Evaluation of oxidative DNA damage and antioxidant defense in patients with nasal polyps

Abstract

Objectives

The presence of inflammatory cells indicates the development of epithelial cell injury in nasal polyposis (NP) and the potential for production of high levels of reactive oxygen and nitrogen species. The aim of our study was to clarify the role of oxidative stress and antioxidant status in the deterioration accompanying NP.

Methods

Twenty patients (11 men) aged 47.2 ± 17.0 years with nasal polyps were included in the study. Twenty healthy subjects (7 men) aged 48.2 ± 15.3 years formed the control group. The erythrocyte activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and plasma nitric oxide (NO) concentrations were measured. An alkaline comet assay was used to determine the extent of blood lymphocyte DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites.

Results

A significant increase of NO (P < 0.05) and non-significant decreases of SOD (P > 0.05), CAT (P > 0.05), and GPx (P > 0.05) were seen in NP patients compared to healthy controls. The level of blood lymphocyte oxidative DNA damage in NP patients was significantly higher compared to the control group (P = 0.01).

Discussion

The blood lymphocyte DNA damage level increased in patients with NP. Elevated DNA damage may be related to overproduction of reactive oxygen and nitrogen species and/or decreased antioxidant protection.     

Intervention of astaxanthin against cyclophosphamide-induced oxidative stress and DNA damage: A study in mice

Astaxanthin, a natural and nutritional red carotenoid pigment, is used as a dietary supplement. The intention of the present study was to investigate the beneficial effects of dietary pigment astaxanthin, against cyclophosphamide-induced oxidative stress and DNA damage. The end points of evaluation of the study included: (a) malondialdehyde, glutathione and superoxide dismutase concentration in liver to detect oxidative stress; (b) normal and modified alkaline comet assays (the latter includes lesion-specific enzymes formamidopyrimidine-DNA glycosylase and endonuclease-III) to detect normal and oxidative stress-induced DNA damage by cyclophosphamide in the mouse bone marrow and the peripheral blood lymphocytes. In addition, micronucleus assay and chromosomal aberration test capable of detecting the DNA damage were also carried out in peripheral blood and bone marrow of mice. Cyclophosphamide (100 mg/kg intra-peritoneal) treatment led to significant increase in liver malondialdehyde and decreased the antioxidant enzymes glutathione and superoxide dismutase. Further, cyclophosphamide also significantly increased the DNA damage as observed from normal and modified comet assays as well as micronucleus and chromosomal aberration assay. Pre-treatment with astaxanthin (12.5, 25 and 50 mg/kg/day for 5 days per oral) resulted in the restoration of oxidative stress markers such as malondialdehyde, glutathione and superoxide dismutase in liver. The amelioration of oxidative stress with astaxanthin pre-treatment correlated well with the decreased DNA damage as evident from normal and modified alkaline comet assays of bone marrow cells and peripheral blood lymphocytes. Further astaxanthin pre-treatment also reduced the frequency of chromosomal breakage and micronucleus formation in the mouse bone marrow cells and peripheral blood reticulocytes. It is thus concluded that pre-treatment with astaxanthin attenuates cyclophosphamide-induced oxidative stress and subsequent DNA damage in mice and it can be used as a chemoprotective agent against the toxicity of anticancer drug cyclophosphamide.     

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.     

Resveratrol and ascorbic acid prevent DNA damage induced by cryopreservation in human semen

Cryopreservation of human semen can cause DNA damages, which compromise the fertilization and normal embryo development. The present study showed that the antioxidant resveratrol prevents these damages both in fertile and infertile men. The addition of ascorbic acid before cryopreservation can reduce DNA damages only in infertile men. Although further studies are needed, the present work showed that resveratrol could be considered in human cryopreservation procedures to avoid/minimize DNA damages and preserve sperm integrity.     

Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.     

DNA damage evaluated by the comet assay in lymphocytes of children with Cs internal contamination caused by the Chernobyl accident

The comet assay is one of the most versatile and popular tools for evaluating DNA damage. Its sensitivity to low dose radiation has been tested in vitro, but there are limited data showing its application and sensitivity in chronic exposure situations. The influence of the internal contamination caused by the Chernobyl accident on the level of DNA damage was evaluated by the comet assay on lymphocytes of 56 Ukrainian children. The study was performed during 2003 on children with demonstrable ¹³⁷Cs internal contamination caused by food consumption. The children were selected for the study immediately after a ¹³⁷Cs whole body counter measurement of internal contamination. The minimal detectable amount of ¹³⁷Cs was 75 Bq. The control group included 29 children without detectable internal contamination, while in the exposed group 27 children with measured activity between 80 and 4037 Bq and committed effective dose between 54 and 3155 μSv were included. Blood samples were taken by a finger prick. The alkaline version of the comet assay was used, in combination with silver stained comets and arbitrary units (AU), for comet measurement. Factors such as disease, medical treatment, surface contamination of children's living location, etc., were considered in the study. Non-significant differences (p > 0.05) in DNA damage in control (9.0 ± 5.7 AU) versus exposed (8.5 ± 4.8 AU) groups were found. These results suggest that low doses of ¹³⁷Cs internal contamination are not able to produce detectable DNA damage under the conditions used for the comet assay in this study. Further studies considering effects of high exposure should be performed on chronically exposed people using this assay.     

Measuring oxidative damage to DNA and its repair with the comet assay

Background

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases.

Scope of review

With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells.

Major conclusions

There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay.

General significance

In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Carbohydrate restriction does not change mitochondrial free radical generation and oxidative DNA damage

Many previous investigations have consistently reported that caloric restriction (40%), which increases maximum longevity, decreases mitochondrial reactive species (ROS) generation and oxidative damage to mitochondrial DNA (mtDNA) in laboratory rodents. These decreases take place in rat liver after only seven weeks of caloric restriction. Moreover, it has been found that seven weeks of 40% protein restriction, independently of caloric restriction, also decrease these two parameters, whereas they are not changed after seven weeks of 40% lipid restriction. This is interesting since it is known that protein restriction can extend longevity in rodents, whereas lipid restriction does not have such effect. However, before concluding that the ameliorating effects of caloric restriction on mitochondrial oxidative stress are due to restriction in protein intake, studies on the third energetic component of the diet, carbohydrates, are needed. In the present study, using semipurified diets, the carbohydrate ingestion of male Wistar rats was decreased by 40% below controls without changing the level of intake of the other dietary components. After seven weeks of treatment the liver mitochondria of the carbohydrate restricted animals did not show changes in the rate of mitochondrial ROS production, mitochondrial oxygen consumption or percent free radical leak with any substrate (complex I- or complex II-linked) studied. In agreement with this, the levels of oxidative damage in hepatic mtDNA and nuclear DNA were not modified in carbohydrate restricted animals. Oxidative damage in mtDNA was one order of magnitude higher than that in nuclear DNA in both dietary groups. These results, together with previous ones, discard lipids and carbohydrates, and indicate that the lowered ingestion of dietary proteins is responsible for the decrease in mitochondrial ROS production and oxidative damage in mtDNA that occurs during caloric restriction.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.     

Melatonin does not prevent the protection of ischemic preconditioning in vivo despite its antioxidant effect against oxidative stress

Free radicals are involved in the protective mechanism of preconditioning (PC), whereas antioxidant compounds abolish this benefit. Melatonin is a hormone with antioxidant properties. The aim of our study was to evaluate the effect of melatonin on infarct size in ischemic preconditioning in vivo. We randomly divided 33 male rabbits into four groups and subjected them to 30 min of myocardial ischemia and 3 h of reperfusion with the following prior interventions: (i) no intervention, (ii) iv melatonin at a total dose of 50 mg/kg, (iii) PC with two cycles of 5 min ischemia and 10 min reperfusion, and (iv) combined melatonin and PC. In a second series of experiments, another antioxidant agent N-acetylcysteine (NAC) was used in a control and in a PC group. Myocardial infarct size was determined and blood samples were drawn at different time points for the determination of lipid peroxidation products, total superoxide dismutase (SOD) activity, and (1)H-NMR spectra to evaluate the changes in the metabolic profile. Melatonin showed no effect on myocardial infarct size in the group of sustained ischemia (42.9 +/- 3.6% vs 47.4 +/- 4.9%) and it did not attenuate the reduction of myocardial infarct size in the PC group (13.6 +/- 2.4% vs 14.0 +/- 1.7%). A similar effect was found in NAC-treated groups (44.8 +/- 3.4% vs 14.3 +/- 1.3%). Lipid peroxidation product levels were significantly elevated in the control and PC groups, whereas melatonin decreased them in both groups. The SOD activity was enhanced in the PC group compared to controls; melatonin kept SOD activity unchanged during ischemia/reperfusion and enhanced its activity when it was combined with PC. Melatonin did not change the metabolic profile of the control and PC groups. Melatonin does not prevent the beneficial effect of ischemic PC on infarct size despite its antioxidant properties.     

Investigation of inflammation,oxidative stress,and DNA damage in COVID-19 patients

COVID-19 disease, which spreads worldwide, is a disease characterized by widespread inflammation and affects many organs, especially the lungs. The resulting inflammation can lead to reactive oxygen radicals, leading to oxidative DNA damage. The pneumonia severity of 95 hospitalized patients with positive RT-PCR test was determined and divided into three groups: mild, moderate, and severe/critical. Inflammation markers (neutrophil–lymphocyte ratio, serum reactive protein, procalcitonin, etc.) were determined, and IL-10 and IFN-γ measurements were analyzed using the enzyme-linked immunosorbent assay method. In evaluating oxidative damage, total thiol, native thiol, disulfide, and ischemia-modified albumin (IMA) levels were determined by measuring spectrophotometrically. The comet assay method’s percentage of tail DNA obtained was used to determine oxidative DNA damage. As a result, when the mild and severe/critical groups were compared, we found that total thiol, native thiol, and disulfide levels decreased significantly in the severe/critical group due to the increase in inflammation markers and cytokine levels (p < 0.05). We could not detect any significance in IMA levels between the groups (p > 0.05). At the same time, we determined an increase in the tail DNA percent level, that is, DNA damage, due to the increased oxidative effect. As a result, we determined that inflammation and oxidative stress increased in patients with severe pneumonia, and there was DNA damage in these patients.     

Phorate triggers oxidative stress and mitochondrial dysfunction to enhance micronuclei generation and DNA damage in human lymphocytes

Herein, we studied phorate for its toxicological effects in human lymphocytes. Phorate treatment for 3 h has induced significant increase in the lymphocytic DNA damage. Compared to control, comet data from highest concentration of phorate (1000 µM) showed 8.03-fold increase in the Olive tail moment (OTM). Cytokinesis blocked micronucleus (CBMN) assay revealed 6.4-fold increase in binucleated micronucleated (BNMN) cells following the exposure with phorate (200 µM) for 24 h. The nuclear division index (NDI) in phorate (200 µM) treated cells reduced to 1.8 vis-à-vis control cells showed NDI of 1.94. Comparative to untreated control, 60.43% greater DCF fluorescence was quantitated in lymphocytes treated with phorate (500 µM), affirming reactive oxygen species (ROS) generation and oxidative stress. Flow cytometric data of phorate (200 µM) treated lymphocytes showed 81.77% decline in the fluorescence of rhodamine 123 (Rh123) dye, confirming the perturbation of mitochondrial membrane potential (ΔΨm). Calf thymus DNA (ct-DNA) treated with phorate (1000 µM) exhibited 2.3-fold higher 8-Hydroxy-2′-deoxyguanosine (8-oxodG) DNA adduct formation, signified the oxidative DNA damage. The alkaline unwinding assay revealed 4.0 and 6.5 ct-DNA strand breaks when treated to phorate and phorate-Cu (II) complex. Overall, the data unequivocally suggests the cyto- and genotoxic potential of phorate in human lymphocytes, which may induce comparable toxicological consequences in persons occupationally or non-occupationally exposed to insecticide phorate.     

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~ 250 μl volumes, at − 80 °C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at − 80 °C, unless a cryopreservative is present. Our “small volume” approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

Assessment of DNA damages in lymphocytes of agricultural workers exposed to pesticides by comet assay in a cross-sectional study

Purpose: To assess the predictive power of the comet assay in the context of occupational exposure to pesticides.

Materials and methods: The recruited subjects completed a structured questionnaire and gave a blood sample. Exposure to pesticides was measured by means of an algorithm based on Dosemeci’s work (Agricultural Health Study). Approximately 50 images were analyzed for each sample via fluorescence microscopy. The extent of DNA damage was estimated by tail moment (TM) and is the product of tail DNA (%) and tail Length.

Results: Crude significant risks (odds ratios, ORs) for values higher than the 75th percentile of TM were observed among the exposed subjects (score?>?1). The frequency of some confounding factors (sex, age and smoking) was significantly higher among the exposed workers. A significant dose–effect relationship was observed between TM and exposure score. Significant high-risk estimates (ORs), adjusted by the studied confounding factors, among exposure to pesticides and TM, % tail DNA and tail length were confirmed using unconditional logistic regression models.

Conclusions: The adjusted associations (ORs) between the comet parameters and exposure to pesticides were significant. The sensitivity of the comet test was low (41%), the specificity (89%) and the predictive positive value (0.77) were found acceptable.     

Antioxidative effects of fluvastatin and its metabolites against oxidative DNA damage in mammalian cultured cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on reactive oxygen species (ROS) and on oxidative DNA damage in vitro, as well as the effects of the main fluvastatin metabolites (M2, M3, and M4) and other inhibitors of the same enzyme, pravastatin and simvastatin. The hydroxyl radical and the superoxide anion scavenging activities of fluvastatin and its metabolites were evaluated using an electron spin resonance spectrometer. Fluvastatin and its metabolites showed superoxide anion scavenging activity in the hypoxanthine-xanthine oxidase system and a strong scavenging effect on the hydroxyl radical produced from Fenton's reaction. Protective effects of fluvastatin on ROS-induced DNA damage of CHL/IU cells were assessed using the single-cell gel electrophoresis assay. CHL/IU cells were exposed to either hydrogen peroxide or t-butylhydroperoxide. Fluvastatin and its metabolites showed protective effects on DNA damage as potent as the reference antioxidants, ascorbic acid, trolox, and probucol, though pravastatin and simvastatin did not exert clear protective effects. These observations suggest that fluvastatin and its metabolites may have radical scavenging activity and the potential to protect cells against oxidative DNA damage. Furthermore, ROS are thought to play a major role in the etiology of a wide variety of diseases such as cellular aging, inflammation, diabetes, and cancer development, so fluvastatin might reduce these risks.     

Assessment of DNA strand breakage by comet assay in diabetic patients and the role of antioxidant supplementation

Diabetes patients often show increased production of reactive oxidative species (ROS) together with vascular complications. The presence of these ROS may lead to increased DNA damage in peripheral blood lymphocytes that may be revealed by the comet assay. To test whether DNA is damaged in diabetes, peripheral blood samples were taken from 30 control individuals and 63 diabetic patients (15 insulin dependent (IDDM) and 48 non-insulin dependent (NIDDM)) and the alkaline comet assay was used to evaluate background levels of DNA damage. Significant differences were detected between control and diabetic patients in terms of frequencies of damaged cells. The extend of DNA migration was greater in NIDDM patients by comparison with IDDM patients which might indicate that IDDM patients are handling more oxidative damage on a regular basis. Smoker individuals had higher frequencies of cells with migration by comparison with the non-smokers in both groups. Also, clear differences between patients on placebo and on Vitamin E supplementation for 12 weeks were observed on the basis of the extend of DNA migration during single cell gel electrophoresis.     

Relationship between DNA damage, total antioxidant capacity and coronary artery disease

OBJECTIVE: It has been known that there was a relation between the levels of DNA damage and the severity of the coronary artery disease (CAD). However, little is known about association of DNA damage with total antioxidant capacity (TAC) and CAD. The aim of this study is to evaluate the relationship between DNA damage, TAC and CAD. METHODS: We used the comet assay to measure DNA damage from 53 patients with angiographically documented CAD and 42 patients with angiographically documented normal coronary vessel. The extent and severity of CAD was calculated to Gensini score index. TAC of plasma was determined using a novel automated measurement method. RESULTS: Mean values of DNA damage were significantly higher in CAD patients than in the control group (p<0.001). There was a positive correlation between Gensini score index and DNA damage (r=0.590, p<0.001). Additionally, significantly positive correlations between score of DNA damage, and diabetes, smoking, obesity and hyperlipidemia were found (p<0.05). There was also a negative correlation between TAC and DNA damage (r=-0.711, p<0.001). The DNA damage was significantly higher in diabetic, smoker, hyperlipidemic and obese individuals than those without these conditions (p=0.001, p=0.006, p=0.001, p=0.004, respectively). CONCLUSIONS: These data indicate that the level of DNA damage is increased and TAC level is decreased in CAD. DNA damage is correlated with the severity of the CAD, and levels of TAC.