3-nitrotyrosine and Cataract

To quantify the levels of nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine in cataractous lenses of smokers and smokers who chewed tobacco in comparison with non-smokers and non-smokers who chewed tobacco.. A total of 80 cataractous lenses from smokers, non-smokers, smokers with tobacco chewing habit, and non-smokers with tobacco chewing habit were collected from the patients who had enrolled in the Department of Ophthalmology, Mahatma Gandhi Medical College & Research Institute, Puducherry.. Levels of nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine were quantified using commercially available enzyme-linked immunosorbent assay kits.. The mean concentrations of lens nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine are as follows: (a) smokers-112.01, 59.57, and 88.91 µmol/L; (b) smokers who chewed tobacco-175.15, 93.95, and 128.72 µmol/L; (c) non-smokers-76.15, 40.65, and 70.20 µmol/L; and (d) non-smokers who chewed tobacco-96.56, 52.87, and 83.88 µmol/L, respectively.. Nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine at high levels are the major causative agents for cataractogenesis. The results of this study suggest that smoking and tobacco chewing habit generate nitrosative stress that could enhance the pathogenesis for early cataractogenesis.

Topics: Cataract; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lens, Crystalline; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrosative Stress; Non-Smokers; Smokers; Smoking; Tyrosine

Oxidative and nitrosative stress underlies cataractogenesis, and therefore, various antioxidants have been investigated for anticataract properties. Several vitamin E analogs have also been studied for anticataract effects due to their antioxidant properties; however, the anticataract properties of tocotrienols have not been investigated. In this study, we investigated the effects of topically applied tocotrienol on the onset and progression of cataract and lenticular oxidative and nitrosative stress in galactosemic rats.. In the first part of this study, we investigated the effects of topically applied microemulsion formulation of tocotrienol (TTE) using six concentrations ranging from 0.01% to 0.2%. Eight groups of Sprague-Dawley rats (n = 9) received distilled water, vehicle, or one of the six TTE concentrations as pretreatment topically twice daily for 3 weeks while on a normal diet. After pretreatment, animals in groups 2-8 received a 25% galactose diet whereas group 1 continued on the normal diet for 4 weeks. During this 4-week period, topical treatment continued as for pretreatment. Weekly slit-lamp examination was conducted to assess cataract progression. At the end of the experimental period, the animals were euthanized, and the proteins and oxidative stress parameters were estimated in the lenses. In the second part of the study, we compared the anticataract efficacy of the TTE with the liposomal formulation of tocotrienol (TTL) using five groups of Sprague-Dawley rats (n = 15) that received distilled water, TTE, TTL, or corresponding vehicle. The mode of administration and dosing schedule were the same as in study 1. Weekly ophthalmic examination and lens protein and oxidative stress estimates were performed as in study 1. Lens nitrosative stress was also estimated.. During the 4-week treatment period, the groups treated with 0.03% and 0.02% tocotrienol showed slower progression of cataract compared to the vehicle-treated group (p<0.05), whereas the group treated with 0.2% tocotrienol showed faster progression of cataract compared to the vehicle-treated group (p<0.05). The lenticular protein content, malondialdehyde, superoxide dismutase, and catalase levels were normalized in the groups that received 0.03% and 0.02% tocotrienol. The lenticular reduced glutathione also showed a trend toward normalization in these groups. In contrast, the group treated with 0.2% tocotrienol showed increased lenticular oxidative stress. When the microemulsion and liposomal formulations were compared, the effects on cataract progression, lens oxidative and nitrosative stress, and lens protein content did not show significant differences.. Topically applied tocotrienol within the concentration range of less than 0.05% and more than 0.01% tends to delay the onset and progression of cataract in galactose-fed rats by reducing lenticular oxidative and nitrosative stress. However, topical tocotrienol at a concentration of 0.2% and higher aggravates cataractogenesis in galactose-fed rats by increasing lens oxidative stress. The anticataract efficacy of 0.03% microemulsion of tocotrienol did not differ from its liposomal formulations at the same concentration.

Topics: Administration, Topical; Animals; Anterior Eye Segment; Catalase; Cataract; Disease Progression; Emulsions; Eye Proteins; Galactosemias; Glutathione; Lens, Crystalline; Liposomes; Malondialdehyde; Nitric Oxide Synthase Type II; Oxidation-Reduction; Particle Size; Rats; Rats, Sprague-Dawley; Static Electricity; Stress, Physiological; Superoxide Dismutase; Tocotrienols; Tyrosine; Viscosity

The Na⁺-H⁺-exchanger-1 (NHE-1) controls intracellular pH and glycolytic enzyme activities, and its expression and activity are increased by diabetes and high glucose. NHE-1-dependent upregulation of the upper part of glycolysis, under conditions of inhibition (lens) or insufficient activation (retina) of glyceraldehyde 3-phosphate dehydrogenase, underlies diversion of the excessive glycolytic flux towards several pathways contributing to oxidative stress, a causative factor in diabetic cataractogenesis and retinopathy. This study evaluated the role for NHE-1 in diabetic cataract formation and retinal oxidative stress and apoptosis. Control and streptozotocin-diabetic rats were maintained with or without treatment with the NHE-1 inhibitor cariporide (Sanofi-Aventis, 10 mgkg-1d-1) for 3.5 months. In in vitro studies, bovine retinal pericytes and endothelial cells were cultured in 5 or 30 mM glucose, with or without 10 µM cariporide, for 7 days. A several-fold increase of the by-product of glycolysis, α-glycerophosphate, indicative of activation of the upper part of glycolysis, was present in both rat lens and retina at an early (1-month) stage of streptozotocin-diabetes. Cariporide did not affect diabetic hyperglycemia and counteracted lens oxidative-nitrative stress and p38 MAPK activation, without affecting glucose or sorbitol pathway intermediate accumulation. Cataract formation (indirect ophthalmoscopy and slit-lamp examination) was delayed, but not prevented. The number of TUNEL-positive cells per flat-mounted retina was increased 4.4-fold in diabetic rats (101 ± 17 vs. 23 ± 8 in controls , P<0.01), and this increase was attenuated by cariporide (45 ± 12, P<0.01). Nitrotyrosine and poly(ADP-ribose) fluorescence and percentage of TUNEL-positive cells were increased in pericytes and endothelial cells cultured in 30 mM glucose, and these changes were at least partially prevented by cariporide. In conclusion, NHE-1 contributes to diabetic cataract formation, and retinal oxidative-nitrative stress and apoptosis. The findings identify a new therapeutic target for diabetic ocular complications.

Topics: Aldehydes; Animals; Apoptosis; Blood Glucose; Blotting, Western; Cataract; Cattle; Diabetes Complications; Extracellular Signal-Regulated MAP Kinases; Fasting; Guanidines; In Situ Nick-End Labeling; Lens, Crystalline; Male; Nitrosation; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Retina; Sodium-Hydrogen Exchangers; Sulfones; Tyrosine

The pathophysiology of the early events leading to diabetic retinopathy is not fully understood. It has been suggested that Inflammatory processes are involved in the development of the disease; however, the concentrations of tissue retinal inflammatory mediators and their possible alteration in diabetic retinopathy have not been described. The aim of this work was to study T-helper cell cytokine and chemokine profiles, and tyrosine nitration in retinal tissue of diabetic rats.. Cytokines (interleukin IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-10, TNFa, GM-CSF, IFN-g), chemokines (MIP-1a, MIP-2, MIP-3a, MCP-1, GRO/KC, RANTES, Fractalkine), and tyrosine nitration were measured in retinal homogenate obtained from Long-Evans rats after 5 months of experimental diabetes.. The T-helper type 1 cytokines IL-2 and INF-gamma, in addition to NO production (measured as nitrotyrosine), were found to be significantly elevated in diabetic rat retina homogenates. None of the other cytokines and chemokines studied were affected by the diabetic condition.. Immunoregulatory cytokines belonging to the Th-1 group (IL-2 and IFN-gamma) were increased in the retina of experimental diabetic rats. Moreover, the nitrotyrosine formation (as an expression of increased NO production) was significantly elevated in the diabetic retina, supporting the concept of an inflammatory element in the development of diabetic retinopathy.

Topics: Animals; Cataract; Chemokines; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Disease Models, Animal; Interferon-gamma; Interleukin-10; Interleukin-1alpha; Interleukin-1beta; Interleukin-2; Interleukin-4; Interleukin-6; Interleukin-8; Male; Nitric Oxide; Rats; Rats, Long-Evans; Retina; T-Lymphocytes, Helper-Inducer; Tumor Necrosis Factor-alpha; Tyrosine

This study was aimed at evaluating the potent and specific aldose reductase inhibitor fidarestat, on diabetes-associated cataract formation, and retinal oxidative-nitrosative stress, glial activation, and apoptosis. Control and streptozotocin-diabetic rats were treated with or without fidarestat (16 mg kg(-1)d(-1)) for 10 weeks after an initial 2-week period without treatment. Lens changes were evaluated by indirect ophthalmoscopy and portable slit lamp. Nitrotyrosine, poly(ADP-ribose), and glial fibrillary acidic protein expression were assessed by immunohistochemistry. The rate of apoptosis was quantified in flat-mounted retinas by TUNEL assay with immunoperoxidase staining. To dissect the effects of high glucose exposure in retinal microvascular cells, primary bovine retinal pericytes and endothelial cells were cultured in 5 or 30 mM glucose, with or without fidarestat (10 microM) for 3-14 days. Apoptosis was assessed by TUNEL assay, nitrotyrosine and poly(ADP-ribose) by immunocytochemistry, and Bax and Bcl-2 expression by Western blot analyses. Fidarestat treatment prevented diabetic cataract formation and counteracted retinal nitrosative stress, and poly(ADP-ribose) polymerase activation, as well as glial activation. The number of TUNEL-positive nuclei (mean +/- SEM) was increased approximately 4-fold in diabetic rats vs. controls (207+/-33 vs. 49+/-4, p<0.01), and this increase was partially prevented by fidarestat (106+/-34, p<0.05 vs. untreated diabetic group). The apoptotic cell number increased with the prolongation of exposure of both pericytes and endothelial cells to high glucose levels. Fidarestat counteracted nitrotyrosine and poly(ADP-ribose) accumulation and apoptosis in both cell types. Antiapoptotic effect of fidarestat in high glucose-exposed retinal pericytes was not associated with the inhibition of Bax or increase in Bcl-2 expression. In conclusion, the findings, i) support an important role for aldose reductase in diabetes-associated cataract formation, and retinal oxidative-nitrosative stress, glial activation, and apoptosis, and ii) provide a rationale for the development of aldose reductase inhibitors, and, in particular, fidarestat, for the prevention and treatment of diabetic ocular complications.

Topics: Aldehyde Reductase; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Blood Glucose; Blotting, Western; Cataract; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Glial Fibrillary Acidic Protein; Imidazolidines; Immunohistochemistry; In Situ Nick-End Labeling; Neuroglia; Ophthalmoscopy; Oxidative Stress; Poly(ADP-ribose) Polymerases; Rats; Retina; Streptozocin; Tyrosine

Cataracts is considered be formed because of an abnormal glucose metabolic pathway or oxidative stress. We explored the damaging role of ONOO- and antagonism of cholecystokinin octapeptide-8 (CCK-8) in diabetic cataractal rat lenses.. A diabetic cataractal animal model was established by peritoneal injection of streptozotocine (STZ). Thirty-six normal SD rats were taken as control group; seventy-two were given STZ (45 mg/kg) and then divided into STZ group and CCK-8 group (peritoneal injection CCK-8). STZ induced diabetic rats were treated with CCK-8 for 60 days. Lenses were examined with slit lamp at 20, 40 and 60 days. Immunofluorescent staining and Western blot analysis were used for determining nitrotyrosine (NT, a marker for ONOO-). PT-PCR and gene array analysis were used for determining the expression of inducible nitric oxide synthetase mRNA (iNOS mRNA) in lens epithelium (LEC).. STZ group rats developed lens opacity by 20 days that reached a high level by 60 days after STZ injection. CCK-8 group rats delayed the cataract formation. CCK-8 group rats delayed the cataract formation. There was no distinct expression of NT and iNOS mRNA in control group. In STZ group, there were distinct expression of NT and upregulation of iNOS mRNA; however, CCK-8 group showed weak expression of NT and downregulation of iNOS mRNA.. NT, which may be a new form of oxidative stress, was expressed in diabetic rat LEC although CCK-8 could reverse NT damage in LEC. The results suggested that CCK-8 might be a useful therapeutic agent against diabetic cataract. The antagonizing mechanism of CCK-8 may be related to direct antagonism of ONOO- as well as its inhibition of the expression of iNOS mRNA for production of NO and therefore decrease in the formation of ONOO-.

Topics: Animals; Blotting, Western; Cataract; Diabetes Mellitus, Experimental; Fluorescent Antibody Technique; Male; Nitric Oxide Synthase Type II; Oxidation-Reduction; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sincalide; Streptozocin; Tyrosine

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 b

Topics: Animals; Apoptosis; Cataract; Diabetes Mellitus, Experimental; Epithelial Cells; Isoflavones; Lens, Crystalline; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Rats; Tyrosine