The protective effects of vitamin C on the DNA damage, antioxidant defenses and aorta histopathology in chronic hyperhomocysteinemia induced rats

Experimental and Toxicologic Pathology 66 (2014) 407–413

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The protective effects of vitamin C on the DNA damage, antioxidant defenses and aorta histopathology in chronic hyperhomocysteinemia induced rats Murat Boyacioglu a,∗ , Selim Sekkin a , Cavit Kum a , Deniz Korkmaz b , Funda Kiral c , Hande Sultan Yalinkilinc a , Mehmet Onur Ak a , Ferda Akar a a

Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Adnan Menderes University, Isikli, Aydin, Turkey Department of Histology and Embriology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey c Department of Biochemistry, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey b

a r t i c l e

i n f o

Article history: Received 20 January 2014 Accepted 16 June 2014 Keywords: Hyperhomocysteinemia Vitamin C Comet assay Lymphocyt Oxidative stress Rats

a b s t r a c t The aim of this study was to investigate the protective effect of vitamin C towards hyperhomocysteinemia (hHcy) induced oxidative DNA damage using the comet assay. The increase in plasma homocysteine levels is an important risk factor for vascular and cardiovascular diseases through free radical production. This study was also conducted to investigate the histopathological changes in the thoracic aorta and the oxidant/antioxidant status in heart, liver and kidney tissues. Twenty-four adult male Wistar rats were divided as control, hHcy and hHcy + vitamin C group. Chronic hHcy was induced by oral administration of l-methionine (1 g/kg/day) for 28 days. Vitamin C was given 150 mg/kg/day within the specified days. DNA damage was measured by use of the comet assay in lymphocytes. Levels of malondialdehyde (MDA) and glutathione (GSH) as well as catalase (CAT) and superoxide dismutase (SOD) activities were determined in heart, liver and renal tissues. Results show that l-methionine administration significantly increased % Tail DNA and Mean Tail Moment in hHcy group as compared with other groups. Vitamin C treatment significantly decreased the high MDA levels and increased activity of antioxidant enzymes in tissues. Aortic diameter and thickness of aortic elastic laminae were significantly lower in hHcy + vitamin C group. Comet assay can be used for the assessment of primary DNA damage caused by hHcy. Histopathological findings showed that vitamin C may have a preventive effect in alleviating the negative effects of hHcy. Vitamin C might be useful in the prevention of endothelial dysfunction caused by hHcy. © 2014 Elsevier GmbH. All rights reserved.

1. Introduction Homocysteine (Hcy) is a sulfur containing amino acid whose metabolism stands at the intersection of two pathways: remethylation to methionine, which requires folate and vitamin B12 and transsulfuration to cystathionine, which requires vitamin B6 (Selhub, 1999). The exact pathogenesis of hyperhomocysteinemia (hHcy) is controversial, but several mechanisms have been proposed (Ueland et al., 2000; Weiss, 2005). However, Hcy dependent oxidative stress and/or Hcy induced protein structure modifications play an important role in biotoxicity of Hcy (Malinowska et al.,

∗ Corresponding author. Tel.: +90 256 247 07 00; fax: +90 256 247 07 20. E-mail addresses: [email protected], [email protected] (M. Boyacioglu). http://dx.doi.org/10.1016/j.etp.2014.06.004 0940-2993/© 2014 Elsevier GmbH. All rights reserved.

2012). High plasma Hcy concentrations may increase in different pathophysiological conditions (renal failure, rheumatoid arthritis and B-vitamins deficiencies etc.), which is considered as a risk factor for cardiovascular diseases (Linnebank et al., 2011; Mahalle et al., 2013). Regarding these possible mechanisms, different pharmacological agents have been evaluated for the prevention of hHcy in many trials (Noll et al., 2009; Yang et al., 2010; Kolling et al., 2011; Liu et al., 2013). The elevation of plasma Hcy levels may contribute to ischemic changes and oxidative stress. hHcy produces changes in structure and function of blood vessels and oxidative stress appears to play a major role in mediating these changes (Higashi et al., 2009; Osto and Cosentino, 2010). Ferrari (2007) demonstrated that impaired capacity to scavenge free radicals and reactive oxygen species (ROS) as a consequence of decreased levels of antioxidant cellular defense systems or excessive free radical production is common in brain, liver, heart and other tissues. Furthermore, Pexa

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et al. (2009) demonstrated that the Hcy tissue content was higher in the liver and kidney after Hcy enriched diet in rats. ROS are responsible for the cardiovascular diseases and atherosclerosis (Victor et al., 2009). It increases formation of superoxide radicals, lipid peroxidation and protein oxidation, which can directly promote damage of cellular organelles and DNA (Wiseman and Halliwell, 1996; Imlay, 2003). Cellular defense against ROS are conducted through intracellular systems such as antioxidant enzymes or reducing agents such as vitamin C (Filip et al., 2010). Antioxidants are known as potential scavengers of ROS, so they protect biological membranes from oxidants. However, the balance between free radicals and synthesis of antioxidant defenses can be broken by chemicals. Vitamin C is a water-soluble antioxidant found in the cytosol and extracellular fluid that can interact directly with free radicals, thus preventing oxidative and DNA damage (Padayatty et al., 2003). Vitamin C is a powerful antioxidant and free radical scavenger (Du et al., 2012). Furthermore, results show a strong relationship between low vitamin C levels and cardiovascular diseases (Deicher et al., 2005). Comet assay (single cell gel electrophoresis) is a current screening method for the assessment of degree of DNA damage such as strand breaks resulting from alkaline labile sites and impairment of DNA repair mechanisms (Tice et al., 2000). It is a visual, sensitive and noninvasive technique for quantitating DNA damage and repair in vivo and in vitro in eukaryotic cells. There are many advantages and utilities of this technique (Fairbairn et al., 1995; Anderson et al., 1998). Numerous studies have investigated the antioxidant effect of vitamin C in tissues. However, information regarding the protective effect of vitamin C on the DNA damage and tissues in chronic hHcy induced rat model is scanty, and further research is required. The purpose of this study was to investigate the effect of vitamin C on DNA damage in lymphocytes, malondialdehyde (MDA) and glutathione (GSH) levels and catalase (CAT) and superoxide dismutase (SOD) activities in the heart, liver and in the renal tissues of chronic hHcy rat model. In addition, the effectiveness of vitamin C for the prevention of hHcy was demonstrated histopathologically.

2. Materials and methods 2.1. Animals and experimental protocol A total of 24 healthy adult male Wistar rats (12 weeks old weighing between 214 and 232 g) were used in the study. The animals were obtained from the Adnan Menderes University, Faculty of Veterinary Medicine, Experimental Research Centre, Aydin, Turkey. They were suspended in screen-bottomed stainless steel cages at 22–24 ◦ C in a room with a 12/12 h light/dark cycle. Rats were randomly divided into three groups (n = 8 rats per group), the control, hHcy and hHcy + vitamin C groups. The rats received a commercial rodent diet and had free access to tap water. After 10 days of acclimatization, rats were weighed. Chronic hHcy group rats were treated with 1 g/kg/day l-methionine (Ungvari et al., 1999). hHcy + vitamin C group rats were treated with 1 g/kg/day l-methionine and 150 mg/kg/day vitamin C (Bagi et al., 2003). The substances were administered to rats by daily oral gavage for 28 days. Control animals received 0.9% NaCl solution in the same volumes as those applied to vitamin C treated rats by daily oral gavage within the specified days. l-Methionine was dissolved in 150 mM phosphate buffer (pH 7.4). Body weight of the rats was monitored weekly. l-methionine and vitamin C intake was calculated each week during the 4 week trial. Blood samples were collected from the heart by cardiac puncture after thiopental injection at the end of the experiment, and transferred heparinized tubes. Animals were sacrificed by cervical dislocation and the thoracic aorta was

immediately removed for histopathological evaluation. The heart, liver and right kidney tissues were dissected for measuring MDA and GSH levels and CAT and SOD activities. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocol was approved by the Animal Ethics Committee of the University of Adnan Menderes (2013/008). 2.2. Measurement of serum Hcy levels The concentration of Hcy in serum samples was determined by a commercial ELISA test kit (CK-E30175, Hangzhou Eastbiopharm Co. Ltd., Yile road, China). 2.3. Lymphocyte isolation and DNA analysis by comet assay 2.3.1. Lymphocyte isolation Lymphocytes were isolated from the fresh blood samples in accordance with a modified procedure described by Collins et al. (1997). Blood samples were mixed v/v (1/1) with PBS solution (P4417, Sigma-Aldrich, Germany) for the determination of DNA fragmentation of blood lymphocytes. To prevent further DNA damage, blood samples were rotated and kept in a dark cold chamber for a maximum of 90 min before the lymphocyte isolation procedure. Lymphocytes were isolated with histopaque-1077 (10771, Sigma-Aldrich, Germany). Lymphocytes were suspended in freezing medium (PBS with 10% fetal calf serum and 15% DMSO) and slowly frozen in aliquots of in 1 ml at −80 ◦ C (NU 9668E, Nuaire, Japan). For experiments, an aliquot of each frozen cell was thawed (WNB10, Memmert, Germany) and immediately diluted with cold PBS, centrifuged (Universal 320R, Hettich, Germany), and the pellet re-suspended in cold PBS. 2.3.2. DNA analysis by comet assay The DNA strand breaks were detected with comet assay. The comet assay was applied with several modifications as previously described by Singh et al. (1988) and Collins et al. (1997). Conventional end-frosted slides were pre-coated with 1% normal melting agarose and allowed to dry overnight. Cells were re-suspended in PBS at a concentration of 1 × 106 cells/ml. Forty microliters of this suspension were mixed with 140 ␮l of 0.6% pre-warmed (37 ◦ C) low melting point agarose and two drops of 70 ␮l of this mixture were placed on a microscope slide. A cover slip (24 × 60 mm) was put on the drops and the gels were allowed to solidify for 5 min at 4 ◦ C. All steps were performed under minimal yellow light at working room temperature adjusted to 22 ◦ C ± 2. Once the gels had become solidified, the cover slip was removed and the slides were dipped into freshly prepared lysis solution jars at 4 ◦ C (2.5 M NaCl, 0.1 M disodium EDTA and 0.01 M Trizma base, pH = 10.5. Triton X-100 1% and DMSO 10% added just before use, for a minimum of 1 h at 4 ◦ C) for at least 1 h. The positive control slide cells was dipped into H2 O2 (50 and 100 ␮M in PBS) solution for 5 min at 4 ◦ C, then washed with cold PBS and introduced into a lysis solution in a separate jar for at least 1 h at 4 ◦ C. After lysis, slides were aligned in a horizontal gel electrophoresis tank (CSL-COM20, Cleaver Scientific, UK) connected to recirculating cooler (FL300, Julabo, Germany) set at 4 ◦ C filled with freshly made alkaline electrophoresis solution (300 mM NaOH, 1 mM disodium EDTA, pH > 13) to a level of 0.25 cm over the slides. Slides were equilibrated and DNA was allowed to unwind for 40 min in the alkaline solution and electrophoresis (CS-300V, Cleaver Scientific, UK) was then carried out at approximately 1 V/cm for 20 min. After electrophoresis, the slides were washed two times for 10 min. with neutralizing buffer (0.4 M TrisHCl buffer, pH 7.5). After washing, the slides were fixed with 50%, 75% and 90% ethanol (for 5 min each) then they were allowed to

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dry at room temperature prior to staining with 70 ␮l of ethidium bromide solution (20 ␮g/ml). For visualization of DNA damage, slides were examined at 400fold magnification under fluorescence microscope (DM3000, Leica, Germany). Measurements of tail intensity and tail moment of ‘comets’ were made by a computer-based image analysis system (Comet Assay IV, Perceptive Instruments, UK) for 100 randomly selected cells, i.e. 50 cells from each of two gels from each sample. Scoring was performed by moving the slide in a zig-zag motion to avoid scoring the same cells twice. % Tail DNA and Mean Tail Moment parameters were used to assess the DNA damage. % Tail DNA was defined as the percentage of DNA that had migrated from the head of the comet into the tail. Mean Tail Moment was defined as the product of the proportion of tail intensity and the displacement of tail center of mass relative to the centre of the head (Olive et al., 1992). The mean value of these parameters was calculated and used for the evaluation of DNA damage. 2.4. Determination of MDA, GSH, CAT and SOD levels in tissues Dissected heart, liver and kidney tissues were immediately rinsed in ice-cold phosphate-buffered saline. Tissues were homogenized (2000 rpm/min for 1 min, 1/10 w/v) using a stirrer (IKA Overhead Stirrer; IKA-Werke GmbH & Co. KG, Staufen, Germany) in 10% 150 mM phosphate buffer (pH 7.4) in an ice bath. The homogenate was centrifuged (Nüve-Bench Top Centrifuge, NF 800R, Nüve, Ankara, Turkey) at 6000 g for 10 min at 4 ◦ C. The supernatants were frozen at −80 ◦ C (Glacier Ultralow Temperature Freezer, Japan) until analyzed. Protein concentrations in supernatants were measured by a spectrophotometer (Shimadzu UV-1601, Kyoto, Japan) using commercially available kits by the Biuret method (Archem Diagnostic Ind. Ltd., Istanbul, Turkey) and the results are expressed as mg/ml protein. The tissue homogenate was used for the lipid peroxidation estimation, which was applied by measuring the formation of thiobarbutric acid reactive substances (TBARS) according to the method of Yoshioka et al. (1979). Absorbance was measured by using a spectrophotometer at 532 nm. The concentration of MDA was calculated by the absorbance complex (absorbance coefficient ε=1.56 × 105 /M/cm) and expressed as nmol/mg tissue protein. GSH levels were spectrophotometrically determined at 412 nm using the method described by Tietze (1969). The results were determined by comparison with an aqueous standard solution of GSH (Sigma Chemical Co., St. Louis, Missouri, USA) and expressed as mg/g tissue protein. CAT activity was determined by measuring the decomposition of hydrogen peroxide at 240 nm, according to the method of Bergmeyer et al. (1974) and was expressed as k/mg tissue protein. SOD activity was determined according to the method of Sun et al. (1988) and the absorbance was measured at 560 nm by a spectrophotometer. The principle of this method is based on the inhibition of nitro blue tetrazolium reduction by the xanthine on xanthine oxidase system as a superoxide generator. SOD activity was then measured by the degree of inhibition of this reaction and the results are shown as U/mg tissue protein. All these enzyme activity assays were analyzed in duplicate, and were averaged. 2.5. Histopathological evaluation The thoracic aortas of rats were fixed in buffered 10% formalin solution for 24 h, dehydrated in a series of graded alcohol, cleared in xylene and embedded in paraffin. Paraffin sections were cut at intervals of 49 ␮m and thickness of 7 ␮m. Tissue sections taken serially were placed on slides. Paraffin section was stained by the Orcein–Giemsa method (Pinkus and Hunter, 1960; Krobock et al., 1978) for elastic laminae and Crossman’s Modified triple

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Table 1 Effect of vitamin C on % Tail DNA and Mean Tail Moment parameters in chronic hHcy induced rats (n = 8). Parameters

% Tail DNA Mean Tail Moment

Experimental groups

P

Control

hHcy

hHcy + vitamin C

28.35 ± 3.14b 7.90 ± 0.90b

48.20 ± 1.30a 19.04 ± 1.69a

26.11 ± 2.07b 7.00 ± 1.31b

*** ***

hHcy: hyperhomocysteinemia. a,b Different letters indicate statistically significant differences in the same row. *** P < 0.001.

stain (Crossman, 1937) for measurement of vessel diameter. Three serial sections were taken from each rat. The vessel diameter was measured in three different sections. Thickness of aortic elastic lamina in aorta was measured in six different parts of the each vessel. Microscopical measurement by the Leica DMLB (Germany) of light microscopy and associated with Leica DC200 CCD camera (Germany) and Q-win standard software of image analysis (Version 2.8). 2.6. Statistical analysis All parameters were checked for normal distribution with the Shapiro–Wilk test and for homogeneity of variance with Levene’s test. The data were compared among groups using Kruskal–Wallis analysis of variance (ANOVA) or one-way ANOVA according to whether data were normally distributed or not. Post hoc multiple comparisons were performed using the Mann–Whitney U test with Bonferroni corrected or Duncan’s test. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software (Version 11.5). Differences were considered statistically significant if P < 0.05. All data were expressed as the mean and standard error (Conover, 1980). 3. Results Body weights did not differ significantly among treatments at the end of the experiment (data not given). Serum Hcy levels of the hHcy (29.80 ± 2.06 ␮mol/L) and hHcy + vitamin C (32.15 ± 1.28 ␮mol/L) groups were higher than the control (5.91 ± 0.49 ␮mol/L) group (P < 0.001). There was no significant difference between serum Hcy levels of hHcy and hHcy + vitamin C group (P > 0.05). Vitamin C administration was not able to decrease serum Hcy levels significantly and it didn’t prevent the manifestation of hHcy. Data of DNA damage recorded in peripheral blood samples were presented in Table 1. They show the distribution of the % Tail DNA and Mean Tail Moment measured in individual samples. When compared with the control and hHcy + vitamin C groups, % Tail DNA (P < 0.001) and Mean Tail Moment (P < 0.001) of isolated lymphocytes were significantly higher in the hHcy group. The % Tail DNA and Mean Tail Moment of isolated lymphocytes are shown in Table 1. Examples of evaluated cells are given in Fig. 1. Vitamin C play a vital role in the protection of cells against oxidative stress. Influence of vitamin C on SOD and CAT activities and GSH and MDA levels in heart, liver and renal tissues of chronic hHcy induced rats are given in Fig. 2. When compared with the hHcy group, SOD (P < 0.001) and CAT (P < 0.05) activities and GSH (P < 0.01) level in the hHcy + vitamin C group were higher, and MDA level was lower (P < 0.01) in heart tissue. Compared with the other groups, in hHcy group, CAT activty and GSH level decreased and MDA level increased significantly in liver tissue (P < 0.01, P < 0.001 and P < 0.01). However, SOD activity was unaffected (P > 0.05) in liver tissue of the hHcy + vitamin C group. On the other hand, compared with the hHcy group, SOD (P < 0.001) and CAT (P < 0.001)

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Fig. 1. The fluorescence microscopy images of lymphocytes. (a) Control group, (b) hHcy group and (c) hHcy + vitamin C group (n = 8 in each group). hHcy: hyperhomocysteinemia, white arrows: head of lymphocytes, arrowhead: tail migration of lymphocytes (bar: 20 ␮m).

Fig. 2. SOD (A) and CAT (B) activities and GSH (C) and MDA (D) levels in heart, liver and renal tissues of chronic hHcy induced rats (n = 8). Each value represents mean ± SE. * Denotes P < 0.05, ** P < 0.01 or *** P < 0.001 compared with control data, # denotes P > 0.05 compared with data from control group and hHcy treated rats. hHcy: hyperhomocysteinemia, SOD: superoxide dismutase activity, CAT: catalase, GSH: glutathione, MDA: malondialdehyde.

activities and GSH (P < 0.001) level increased and MDA (P < 0.001) level decreased in the hHcy + vitamin C group significantly in renal tissue. The aortic diameter and thickness of aortic elastic laminae were higher in hHcy group (P < 0.05 and P < 0.001, respectively). The histopathological sections of the groups are shown in Fig. 3. There was no significant difference between aortic diameter and thickness of aortic elastic laminae of control and hHcy + vitamin C group (P > 0.05) (Table 2).

4. Discussion In this study we have performed induced chronic hHcy in rats. The most important finding of this study was the demonstration of

Table 2 The aortic diameter and thickness of aortic elastic laminae in chronic hHcy induced rats (n = 8). Parameters (␮m)

Diameter Thickness

Experimental groups

P

Control

hHcy

hHcy + vitamin C

5325.75 ± 117.32b 73.67 ± 1.32b

6138.70 ± 338.22a 93.12 ± 3.54a

5361.68 ± 173.00b 79.74 ± 2.17b

* ***

hHcy: hyperhomocysteinemia. a,b Different letters indicate statistically significant differences in the same row. * P < 0.05. *** P < 0.001.

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Fig. 3. Image of thickness of aortic elastic lamina in experimental groups (n = 8). Rat aorta stained Orcein–Giemsa method for elastic fibres. (a) Control group, (b) hHcy group and (c) hHcy + vitamin C. hHcy: hyperhomocysteinemia, white arrows: elastic lamina of aorta, L: lumen, ct: connective tissue (bar: 10 ␮m, ×40).

a significant decrease in the incidence of DNA damage by means of vitamin C administration in l-methionine exposured rats. The disturbance in the delicate oxidant–antioxidant balance results many pathophysiologic conditions related to lipid peroxidation, protein degragation and DNA damage. Oxidative damage to DNA caused by oxygen-derived species including free radicals is the most frequent type encountered by aerobic cells (Valko et al., 2004). Picerno et al. (2007) showed that Hcy-treated human peripheral blood lymphocytes in the culture presented increase in DNA damage and in micronucleus frequency, altering immune function. DNA is one of the most biologically significant target of oxidative attack. Parameters of % Tail DNA and Mean Tail Moment were used for the evaluation of DNA damage. In our experimental conditions, we have not tested whether vitamin C induced DNA damage or not. However, vitamin C has exhibited the strongest protection against l-methionine induced DNA damage and vitamin C did not act as a pro-oxidant in our study (Table 1). A number of publications have shown that, antioxidants protect the integrity of DNA from genotoxicants and are capable of eliminating ROS generated oxidative stress (Suhail et al., 2012; Aydin et al., 2013). Our findings indicate that exposure to l-methionine may induce genotoxic effect in lymphocytes, indicating a potential health risk for rats. Hydroxyl radicals, which are the most toxic oxygen metabolities are believed to be one of the most potent causes of DNA damage by means of the Fenton reaction (Valko et al., 2004). Ascorbic acid acts as an antioxidant in biological fluids by scavenging superoxide anion (O•2 ), hydroxyl radical (OH• ) and various lipid hydroperoxides (Duarte and Lunec, 2005). Vitamin E plays an important role in protection against oxidative stress, mostly by lipid peroxylradical scavenging. Vitamin C can also interact with the tocopheroxyl radical and regenerate reduced tocopherol (Du et al., 2012). It was previously shown that DNA damage were decreased by vitamin C supplementation (Kahl et al., 2012; Szeto et al., 2013). The present study results are concordant with these data. The antioxidant enzyme activities in hHcy + vitamin C group were significantly increased in heart tissue. ROS mediated cytotoxic effects of Hcy on endothelial cells have already been demonstrated (Verhoef et al., 1997). As previously shown, folate regulate metabolism of homocysteine. Previous study indicated that lowfolate diet was significantly decreased CAT activity and GSH level, and increased TBARS in heart tissue (Pravenec et al., 2013). TBARS are formed as a by-product of lipid peroxidation and are markers of oxidative stress. Hempel (1998) previously described that hHcy may sensitize endothelial cells to oxidative stress, thereby reducing endothelial cell glutathione levels. In our study, SOD and CAT activities and GSH level in the hHcy group were decreased, and MDA level was increased in heart tissue. Intracellular Hcy can be irreversibly degraded to cysteine through the transsulfuration pathway, which is mainly limited to

cells of the liver and kidneys (De Bree et al., 2002). In addition, high methionine or low-folate administration might deteriorate the oxidant/anitoxidant status of liver or renal tissues (Yamada et al., 2012; Pravenec et al., 2013). Therefore, this study investigated also the protective effect of vitamin C in the liver and kidneys. CAT activity (P < 0.01) and GSH level were higher (P < 0.001), and MDA level was lower (P < 0.01) in hHcy + vitamin C groups as compared to the hHcy group, however significant alteration was not shown in liver tissue SOD activities (P > 0.05). Sauls et al. (2004) previously demonstrated that adult rabbits made hyperhomocysteinemic by chronic injection of Hcy developed elevated levels of lipid peroxidation products in liver. But, Mendes et al. (2014) showed that CAT and SOD activities were not different between control and methionine groups in liver tissues of hHcy rat model, which methionine was given 100 mg/kg/day for 8 weeks. They demonstrated that no significant alteration of CAT and SOD may be associated with adaptation of antioxidant enzymatic system. Robin et al. (2004) reported that oxidant and antioxidant balance has been changed in the liver of rats following high methionine diet. Lower SOD and CAT activities in hHcy group in our study may be dependent on insufficient adaptation time of antioxidant enzymatic system and/or high dose intake of methionine. It was reported that SOD localized in the cytosol (SOD1) and in the mitochondria (SOD2) of cells (Ghezzi et al., 2005), indicating that lipid peroxidation damaged the cell membrane leading to an increase in MDA, but did not damage cell components such as mitochondria (Fridovich, 1995). Increased MDA in tissue may depend on overproduction of ROS, resulting in oxidative stress. Similar results were found in the present study. The attenuation of the increase in antioxidant enzyme activities by vitamin C might be conveyed by restoring energy metabolism via enhancement of mitochondrial ␤-oxidation. Furthermore, it should be noted that SOD converts superoxide to less cytotoxic hydrogen peroxide, which then decomposes into water via the enzymes CAT and glutathione peroxidase. Compared with hHcy group, SOD (P < 0.001) and CAT (P < 0.001) activities and GSH (P < 0.001) level in control and hHcy + vitamin C groups increased significantly in renal tissue. MDA level also increased in response to l-methionine administration via oxidative stress in renal tissues of rats in the hHcy group (P < 0.001). Of interest, researchers suggested a relation between hHcy and free radical generation (Matte et al., 2009; Machado et al., 2011). Increased CAT activity might have been sustained to counteract fast generating superoxide radicals, or GSH may have protected the cells from reactive free radicals and peroxides. Application of l-methionine, increases renal free radical production through postischemic oxidative stress. Abais et al. (2014) demonstrated that in renal tissue SOD-dependent O•− 2 production were higher in mice fed the folatefree diet than the normal diet in hyperhomocysteinemic mice. The

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aim of this study was to investigate the application of the comet assay to check DNA damage induced by hHcy conditions. However, oxygen free radicals, caused by hHcy, affected not only heart tissue, but also liver and renal tissues. As previously shown, vitamin C was found effective in the protection of tissues. Results showed that Hcy reduced enzymatic antioxidant potential in tissues, and vitamin C administration prevented such effects. Moreover, oxygen radicals may play an important role in this particular hHcy model. All data from the present trial suggest that vitamin C may have an important role in preventing hHcy and that its antioxidant properties seem to play the major role. In vivo oxidative stress of hyperhomocysteinemic rats reduced with the antioxidant vitamin C treatment (Bagi et al., 2003). Our results show that vitamin C administration stimulated SOD and CAT activities and raised GSH levels in tissues. The most dramatic results have been observed in hHcy group. The aortic diameter and thickness of elastic lamina increased in this group (P < 0.05 and P < 0.001, respectively). The areas of atherosclerotic lesions were increased in apolipoprotein E deficiency hyperhomocysteinemic mice (Jiang et al., 2012). The intima of descending aorta in rats receiving methionine rich diet was thickened (Yang et al., 2010). Moreover, hHcy promotes low density lipoprotein (LDL) oxidation and internalization, which in turn is the initial step of atherosclerosis (Murawska-Cialowicz et al., 2008). But, high dietary intake of antioxidative vitamins could be a protective factor against atherosclerosis and the pro-oxidative effect of Hcy on LDL (Seo et al., 2010). In our study aortic injury is restored by vitamin C. The prominent finding of this study is the demonstration of no significant difference between the control and hHcy + vitamin C groups by means of more serious histological findings like aortic diameter and thickness of elastic lamina. These histological findings were significantly higher in the hHcy group. Presence of relation between lymphocyte DNA damage and thickness of elastic lamina may be plausible, because both atherosclerosis and reduced antioxidant parameters are related with increased DNA damage. Serum Hcy concentration was increased after methionine administration (Sharma et al., 2013). Our results showed that the serum Hcy concentration was over the basal concentration. However, vitamin C did not decrease the elevated Hcy serum levels after chronic Hcy administration in rats. These results of the present study are in agreement with some previous studies (Cascalheira et al., 2008; Machado et al., 2011). In addition, rats were simultaneously given l-methionine and vitamin C by the same route of oral administration. There is a very low possibility that the two substances react with each other in the stomach and are then inactivated, before absorption. In conclusion, comet assay can be used for the assessment of primary DNA damage caused by hHcy. As a result, supplementation of vitamin C, reduced oxidative DNA damage in chronic hHcy induced rat model. Additionally, vitamin C decreased oxidative stress during the normal cellular metabolism by increasing the total antioxidant activity and free radical scavenging potential. When the effects of vitamin C taken into account, it could be thought to have a strong potential for being used as supplementation in atherosclerotic cardiovascular diseases. References Abais JM, Xia M, Li G, Gehr TW, Boini KM, Li PL. Contribution of endogenously produced reactive oxygen species to the activation of podocyte NLRP3 inflammasomes in hyperhomocysteinemia. Free Radic Biol Med 2014;67:211–20. Anderson D, Yu TW, McGregor DB. Comet assay responses as indicators of carcinogen exposure. Mutagenesis 1998;136:539–55. Aydin S, Tokac M, Taner G, Arikok AT, Dundar HZ, Ozkardes AB, et al. Antioxidant and antigenotoxic effects of lycopene in obstructive jaundice. J Surg Res 2013;182:285–95. Bagi Z, Cseko C, Toth E, Koller A. Oxidative stress-induced dysregulation of arteriolar wall shear stress and blood pressure in hyperhomocysteinemia is prevented by chronic vitamin C treatment. Am J Physiol Heart Circ Physiol 2003;285:2277–83.

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