argpyrimidine, a methylglyoxal-derived advanced glycation end product, increases apoptosis of lens epithelial cells both in vitro and in vivo.
Junghyun KIM ; Ohn Soon KIM ; Chan Sik KIM ; Eunjin SOHN ; Kyuhyung JO ; Jin Sook KIM
Experimental & Molecular Medicine 2012;44(2):167-175
The formation of advanced glycation end products (AGEs) has been considered to be a potential causative factor of injury to lens epithelial cells (LECs). Damage of LECs is believed to contribute to cataract formation. The purpose of this study was to investigate the cytotoxic effect of AGEs on LECs both in vitro and in vivo. We examined the accumulation of argpyrimidine, a methylglyoxal-derived AGE, and the expression of apoptosis-related molecules including nuclear factor-kappaB (NF-kappaB), Bax, and Bcl-2 in the human LEC line HLE-B3 and in cataractous lenses of Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes. In cataractous lenses from twenty-one-week-old ZDF rats, LEC apoptosis was markedly increased, and the accumulation of argpyrimidine as well as subsequent activation of NF-kappaB in LECs were significantly enhanced. The ratio of Bax to Bcl-2 protein levels was also increased. In addition, the accumulation of argpyrimidine triggered apoptosis in methylglyoxal-treated HLE-B3 cells. However, the presence of pyridoxamine (an AGEs inhibitor) and pyrrolidine dithiocarbamate (a NF-kappaB inhibitor) prevented apoptosis in HLE-B3 cells through the inhibition of argpyrimidine formation and the blockage of NF-kappaB nuclear translocalization, respectively. These results suggest that the cellular accumulation of argpyrimidine in LECs is NF-kappaB-dependent and pro-apoptotic.
Animals
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Apoptosis/*drug effects
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Cell Line
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Epithelial Cells/*cytology/*drug effects
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Glycosylation End Products, Advanced/*pharmacology
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Lens, Crystalline/*cytology
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Male
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Ornithine/*analogs & derivatives/pharmacology
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Pyrimidines/*pharmacology
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Pyruvaldehyde/*chemistry
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Rats
8.IGF-1 Counteracts TGF-beta-Mediated Enhancement of Fibronectin for in Vitro Human Lens Epithelial Cells.
So Hyang CHUNG ; Sun Ah JUNG ; Young Jae CHO ; Joon H LEE ; Eung Kweon KIM
Yonsei Medical Journal 2007;48(6):949-954
PURPOSE: To determine whether insulin-like growth factor (IGF-1) affects transforming growth factor (TGF-beta)- mediated fibronectin accumulation in human lens epithelial cell line (HLE B-3) cells. MATERIALS AND METHODS: HLE B-3 cells were incubated for 24 hours with TGF-beta (10ng/ mL), IGF-1 (10ng/mL), or both. Expression of the fibronectin gene was determined using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Fibronectin levels were examined using western blot analysis and immunofluorescence staining. RESULTS: Expression of the fibronectin gene was not different between the TGF-beta/IGF-1 treated group and the TGF-beta treated group (p= 0.116). However, western blot analysis demonstrated decreased fibronectin levels in human lens epithelial cells treated with TGF-beta and IGF-1 compared to those treated with TGF-beta only (p < 0.01). Immunofluorescence staining disclosed inhibition of TGF-beta-induced fibronectin in the presence of IGF-1. CONCLUSION: This study suggests that IGF-1 counteracts TGF-beta-mediated fibronectin accumulation in human lens epithelial cells.
Cell Line
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Epithelial Cells/cytology/*drug effects/metabolism
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Fibronectins/*genetics/metabolism
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Fluorescent Antibody Technique
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Gene Expression Regulation/drug effects
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Humans
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Insulin-Like Growth Factor I/*pharmacology
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Lens, Crystalline/cytology
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Reverse Transcriptase Polymerase Chain Reaction
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Transforming Growth Factor beta/*pharmacology
9.Study on anti-cataract effect of gigantol combined with syringic acid and their mechanism.
Hongxing DIAO ; Yanqun YI ; Hui QI ; Xinxin GAO ; Hua FANG ; Xiaoyong WEI ; Qiong GU ; Ling WANG ; Zhengtao WANG ; Lianquan GU
China Journal of Chinese Materia Medica 2012;37(16):2429-2434
OBJECTIVETo study the anti-cataract effect of gigantol combined with syringic acid and their action mechanism.
METHODH202-induced lens oxidative injury in vitro rat model was establish to observe the impact of gigantol combined with syringic acid on lens transparency under a dissecting microscope. D-galactose-induced cataract rat model was established to observe the impact of gigantol combined with syringic acid on lens transparency under a slit-lamp. UV spectrophotometry was adopted to detect the inhibitory activity of gigantol combined with syringic acid against AR. Molecular docking method was used to detect binding sites, binding types and pharmacophores of gigantol combined with syringic acid in prohibiting aldose reductase.
RESULTBoth in vitro and in vivo experiments showed a good anti-sugar cataract activity in the combination of gigantol and syringic acid and a better collaborative effect than single component-gigantol and syringic acid and positive control drug Catalin. Molecular docking and dynamic simulation showed their collaborative AR-inhibiting amino acid residue was Asn160 and the major acting force was Van der Waals' force, which formed common pharmacophores.
CONCLUSIONGigantol combined with syringic acid shows good anti-cataract, their action mechanism is reflected in their good collaborative inhibitory effect on AR.
Aldehyde Reductase ; antagonists & inhibitors ; Animals ; Bibenzyls ; Cataract ; drug therapy ; enzymology ; Drug Synergism ; Female ; Gallic Acid ; analogs & derivatives ; pharmacology ; Guaiacol ; analogs & derivatives ; pharmacology ; Humans ; In Vitro Techniques ; Lens, Crystalline ; drug effects ; enzymology ; Male ; Rats ; Rats, Wistar
10.Puerarin decreases lens epithelium cell apoptosis induced partly by peroxynitrite in diabetic rats.
Li-Na HAO ; Yi-Qun LING ; Xiu-Mei LUO ; Yu-Xiang MAO ; Qi-Yan MAO ; Shou-Zhi HE ; Yi-Ling LING
Acta Physiologica Sinica 2006;58(6):584-592
The present study was designed to observe if puerarin decreases lens epithelium cell (LEC) apoptosis induced partly by peroxynitrite (ONOO(-)). One hundred and eight rats were randomly divided into control group (n=36), streptozotocin (STZ) group (n=36) and STZ + puerarin group (n=36). The rats in the control group intraperitoneally (i.p.) received 0.5 ml of saline. The rats in STZ group and STZ + puerarin group received intraperitoneal injection of STZ (45 mg/kg). Three days later, the rats in STZ + puerarin group were given puerarin (140 mg/kg per day, i.p.). On days 20, 40 and 60 of the experiment, morphologic changes of lenses were observed with slit lamp. Then the animals were sacrificed for further analysis. The amount and percentage of apoptotic LECs were determined by flow cytometry. Nitrotyrosine (NT, the foot print of ONOO(-)) was examined by immunohistochemistry. Apoptosis-related genes (iNOS, etc.) were analyzed by gene array. The results showed that in the control group, all the lenses were clear. In STZ group, gradually severe opacity of the lens was observed on days 20, 40 and 60. But in STZ + puerarin group, mild opacity of the lens was observed on day 20 and more severe on day 40, but markedly decreased on day 60. In the control group, mild apoptosis of LECs was observed. In STZ group, time-dependent increase in apoptosis of LECs was observed. In STZ + puerarin group, mild apoptosis of LECs was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of NT in the lens in the control group, but an increased expression of NT in STZ group. In STZ + puerarin group, mild expression of NT was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of iNOS in the lens in the control group, but continuous up-regulation of iNOS expression in STZ group. In STZ + puerarin group, mild expression of iNOS was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. Except the changes of iNOS related to NO production, the other apoptosis-related genes, including BCL-2 and SOD were down-regulated, while NF-kappaB and TNFR1-FADD-caspase signal transduction way were up-regulated in STZ group. The results were opposite in STZ + puerarin group and the control group. These findings show that NT is expressed in diabetic rat lens, which proves that LEC apoptosis in diabetic lens is partly induced by ONOO(-) which may be a new oxidative damage way to form cataract. Puerarin partly decreases LEC apoptosis induced by ONOO(-) and is a potential medicine for therapy of diabetic cataract. The mechanism of puerarin dealing with diabetic cataract may be related to its direct inhibition of LEC apoptosis and antagonism of ONOO(-) in diabetic rats.
Animals
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Apoptosis
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Cataract
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chemically induced
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Diabetes Mellitus, Experimental
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Epithelial Cells
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drug effects
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Isoflavones
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pharmacology
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Lens, Crystalline
;
cytology
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Nitric Oxide Synthase Type II
;
metabolism
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Peroxynitrous Acid
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Rats
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Tyrosine
;
analogs & derivatives
;
metabolism