Effect of Ascorbic Acid (Vitamin C) on H2O2 Induced Oxidative DNA Damage in Human Lymphocytes Estimated by Comet Assay

"Oxidative DNA damage can be induced by reactive oxygen species and free radicals. Reactive Oxygen Species which is induced by oxidative damage plays a key role in DNA damage". The aim of our study is to identify the protective effect of ascorbic acid (AA, vitamin C) on hydrogen peroxide which induced oxidative damage in DNA by using the Comet assay. Lymphocytes pretreated with or without antioxidants, incubated at 37C for 30 minutes, then H2O2 (100μM) was added & incubated again at 37C for 1 hour (60 minutes). Viability of cells was detected by trypan blue stain exclusion method. The decrease in viability brought about by H2O2 when the cells incubated for 60 minutes and the viability was present to be 39±3% from 80±4% and it was highly developed by the found of AA at 100 μM which appeared 72 ± 1%. These findings indicate that the activity of ascorbic acid as an antioxidant is evidenced by its ability to suppress the oxidative effect against H2O2 and protect the lymphocytes. Estimation of comet tail moment and tail length in human lymphocyte treated with 100μM of hydrogen peroxide as positive control showed that 13± 4.5% of the cells showed no DNA damage while the DNA damage from low to very high damage were 19± 3.5%, 12.2± 2.3%, 14± 3.2% and 37± 3.0%, respectively. In contrast, treatment of the cells with 100 μM H2O2in combination with 10, 25,75 Effect of Ascorbic Acid (Vitamin C) on H2O2 Induced Oxidative DNA Damage in Human Lymphocytes Estimated by Comet Assay Hanaa Naji Abdullah, Sura Ali Al Asadi and Mohammed Abduldaim Saleh 204 Vol: 14 No:1, January 2018 DOI : P-ISSN: 2222-8373 E-ISSN: 2518-9255 and 100 μM of AA reduced the percentage of DNA damage according to the concentrations used and they were 9± 2.3%, 4.5± 1.6%, 6± 0.5% and 10± 1.4%, respectively. In addition, H2O2 induced DNA damageat percentage of 78% at concentration of 100 μmol/L withoutthe addition of ascorbic acid. while the treatment of human lymphocyte with ascorbic acid was able to reduce oxidative DNA damage by 17% in comparison with control group.


‫الهيدروجين‬ ‫بيروكسيد‬ ‫بواسطة‬ ‫المحث‬ ‫التأكسدي‬ ‫النووي‬ ‫الحمض‬ ‫تدمير‬ ‫على‬ ‫االسكوربك‬ ‫حامض‬ ‫تأثير‬ ‫اللمفاوية‬ ‫الخاليا‬ ‫في‬ ‫المذنب.‬ ‫فحص‬ ‫بواسطة‬ ‫والمقاسة‬ ‫البشرية‬ ‫عبدهللا‬ ‫ناجي‬ ‫هناء‬
Ascorbic acid is an important antioxidant which prevents other compounds from being oxidized (2).In addition, AA is considered a very sensitive index of oxidative stress (3) which involves production of reactive oxygen species (ROS) and also responsible for aging and neoplasmic progressions (4).Hhydrogen peroxide (H2O2) is widely recognized to produce reactive oxygen species which cause damage to nucleic acids, proteins and disintegrate plasma membrane.It is also responsible for aging and neoplasmic progressions (5).H2O2 is utilized to induce oxidative DNA damage (6) and found to be related to the induction of most cancers in animals (7), carcinogenesis and mutagenesis (8).importance originates from its role in the induction and progress of carcinogenesis (9).Hydrogen peroxide induces its harmful effect endogenously by various physiological strategies during oxidative phosphorylation in time of inflammatory respiratory burst.The hydrogen peroxide is a critical source of oxidative damage in cells, resulting in DNA lesions, together with ss and ds breaks (10).Ascorbic acid (Vitamin C) act as antioxidant and has long been known for its anti-cancer properties (11).The comet assay is a defined genotoxicity assay (in vitro and in vivo) of chemicals (12,13) and it is a simple, rapid, and sensitive method for detecting DNA breaks in individual cells, and is commonly used in genotoxicity assays, primary researches in DNA damage and repair and is also being increasingly more used in human biomonitoring (14,15,16).The aim of the present was to detect the protective effect of AA on H2O2 that induced oxidative damage in DNA by using Comet assay.

Blood collection
Blood samples were obtained by vein puncture and poured into heparinized tubes from apparently healthy male individuals (nonsmokers, non-alcohol ,non-drug consumers, nonexposure to mutagen such as X-ray).After collection, blood samples blood were coded and processed within 2hours.

Isolation of human lymphocytes
Heparinized whole blood (5 ml) was diluted one to one with phosphate buffer saline (PBS), then cautiously layered on top of a lymphocyte separation medium in a centrifuge tubes.After centrifugation for 20 minutes at 2000 rpm.lymphocytes were gradient separated [aqueous solution of Ficoll, 57 g/L; density of 1.077 g/mL], then diluted with PBS & centrifuged again at 1500 RPM for 10 minutes.The cellular pellets were re-suspended in 500 mL of PBS & the cells were counted by Neaubauer chamber and then cell concentrate was adjusted to 5000 cells/mL to be ready for the Comet assay (17).The trypan blue dye exclusion method was used to determine cell viability.However, only cell suspensions with more than 96% viabilities were used to determine DNA damage (18).The viability (%) was calculated by using following equation:

Preparation of Slides
Depending on the method described by Singh and coworkers (19), the comet assay was carried out under alkaline conditions.The slides were with two layers of 1% agarose.While this layer was solidified, a second layer containing 10 μl samples of WBCs was mixed with 75 μl (1% low melting agarose) and placed on the slides.After putting the slides on ice for 10 minutes, they were covered with coverslips and stored at 4ºC.

Cell Lysis
The coverslips have been removedand the slides were immersed of slides in a newly prepercd lysis solution [10 mM Tris-HCl , 100 mM EDTA , 2.5 M NaCl & NaOH [pH 10] with one percent Triton X-100 (Sigma) and 10% DMS were added to the lysed cells and DNA unfolding.After removing the slides from the lysis solution they were placed in electrophoresis tank.

Electrophoresis
The slides were then placed in a horizontal gel electrophoresis tank, dealing with the anode.
The unit turned into packed with TBE buffer, and then electrophoresis was performed at 4°C under dim light conditions.At 25 V (300 mA), ectrophoresis was carried out for 30 minutes.
Staining was done with ethidium bromide stain (20 μg/ml) and slides were covered with cover slips to be stored at 4°C in sealed bins until evaluation.

Statistical analysis
All statistical analyses had been carried out using SPSS software and the results were described as mean ± SD.By using ANOVA test.When the P value was less than 0.05, the difference was considered significant.

Results and Discussion
Comet assay was used for evaluation of the protective effect of AA against the oxidative damage in DNA induced by Hydrogen peroxide (H2O2).Hydrogen peroxide produced oxidative damage in DNA of human lymphocytes.Lymphocytes pretreated without or with antioxidants, incubated at 37ºC for 30 minutes., then H2O2 (100µM/L) was added, incubated at 37ºC for 60 minutes.The viability of the cells was detected using Trypan blue exclusion method, and the proportion of viable cells was calculated as shown in Table 1.
Table1: Estimation of cellular viability stimulated by H2O2 and protection by using ascorbic acid.The results of the current study indicated that the viability of lymphocytes on pretreatment of H2O2, simultaneous with a time period examine was skilled.As shown in Table 1, H2O2 was to reduce the viability of the lymphocytes and after incubation for 60 minutes and the viability was 39±3%.In contrast, the viability of the cells increased after the addition of AA at a concentration of 100 μ Mand became 72 ± 1%.These results indicate the efficiency of ascorbic acid as antioxidant as evidenced by its ability to protect lymphocytes against the harmful effect of H2O2.Consequently, the protective mechanism by AA against the oxidative DNA damage might be due to the ability to scavenge the free radicals especially ROS or hydroxyl radicals.The protective effect of ascorbic acid against pro-oxidants stimulated oxidative DNA damage in lymphocytes became more pronounced at the highes concentration (100µM/L).Our findings disagree with the results of Beevi and his colleagues (20) who reported that H2O2 induced the DNA damage in human and the maximum cell death was observed at a concentration of 25 μM.As it can be seen from Table 2, the Comet tail lengths and tail moments measured in human lymphocyte exposed to hydrogen peroxide at 100µM as a positive control showed that 13± 4.5% of the cells showed no DNA damage while the percentage of DNA damage from low to very high damage were 19± 3.5%, 12.2± 2.3%, 14± 3.2% and 37± 3.0%, respectively.In contrast, lymphocytes treated with 100 µM H2O2 in combination with 10, 25,75 and 100 µM of AA showed lower in the percentages of DNA damage and they were 9± 2.3%, 4.5± 1.6%, 6± 0.5%and 10± 1.4%), respectively (Table 2).In the present study, we found that vitamin C had significant protective effect against the H2O2 induced genotoxicity.We observed that the human lymphocytes exposed to 100µM of H2O2 had shown increase in comet frequency.The results of the present study confirm that the alkaline comet assay is a highly sensitive technique to detect DNA damage induced by H2O2.The increase in the comet tail length and tail moment of the H2O2 treated lymphocytes may be caused by DNA strand breaks induction.
Our results disagree with that of Bhat and his colleagues (21) and Harréus and his colleagues (22) who found in their tests that AA is capable of generating large DNA degradation at concentrations of 100 -200 µM.
In the current study we showed that the comet assay can be used to give reproducible results in estimating the extent of oxidative DNA damage to human lymphocytes.It thus provide a good assay for the determination of the potency of the antioxidant agents tested with high confidence.
Several studies have targeted modulating the effect of antioxidant Vitamins, including diet Vit.
C on DNA damage (in vivo & in vitro) cells, brought about through the factors which triggered the free radical reactions (23).Nevertheless, in comparison with the positive control, when the lymphocytes were exposed to ascorbic acid in the presence of hydrogen peroxide, we detected a low-level damage of DNA in cells prompted by using H2O2.In addition, high concentrations of AA resulted in reduced damage of DNA in the tails of comet.However, ascorbic acid has been found to be involved within the Fenton reaction that resulted in OH radical generation, and thus responsible for in vitro oxidative damage of DNA (24).
In another experiment, the antioxidant effect of ascorbic acid was assessed at 100 µmol /L and, the results are shown in Figure 1.Hydrogen peroxide induced 78% DNA damage at concentration (100 µmol/L) without the addition of ascorbic acid, while when human lymphocyte were treated with ascorbic acid, the was oxidative DNA damage was reduced by 17% in comparison with control group 0%.Our results showed that the protective effects of ascorbic acid was significant and that the interaction between them was insignificant.by parts initiating free-radical reactions (27).Genotoxicity testing (comet assay) gives human a hazard appraisal.An expansion in the genotoxic harm is related with an expanded general danger of malignancy (27,28).The results of the present investigation demonstrated that the ascorbic corrosive is sufficiently powerful to decrease the genotoxic harm of H2O2 and subsequently lessening the odds of creating growths, as the high level of H2O2 prompts the tumor induction in animals because of the DNA harm (29).Antioxidant vitamins can inert profoundly receptive particles, for example, free radicals, which are produced within different biochemical procedures in the cells (30).Vitamins behave as cell antioxidants and free radical foragers, and consequently act as anticarcinogenic, anticlastogenic and antimutagenic operators.Of these vitamins C and α-tocopherol are among the best-known cancer prevention agents utilized as in vivo animal models (26).Ascorbic acid is a nonenzymatic antioxidant and is in this manner conceivably included in ensuring cells against oxidative stress.Additionally it behaves as a free radical scavenger and its essence helps different systems in diminishing various problematic free radical procedures (31).The co-organization of ascorbic acid with H2O2 diminished lipid peroxidation.The results demonstrate the helpful impacts of ascorbic acid to defeat oxygen dependent cytotoxicity in animals.Despite the fact that the mode of action of ascorbic acid is not completely comprehended, it is trusted that the ascorbic acid act as antioxidant may invigorate the 7-α-hydroxylation of lipids and cholesterol cores in this way improving their corruption to bile acids, which could be discharged from the body (31,32).
Vitamin C has a significant nucleophilic character and it has been proposed that ascorbate may ensure against electrophilic assault on cell DNA and cell layers by catching receptive specialists (29) or that ascorbyl anion radical, with the high degree of unpaired electron delocalisation, is in charge of the searching of free radicals or ascorbic acid may focus its role as a chain breaking inhibitor of the peroxidation procedure by rummaging middle person peroxyl and alkoxyl radical (33,34).

Conclusion
The results of the present study confirmed the protective effect of ascorbic acid which is evidenced by its ability to act as an antioxidant and protect the lymphocyte against the was more efficient on oxidants induced lymphocyte oxidative DNA damage, especially at a high concentration (100µM/L).
Cells were incubated in a dark incubator with different concentrations of ascorbic acid 10,25,50,75and 100µM (Sigma, Aldrich®) at 37ºC for 30 minutes together with the untreated control samples.Samples were then spun at 200 r.p.m for 3 minutes at 48ºC.Cells were centrifuged and washed for two times with PBS (0.01 Mol) at 200 r.p.m for 3 minutes at 48ºC after pretreatment.Cells had been suspended in PBS with 100 µMol H2O2/L for 5 minutes on ice at dark.Samples were then centrifuged at 200 rpm for 3 minutes at 48ºC.Control samples had been incubate with PBS only without hydrogen peroxide.

Fifty
captured comets from each slide were examined at 400× by fluorescence microscope (Comet Assay II; UK).Tail moment (TM, length of DNA migration) was estimated in order to quantify the DNA damage tail length (TL, length of DNA migration).Mean and standard errors (SE) are used for analysis results for the fifty cells (twenty five cells/ slide).

Figure 1 :
Figure 1: DNA damage after treatment with hydrogen peroxide (100 µmol/L) in the comet assay.

Figure 1 :mean
Figure 1: Antioxidant activity of Ascorbic acid (100 µmol /L) on human lymphocyte in the comet assay.

Table 2 :
Percentage of oxidative DNA damage in human lymphocytes by using comet assay.