Oxidative DNA damage in the human trabecular meshwork: clinical correlation in patients with primary open-angle glaucoma (original) (raw)
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Methodology for Evaluating Oxidative DNA Damage and Metabolic Genotypes in Human Trabecular Meshwork
Toxicology Mechanisms and Methods, 2003
In vitro studies have suggested the possible role of oxidative DNA damage in degenerative eye diseases such as glaucoma. We propose a method aimed at evaluating the oxidative molecular damage directly in the human trabecular meshwork (HTM) collected during surgery from patients affected by glaucoma. In the same DNA samples, we evaluated two genes involved in the cellular defense against oxidative stress, the glutathione S-transferase-encoding genes GSTM1 and GSTT1. DNA was extracted, using a high-performance phenol/chloroform procedure, from the HTM collected during surgery from nine glaucoma patients and five controls. Oxidative DNA damage was evaluated by determining the levels of 8-hydroxy-2 -deoxyguanosine (8-OHdG) by means of 32 P postlabeling, thin-layer chromatography, and electronic autoradiography. GSTM1 and GSTT1 polymorphisms were determined by polymerase chain reaction (PCR) and agarose electrophoresis. Sufficient DNA amounts were obtained from all examined specimens. 8-OHdG was detected in all samples, with a level of 4.0 ± 6.5 (mean ± SD) 8-OHdG molecules/10 5 normal nucleotides in the glaucoma patients and a level of 2.6 ± 2.2 in the controls. These results were obtained by using DNA amounts as low as 0.11 µg. The genotype status of GSTM1 and GSTT1 was successfully determined in all patients by analyzing an aliquot of the same DNA used for the 8-OHdG evaluation. This method allows for the use of samples collected from living subjects during ocular surgery in order to study the role of oxidative DNA damage in the pathogenesis of degenerative eye diseases such as glaucoma.
Journal of Advances in Medicine and Medical Research, 2021
Aim: The study aimed to estimate plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, a marker of oxidative deoxyribonucleic acid (DNA) damage, and evaluate their potential association with primary angle-closure glaucoma (PACG) and related clinical markers. Study Design: This is retrospective case-control study. Place and Duration: The study was performed on samples recruited from King Abdulaziz University Hospital, Riyadh, Saudi Arabia between August 2018 through September 2019 in the participants of Saudi origin. Methodology: Plasma 8-OHdG levels were measured in duplicates using automated ELISA analyzer in 43 PACG patients and 45 non-glaucomatous controls. Results: The mean levels of 8-OHdG were 13.59 ng/mL [±4.50] in PACG patients compared to 16.95 ng/mL [±10.66] in controls. The differences did not achieve statistical significance (P = 0.06). Similarly, no significant differences (P = 0.42) were observed in the median levels of 8-OHdG between PACG and controls. Furthermore, ...
Evaluation of oxidative stress markers in pathogenesis of primary open-angle glaucoma
Experimental and Molecular Pathology, 2011
Primary open-angle glaucoma (POAG) is the leading cause of blindness in the industrial countries. It is reported that oxidative stress might be an important risk factor in the pathogenesis of POAG. Forty subjects including 20 patients with open-angle glaucoma (9 men and 12 women, mean age 61.8 ± 12.1 yr) and 20 controls without glaucoma symptoms (9 men and 12 women, mean age 58.1 ± 17.7 yr) were enrolled in our study. The main aim of the work was to evaluate oxidative stress markers in the pathogenesis of open-angle glaucoma. In our work the activity of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) as well as the total antioxidant status (TAS) was estimated. An alkaline comet assay was used to measure DNA damage of strand breaks (SB), oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pirmidines as endonuclease III (Nth) sites. We measured endogenous as well as exogenous DNA damage after 10 μM hydrogen peroxide treatment (H 2 O 2). We did not observe any statistical changes of DNA strand break lesion in examined POAG patients according to healthy subjects (P N 0.05). However, either endogenous (P b 0.01) or exogenous (P b 0.001) levels of oxidative DNA damage in POAG patients were found to be statistically higher than controls. A significant decrease of antioxidant enzymes: CAT (P b 0.001), SOD (P b 0.05), and GPX (P b 0.001) and a non-statistical decrease of TAS status (P N 0.05) in glaucoma patients according to controls were also indicated. In conclusion our data revealed that oxidative stress had a pathogenic role in primary open-angle glaucoma. Therefore, we suggested that the modulation of a pro-oxidant/antioxidant status might be a relevant target for glaucoma prevention and therapy.
Mitochondrial Damage in the Trabecular Meshwork of Patients With Glaucoma
Archives of Ophthalmology, 2010
To analyze the frequency of mitochondrial DNA (mtDNA) damage in patients with primary open-angle glaucoma. Oxidative damage plays a major role in glaucoma pathogenesis. Since no environmental risk factor for glaucoma is recognized, we focused our attention on mitochondria, the main endogenous source of reactive oxygen species. Methods: Mitochondrial damage was evaluated analyzing a common mtDNA deletion by real-time polymerase chain reaction in trabecular meshwork collected at surgery from 79 patients with primary open-angle glaucoma and 156 unaffected matched controls. In the same samples, polymorphisms of genes encoding for antioxidant defenses (GSTM1), repair of oxidative DNA damage (OGG1), and apoptosis (FAS) were tested. Results: Mitochondrial DNA deletion was dramatically increased (5.32-fold; P = .01) in trabecular meshwork of patients with glaucoma vs controls. This finding was paralleled by a decrease in the number of mitochondria per cell (4.83-fold; P Ͻ .001) and by cell loss (16.36-fold; P Ͻ .01). Patients with glaucoma bearing the GSTM1null genotype showed increased amounts of mtDNA deletion and a decreased number of mitochondria per cell as compared with GSTM1-positive subjects. Patients bearing a FAS homozygous mutation showed only a decreased number of mitochondria per cell. Conclusions: Obtained results indicate that mitochondrion is targeted by the glaucomatous pathogenic processes. Some subjects bearing adverse genetic assets are more susceptible to this event. Clinical Relevance: Oxidative damage to the trabecular meshwork exerts a pathogenic role in glaucoma inducing mitochondrial damage and triggering apoptosis and cell loss. This issue may be useful to develop new glaucoma molecular biomarkers and to identify highrisk subjects.
Investigative Opthalmology & Visual Science, 2009
PURPOSE. The anterior chamber of the eye is a highly specialized structure delimited by the cornea, lens, and iris. It contains the aqueous humor, secreted by the ciliary body and drained by the trabecular meshwork. Alteration of aqueous humor homeostasis plays a major role in the pathogenesis of glaucoma. The trabecular meshwork is the target tissue of glaucoma in the anterior chamber, and the development and progression of glaucoma are accompanied by accumulation of oxidative damage in this tissue. This study was conducted to comparatively evaluate the sensitivity to oxidative stress of anterior chamber tissues including the cornea, iris, and trabecular meshwork. METHODS. Cornea, iris, and trabecular meshwork fragments collected from six cornea donors were either left untreated or treated with hydrogen peroxide. Oxidative damage was determined by evaluating nucleotide oxidative modifications (8hydroxy-2Ј-deoxyguanosine) and apurinic alkali-fragile sites by capillary electrophoresis. RESULTS. The results indicated that the basal level of oxidative nucleotide modifications was higher in the cornea than in the iris and trabecular meshwork. The trabecular meshwork was the most sensitive tissue to oxidative damage, as after exposure to hydrogen peroxide both markers of oxidative damage dramatically increased in the trabecular meshwork but not in the cornea and iris. CONCLUSIONS. Because the cornea and iris are directly exposed to light, they possess antioxidant defense mechanisms that are not activated in the trabecular meshwork. The peculiar sensitivity of the trabecular meshwork to oxidative stress is consistent with the damage selectively induced in it, triggering glaucoma's pathogenic cascade.
Genomic and post-genomic effects of anti-glaucoma drugs preservatives in trabecular meshwork
Mutation research, 2015
Oxidative stress plays an important role in glaucoma. Some preservatives of anti-glaucoma drugs, commonly used in glaucoma therapy, can prevent or induce oxidative stress in the trabecular meshwork. The aim of this study is to evaluate cellular and molecular damage induced in trabecular meshwork by preservatives contained in anti-glaucoma drugs. Cell viability (MTT test), DNA fragmentation (Comet test), oxidative DNA damage (8-oxo-dG), and gene expression (cDNA microarray) have been evaluated in trabecular meshwork specimens and in human trabecular meshwork cells treated with benzalkonium chloride, polyQuad, purite, and sofzia-like mixture. Moreover, antimicrobial effectiveness and safety of preservative contents in drugs was tested. In ex vivo experiments, benzalkonium chloride and polyQuad induced high level of DNA damage in trabecular meshwork specimens, while the effect of purite and sofzia were more attenuated. The level of DNA fragmentation induced by benzalkonium chloride was...
Oxidative Stress in Primary Open-angle Glaucoma
Journal of Glaucoma, 2008
Introduction 3. Oxidative stress and the anterior chamber 3.1. General 3.1.1. The aqueous humour acts as a stream of fluid that flushes out free radicals 3.1.2. Water-soluble antioxidants 3.1.3. Fat-soluble antioxidants 3.1.4. Specific enzymes 3.2. Age-related changes 3.3. In primary open-angle glaucoma 4. Oxidative stress and the posterior chamber of the eye 4.1. General 4.1.2. Hormones 4.1.1. Pigments 4.1.3. Water-soluble antioxidants 4.1.4. Fat-soluble antioxidants 4.1.5. Specific enzymes 4.2. Age-related changes 4.3. In primary open-angle glaucoma 5. Systemic markers for oxidative stress in POAG 6. Intensification of oxidative stress in glaucoma due to systemic disease 7. The triggering of immunological processes in glaucoma due to oxidative stress 8. Summary 9. References
Time Course Changes of Oxidative Stress Markers in a Rat Experimental Glaucoma Model
Investigative Ophthalmology & Visual Science, 2010
To evaluate the relationship between oxidative stress markers and increased intraocular pressure in experimental glaucoma. METHODS. In vivo chemiluminescence (CL), total antioxidant capacity (TRAP), nitrite concentration (NC), and lipid peroxidation markers (TBARS) were evaluated. Wistar rats (n ϭ 18 for each time point) underwent operation, and two episcleral veins were cauterized. RESULTS. Decreases of 22%, 35%, and 27% at 7, 15, and 30 days and an increase of 22% at 60 days in CL were observed in glaucomatous eyes. In optic nerve, TBARS values were 6.9 Ϯ 0.5 nmol/mg protein ), 9.4 Ϯ 0.4 nmol/mg protein (15 days), 18.0 Ϯ 1.2 nmol/mg protein (30 days), and 43.1 Ϯ 5.3 nmol/mg protein (60 days) (control, 6.2 Ϯ 0.4 nmol/mg protein; P Ͻ 0.001). NC was 37.0 Ϯ 1.8 M (7 days), 31.4 Ϯ 1.2 M (15 days), 39.6 Ϯ 1.3 M (30 days), and 40.0 Ϯ 1.3 M (60 days) (control, 21.1 Ϯ 1.7 M; P Ͻ 0.001). In glaucomatous vitreous humor, TRAP decreased by 42% at 15 days and 78% at 60 days (control, 414 Ϯ 29 M; P Ͻ 0.001). In glaucomatous aqueous humor, TRAP values were 75 Ϯ 7 M (7 days), 54 Ϯ 4 M (15 days), 25 Ϯ 4 M (30 days), and 50 Ϯ 3 M (60 days) (control, 90 Ϯ 10 M; P Ͻ 0.001). CONCLUSIONS. Reactive species were increased in glaucoma, as evidenced by the increases in CL, TBARS, and NC. The decrease in the antioxidant levels may be a consequence of an increase in oxidative processes. (Invest Ophthalmol Vis Sci.
International Journal of Ophthalmology, 2016
• Toinvestigatethemalondialdehyde (MDA)levels, paraoxonase1 (PON1) activity and 8-hydroxy 2deoxyguanosine(8-OHdG)levelsintheprimaryopenangle glaucoma(POAG)patient.Bloodsamplesfrom52healthy individualsand53patientswithPOAGwereanalyzedfor MDA and 8-OHdG by high-performance liquid chromatography(HPLC)andPON1byspectrophotometry. Thedataobtainedwereanalyzedstatistically.MDAlevels were10.46依8.4and4.70依1.79 滋mol;PON1levelswere121依 39.55and161.62 依60.22U/mL;and8-OHdGvalueswere 1.32 依0.53/10 6 dGand0.47 依0.27/10 6 dGinthePOAG patients and the control group, respectively. The differencewassignificantinMDAlevels,8-OHdGlevels andPON1activityinPOAGpatientsincomparisonwith controls (<0.001).Weconcludedthattheobserved increaseinMDAand8-OHdGlevelsmaybecorrelated withdecreasedPON1activity.Oxidativestressplaysan importantroleinglaucomadevelopment.