Acta Ophthalmol. 2010: 88: 891–895
Purpose: This study examines the levels of oxidative damage in patients with cataract.
Methods: Blood samples were collected from 60 patients with cataract and 60 age- and gender-matched healthy individuals to measure 8-hydroxy 2-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) levels.
Results: A significant difference was observed in leukocyte 8-OHdG levels in patients with cataract in comparison with healthy persons (p < 0.001). Similarly, a significant difference was observed in plasma MDA levels in patients with cataract in comparison with healthy persons (p < 0.001). In addition, a significant correlation was found between levels of 8-OHdG in leukocyte DNA and plasma MDA (r = 0.859, p < 0.001).
Conclusion: This study measured the oxidative DNA damage by measuring the 8-OHdG in the leukocyte DNA in patients with cataract. In addition, the level of MDA – a marker for lipid peroxidation – was measured to determine lipid peroxidation.
Cataract is the opacification of the eye lens, and is the leading cause of blindness worldwide (Watkins 2002). The onset of cataract occurs at least 20 years earlier in developing countries than in the developed world (Hashim & Zarina 2007). Cataract formation has been associated with various risk factors such as age, sex, social class, ultraviolet radiation (UVR), smoking and diabetes mellitus (DM). Despite higher incidence of cataract formation in diabetic patients, old age has been considered by far the greatest risk factor (Spector 1995). Although loss of transparency is generally assumed to be an implication of the normal ageing process, it has been argued recently that the changes within the lens associated with ageing and cataract are distinct and identify oxidative stress as a distinctive property (Truscott 2005). Free radicals are produced continually, mainly by mitochondrial electron transport enzymes such as xanthine oxidase and aldehyde oxidase, and as a result of inflammation, xenobiotic metabolism and hyperoxia (Martinez-Cayuela 1995). The free-radical theory of ageing holds that reactive oxygen species (ROS) cause oxidative damage over the life of the patient. The cumulative and potentially increasing amount of accumulated damage accounts for the dysfunctions and pathologies seen in normal ageing (Irshad & Chaudhuri 2002).
The formation of ROS by photosensitizing mechanisms may be caused by exposure to light. Oxidants are regenerated slowly by many tissues (such as lens tissue), causing an increase in the risk for an accumulation of oxidant-inflicted damage in the tissue components. The lens contains a series of defence mechanisim that may protect it from the harmful effects of oxidations. But the lens has a restricted protein turnover, especially in the nucleus (Ateş et al. 2004). If this defence is breached, oxidative damage may increase.
Previous studies report that exposing lens proteins to UVR or even sunlight in the presence or absence of aromatic amino acids generates modified proteins, which are insoluble, coloured and fluorescent (Zigman et al. 1973). Because crystallins and other proteins in lens fibre do not regenerate and must serve the lens for the lifetime of the person, the lens requires more protection than most tissues from oxidative damage (Alberti et al. 1996).
As an individual ages, abnormally high levels of ROS caused by over-production/inadequate removal may damage cellular proteins, nucleic acids and membrane lipids (Zoric 2003; Williams 2008). Lipid peroxidation products are formed because of ROS-mediated oxidation of cell membrane lipids. Lipid peroxides, a well- established mechanism of cellular injury in humans, are used as an indicator of oxidative damage in cells and tissues. Lipid peroxides are unstable and decompose to form a complex series of compounds, including reactive carbonyl compound, among which malondialdehyde (MDA) is the most abundant (Ohira et al. 2008). Therefore, MDA levels are used widely as an indicator of lipid peroxides (Bonnefoy et al. 2002; Irshad & Chaudhuri 2002).
ROS cause strand breaks in DNA and base modifications, including the oxidation of guanine residues into 8-hydroxy 2-deoxyguanosine (8-OHdG). Thus, 8-OHdG can serve as a sensitive biomarker of oxidative DNA damage (Loft et al. 1993). Recent studies have reported significantly increased levels of 8-OHdG, a specific marker of oxidative damage to DNA in the lymphocytes of patients with type I and type II DM and diseases associated with inflammatory processes and ageing, as well as drugs and carcinogens (Floyd 1990; Dong et al. 2008). In another study, levels of oxidative DNA lesions in biological fluids (e.g. urine, serum, cerebrospinal fluid) and tissues have been reported to be reliable biomarkers of oxidative stress (Kadiiska et al. 2005).
This study evaluates oxidative DNA damage by measuring 8-OHdG in the leukocyte DNA of patients suffering from cataract caused by ageing. In addition, the concentration of MDA – a marker for lipid peroxidation – was measured to determine the extent of lipid peroxidation.
Materials and Methods
The study involved 60 patients with cataract (37 men, 23 women; age range 53–76 years) who attended the ophthalmology clinic for extracapsular cataract surgery and 60 healthy control individuals (30 men, 30 women; age range 55–73 years) who applied to our clinic for an annual check-up. Patients with thyroid function disorders, hypertension, DM, liver, smoking and renal dysfunction, anaemia, osteoporosis and inflammatory arthritis were identified carefully and excluded from the study. Patients with cataract formation secondary to identifiable causes, such as DM, trauma and steroid administration, were also excluded.
After enrolment, all the patients were given a detailed lens examination to determine the updated cataract status. In the examination, the Lens Opacities Classification System II (Chylack et al. 1989), which uses photographic standards to grade cataract type and severity, was used to grade lens status with slit-lamp examination. The patients were classified into pure (nuclear, cortical and posterior subcapsular) and mixed types, depending on the lens status of both eyes. Pure cases had a single type of opacity. The patients with more than one type of opacity were classified as mixed. A patient qualified as a control if: (i) both pupils could be dilated to at least 6 mm and (ii) both lenses were graded as having no opacities or having grade II or less opacities. The patients were informed about the study and they consented to giving blood samples. Venous peripheral blood samples (10 ml) were collected. Samples were stored at −80°C until biochemical analysis.
Isloation and hydrolization of DNA
DNA isolation from blood was performed according to (Miller et al. 1988) with some modifications. Blood (2 ml) with ethylene diamine tetraacetic acid (EDTA) was mixed with 3 ml of erythrocyte lysis buffer, and incubation for 10 min in ice was followed by centrifugation (10 min at 1500 × g). The supernatant was decanted, and the pellet was resuspended thoroughly in sodium dodecyl sulfate (10%, v/v), proteinase K (20 mg/ml) and 1.9 ml leukocyte lysis buffer (4m NaCI, 0.5 m EDTA). The mixture was incubated at 65°C for 1 hr and then mixed with 0.8 ml of 9.5 m ammonium acetate. After centrifugation at 1500 × g for 25 min, the clear supernatant (2 ml) was transferred to a new sterile tube and DNA was precipitated by the addition of 4 ml ice-cold absolute ethanol. DNA samples were dissolved in Tris EDTA buffer (10 mm, pH 7.4) and then hydrolized according to Shigenaga’s method (Shigenaga et al. 1994).
Analysis of 8-OHdG and dG by high-performance liquid chromatography
In the hydrolysed DNA samples, 8-OHdG and dG levels were measured using high-performance liquid chromatography (HPLC) with electrochemical (HPLC-ECD) and variable wavelength detector (HPLC-UV) systems as described previously (Donald 1998). Before analysis with HPLC, the hydrolized DNA samples were redissolved in the HPLC eluent (final volume 1 ml). Final hydrolysate (20 μl) was analysed by HPLC-ECD (HP, Agilent 1100 modular systems with HP 1049A ECD detector; Agilent, Waldbronn, Germany); column, reverse phase-C18 (RP-C18) analytical column (250 mm × 4.6 mm × 4.0 μm; Phenomenex, Torrance, California, USA). The mobile phase consisted of 0.05 m potassium phosphate buffer (pH 5.5) containing acetonitrile (97:3, v/v) with a flow rate of 1 ml/min. The dG concentration was monitored based on absorbance (245 nm) and 8-OHdG based on the electrochemical reading (600 mV). Levels of dG and 8-OHdG were quantified using the standards of dG and 8-OHdG from sigma; the level of 8-OHdG level was expressed as the number of 8-OHdG molecules per 106 dG (Kasai 1997; Tarng et al. 2000).
Analysis of plasma for MDA by HPLC
MDA concentrations in blood plasma sample were measured by HPLC with fluorescent detection (HPLC-FLD), as described previously (Khoschsorur et al. 2000). Briefly, 50 μl of plasma sample was mixed with 0.44 m H3PO4 and 42 mm tiobarbituric acid (TBA), and incubated for 30 min in a boiling water bath. After cooling rapidly on ice, an equal volume of alkaline methanol was added to the sample, shaken vigorously, centrifuged (3000 × g for 3 min) and the aqueous layer removed. Then, 20 μl supernatant was analysed by HPLC (HP, Agilent 1100 moduler systems with FLD detector); column, RP-C18 (5 μm, 4.6 × 150 mm) (Eclipse VDB-C18; Agilent); elution, methanol (40:60, v/v) containing 50 mm KH2PO4 buffer (pH 6.8); flow rate, 0.8 ml/min. Fluorometric detection was performed with excitation at 527 nm and emission at 551 nm. The peak of the MDA-TBA adduct was calibrated as a 1, 1, 3, 3- tetraethoxypropane standard solution, carried out in exactly the same process as with the plasma sample.
Statistical analyses were performed using spss for Windows (version 11.5; SPSS Inc., Chicago, Illinois, USA). The statistical significance was calculated using the one-way anovatest. A p-value of < 0.05 was considered statistically significant. All the results were expressed as mean scores with their standard deviation (mean ± SD).
Of all the patients, 61% were male; 50% of the control group was male. The mean age of the patient group was 65.5 years; the mean age of the control group was 61.4 years. The age and gender distributions of the patient and control groups did not differ significantly (p > 0.05). All the persons in the patient group had bilateral cataract. The distribution of the types of cataracts was as follows: cortical cataract in 15 patients (25.0%), mixed-type cataract in 15 patients (25.0%), nuclear cataract in 15 patients (25.0%) and posterior subcapsular cataract in 15 patients (25.0%).
The level of 8-OHdG in leukocyte DNA of the patients (2.4413 ± 0.6072/106 dG) was higher than that of the control group (1.0684 ± 0.3542/106 dG) (p < 0.001) (Fig. 1). The mean level of plasma MDA of the patient group (1.7151 ± 0.5458 μm) was higher than that of the control group (0.6213 ± 0.2283 μm) (p < 0.001) (Fig. 2). There was also a correlation between the levels of 8-OHdG in leukocyte DNA and plasma MDA (about 25% of the patients) (r = 0.543, p ≤ 0.001). The level of 8-OHdG in leukocyte DNA and plasma MDA levels of the four different cataract types are shown in Table 1. There was no significant difference between the different cataract subgroups when the level of 8-OHdG in leukocyte DNA and plasma MDA levels were compared (p > 0.05).
|Variables (mean ± SD)||Cortical cataract (n = 15)||Nuclear cataract (n = 15)||Posterior subcapsular cataract (n = 15)||Mixed cataract (n = 15)||p-value|
|8-OHdG / 106dG||1.8208 ± 0.6063||1.8863 ± 0.6927||1.6914 ± 0.5||1.4757 ± 0.2256||*|
|MDA (μm)||2.7379 ± 0.4173||2.0654 ± 0.4507||1.9108 ± 0.3935||2.9945 ± 0.4512||*|
Oxygen-free radicals and antioxidant systems have been held responsible for pathological processes in the eye, including glaucoma, age-related macular degeneration, uveitis, retinopathy of prematurity, corneal inflammation and keratitis (Alio et al. 1995; Beatty et al. 2000; Dani et al. 2004; Takagi 2007). Oxidative mechanisms are commonly believed to play an important role in the aetiology of cataract, especially maturity-onset cataract (Babizhayev & Costa 1994). Studies using in vitro models have shown oxidative stress to cause cataract (Spector et al. 1993). In many types of cataracts – including maturity-onset cataract – proteins, lipids and DNA are damaged extensively (Spector 1984; Kleiman & Spector 1993; Babizhayev et al. 2004). Oxidative stress has been correlated with high levels of lipid peroxidation products such as MDA in cataract patients (Micelli-Ferrari et al. 1996; Hashim & Zarina 2007). In the present study, the higher MDA concentrations detected in the cataract patients are similar to the findings of earlier reports (Micelli-Ferrari et al. 1996; Bhatia et al. 2006; Hashim & Zarina 2007).
In this study, the possibility of DNA damage resulting from oxidative damage-induced lipid peroxidation was also investigated. Because DNA damage is repaired well by cellular enzymes – unlike the oxidation damage to other biomolecules that are not repaired and/or have a slow turnover, such as lipids or proteins – the measurement of DNA damage shows the level of oxidative damage clearly (Krapfenbauer et al. 1998). ROS causes strand breaks in DNA and base modifications including the oxidation of guanine residues to 8-OHdG. Therefore, 8-OHdG can be a sensitive biomarker of oxidative DNA damage (Giugliano et al. 1996; Krapfenbauer et al. 1998).
Previous studies have reported significantly raised levels of a specific marker of oxidative damage to DNA – 8-OHdG – in the lymphocytes of patients with type I and type II DM and diseases associated with inflammatory processes and ageing, as well as drugs and carcinogens (Troll & Wiesner 1985; Holmes et al. 1992; Collins et al. 1996; Giugliano et al. 1996).
We confirmed a significantly higher level of leukocyte 8-OHdG in the patients with cataract than in the control patients. These data suggest that 8-OHdG levels are a potentially useful marker of oxidative DNA damage in cataract patients. Furthermore, in a recent study Charissou et al. (2004) examined the relationship between cellular and genomic oxidative damages in freshwater bivalves in realistic conditions of exposure in the field. They showed that oxidative damage leading to lipid peroxidation may be accompanied by an increase in oxidative DNA damage. Oxidative DNA damage was evaluated in terms of 8-OHdG in the eyes of glaucoma patients.
The levels of 8-OHdG were significantly higher in glaucoma patients than in controls; therefore, it was concluded that oxidative damage might represent an important pathogenetic step in primary open-angle glaucoma. Because it could induce human trabecular meshwork degeneration, favouring an intraocular pressure increase, it could prime the glaucoma pathogenetic cascade (Izzotti et al. 2003).
In a more recent study, oxidative damage parameters were measured in type 2 DM with or without retinopathy and the relationship between oxidative damage and patients with type 2 diabetic retinopathy was investigated. The authors found that the concentrations of MDA and 8-OHdG in type II DM are significantly higher than those of control patients and that the concentration of MDA and 8-OHdG in patients with retinopathy is significantly elevated compared with that of patients with DM without retinopathy, indicating severe lipid peroxidation, protein oxidation and oxidative DNA damage in diabetes. Thus, the authors speculated that oxidative damage in diabetes might contribute to the pathogenesis of DM and play an important role in the development of diabetic retinopathy (Pan et al. 2008).
In our study, we found an overall elevation of leukocyte DNA 8-OHdG in patients with cataract. Strikingly, it was correlated with our other marker: higher MDA concentrations were also found in patients with cataract. This study supports the hypothesis of cataract as an oxidative disorder, in accordance with previous studies on this subject. But further studies on this interesting field of research are required in order to support the exact role of oxidant stress in the development of cataract formation.