**4.2.1 Ascorbic acid**

424 Gel Electrophoresis – Advanced Techniques

comprise sunburn inflammation erythema, tanning, and local or systemic immunosuppression. On the other hand, UV irradiation present in sunlight is an environmental human carcinogen. There is considerable evidence that UV is implicated in skin carcinogenesis and the risk of cutaneous cancers has increased during the last decade due to increase of sun exposure. For a long time, ultraviolet B radiation (UVB: 290-320 nm) have been considered to be the more efficient wavelength in eliciting carcinogenesis in human skin. It is today clear that ultraviolet A (UVA, 320-400 nm), especially UVA1 (340-400 nm) also participate to photo-carcinogenesis. It penetrates deeply, but it does not cause sunburn. One of molecular mechanisms in the biological effects of UV is the induction of ROS directly or through endogenous photosensitization reactions. UVA radiation mainly acts *via* this production of ROS and the subsequent oxidative stress seems to play a crucial role in the deleterious effects of UVA. UVA does not damage DNA directly like UVB and UVC, but it can generate highly reactive chemical intermediates, such as hydroxyl and oxygen radicals, which in turn can damage DNA and lead to the formation of 8-oxoGua **(Ridley et al., 2009)**. UVB light can cause direct DNA damage. The radiation excites DNA molecules in skin cells, causing aberrant covalent bonds to form between adjacent cytosine bases, producing a dimer. When DNA polymerase comes along to replicate this strand of DNA, it reads the dimer as "AA" and not the original "CC". This causes the DNA replication mechanism to add a "TT" on the growing strand. This mutation can result in cancerous growths, and is known as a "classical C-T mutation". The mutations caused by the direct DNA damage carry a UV signature mutation that is commonly seen in skin cancers. The mutagenicity of UV radiation can be easily observed in bacterial cultures. This cancer connection is one reason for concern about ozone depletion and the ozone hole. UVB causes some damage to collagen, but at a very much slower rate than UVA. Fortunately, the skin possesses a wide range of inter-linked antioxidant defense mechanisms to protect itself from damage by UV-induced ROS. However, the capacity of these systems is not unlimited; they can be overwhelmed by excessive exposure to UV and then ROS can reach damaging levels. An interesting strategy to provide photoprotection would be to support or enhance one or

There is limited number of studies in literature concerning the protective effect of selenocompounds on UV-caused genotoxicity. In a study by **Emonet-Piccardi et al. (1998)**, the researchers determined the protective effects of NAC (5 mM), SS (0.6 M) or zinc chloride (ZnCl2, 100 M) against UVA radiation in human skin fibroblasts using Comet assay. The cells were incubated with NAC, SS or ZnCl2 and then UVA was applied as 1 to 6 J/cm2 to the cells. The tail moment increased by 45% (1 J/cm2) to 89% (6 J/cm2) in nonsupplemented cells (p<0.01). DNA damage was significantly prevented by NAC, SS and ZnCl2, with similar efficiency from 1 to 4 J/cm2. For the highest UVA dose (6 J/cm2), SS and

In a study assessing the effects of pretreatment of primary human keratinocytes with Se on UV-induced DNA damage, cells were irradiated with UVB from FS-20 lamps and were subjected to Comet assay. Comet tail length due to UVB-induced T4 endonuclease Vsensitive sites (caused by cyclopyrimidine dimers, CPDs) increased to 100% immediately after irradiation (time 0). After 4 h, 68% of the damage remained and after 24 h, 23% of the damage was still present. Treatment with up to 200 nM SM or 50 nM SS had no effect on CPD formation or rates of repair, or on the number of excision repair sites as measured by cytosine arabino furanoside and hydroxyurea treatment. However, both SS and SM

more of these endogenous systems **(Béani, 2001)**.

ZnCl2 were more effective than NAC.

Diet should include components such as vitamins and flavonoids and the antioxidant capacity of body is directly linked to the diet. Vitamins like ascorbic acid (vitamin C, AA) are important antioxidants. About 90% of AA in the average diet comes from fruits and vegetables **(Vallejo et al., 2002)**.

AA is a water soluble dietary antioxidant that plays an important role in controlling oxidative stress **(Vallejo et al., 2002)**. Most importantly, AA is a mild reducing agent. For this reason, it degrades upon exposure to oxygen, especially in the presence of metal ions and light. It can be oxidized by one electron to a radical state or doubly oxidized to the stable form called "dehydroascorbic acid". Typically it reacts with oxidants such as ROS, such as the •OH formed from H2O2. Hydroxyl radical is the most detrimental species, due to its high interaction with nucleic acids, proteins, and lipids. AA can terminate these chain radical reactions by electron transfer. AA is special because it can transfer a single electron, owing to the stability of its own radical ion called "semidehydroascorbate". The oxidized forms of AA are relatively unreactive, and do not cause cellular damage. However, being a good electron donor, high concentrations of AA in the presence of free metal ions can not only promote, but also initiate free radical reactions, thus making it a potentially dangerous pro-oxidative compound in certain metabolic contexts **(Choe and Min, 2006; Blokhina et al., 2003)**.

Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 427

drinking water **(Bartsch and Spiegelhalder, 1996; Bofetta et al., 2008; Jakszyn & Gonzalez,** 

In several studies, AA was found to be protective against NOC-induced genotoxicity using Comet assay. In a study by **Robichová et al. (2004)**, the researchers used three cell lines (HepG2, V79 and VH10) to determine the genotoxic effect of N-Nitrosomorpholine (NMOR). NMOR was found to induce DNA damage in a dose-dependent manner but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by Comet assay confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pretreated with vitamin A, vitamin E and AA the researchers found a significant decrease of % tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E**.** AA did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. In a study by **Arranz et al. (2007)**, HepG2 cells were simultaneously treated with AA and the genotoxic effects of the N-nitrosamines, namely, N-nitrosodimethylamine (NDMA), Nnitrosopyrrolidine (NPYR), N-nitrosodibutylamine (NDBA) or N-nitrosopiperidine (NPIP) were reduced in a dose-dependent manner. At concentrations of 1-5 M AA, the protective effect was higher towards NPYR-induced oxidative DNA damage (78-79%) than against NDMA (39-55%), NDBA (12-14%) and NPIP (3-55%), in presence of Fpg enzyme. However, a concentration of 10 M AA led to a maximum reduction in NDBA (94%), NPYR (81%), NPIP (80%) and NDMA (61%)-induced oxidative DNA damage, in presence of Fpg enzyme. The greatest protective effect of AA (10 M) was higher towards NDBA-induced oxidative DNA damage. The authors concluded that one feasible mechanism by which AA exerted its protective effect could be that it might interact with the enzyme systems catalyzing the metabolic activation of the N-nitrosamines, blocking the production of genotoxic

In our previous studies performed using Comet assay, we have shown that AA was highly protective in HepG2 cells against the genotoxicity of both nitrite and three important NOC, namely NDMA, Nitrosodiethylamine (NDEA) and NMOR **(Erkekoglu et al., 2010c)**. Nitrite was added as 20 µM, NDMA as 10 mM, NDEA as 10 mM and NMOR as 3 mM to the medium for 30 min with or without AA (10 M). When compared to untreated cells, nitrite (p>0.05), NDMA (p<0.05), NDEA (p<0.05), and NMOR (p<0.05) raised the tail intensity up to 1.18-, 3.79-, 4.24-, and 4.16-fold, respectively. AA was able to reduce the tail intensity caused by nitrite, NDMA, NDEA, and NMOR to 34%, 59%, 44%, and 44%, respectively, and these reductions were statistically significant when compared to each individual toxic compound applied group (all, p<0.05). Besides, nitrite, NDMA, NDEA, and NMOR increased the tail moment up to 1.94, 6.04, 6.05, and 5.70, respectively. AA (10 μM) enabled a reduction of 27%, 30%, 23%, and 22% in the tail moment in nitrite, NDMA, NDEA, and NMOR-treated cells, respectively, and these reductions were statistically significant when compared to each individual toxic compound applied group (all, p<0.05) **(Erkekoglu et al.,** 

In an experiment performed on multiple organs of mice, the genotoxicity of endogenously formed N-nitrosamines from secondary amines and sodium nitrite was evaluated in, using Comet assay. Dimethylamine, proline, and morpholine were simultaneously with sodium

**2006; Tricker, 1997)**.

intermediates.

**2010c)**.

AA is able to suppress ROS efficiently *in vivo*; thus, reducing DNA damage to tumor suppressor genes which might explain its anticancer properties **(Crott et al., 1999)**. *In vitro*, AA acts in conjunction with vitamin E, present in lipid membranes, to quench free radicals and prevent lipid peroxidation **(Niki et al., 1995)**.

In the Comet assay, evidence of protection was seen against the effects of H2O2 when AA was present at low concentrations (up to 1 mM); by contrast, there was exacerbation at higher doses (>5 mM) **(Harréus et al., 2005; Anderson et al., 1994; Anderson and Phillips, 1999)**. After 2–4 h after intake, AA provided significant protection to the DNA of isolated lymphocytes when challenged with H2O2 **(Panayiotidis and Collins, 1997)**. Besides, AA was found to be protective against H2O2-induced DNA damage (DNA strand breaks and oxidized purines/pyrimidines) in human hepatoma cells (HepG2 cells) **(Arranz et al., 2007a, Arranz et al., 2007b)**. In intervention studies, supplementation of 100 mg/day to 50–59 yearold men led to a decrease in oxidative base damage and enhanced resistance against oxidative damage **(Duthie et al., 1996)**. In a long-term study, the antioxidant effect of AA was studied by measuring oxidative DNA damage and DNA repair in blood cells with the Comet assay. Male smokers were given AA (2 × 250 mg) daily in the form of plain or slow release tablets combined with plain release vitamin E (2 × 91 mg), or placebo for 4 weeks. The results of this study suggested that long-term AA supplementation at a high dose, i.e. 500 mg, together with vitamin E in moderate dose, i.e. 182 mg, decreased the steady-state level of oxidative DNA damage in lymphocytes of smokers **(Møller et al., 2004)**. In a study performed on gastric epithelial cells SGC-7901, both AA and SS were found to be protective against Helicobacter pylori-induced oxidative stress and genotoxicity **(Shi and Zheng, 2006)**.

AA was also tested for its protective effects against the genotoxicity of several toxic chemicals, drugs and metals. Using peripheral blood lymphocytes, AA as well as vitamin E were found to be protective against benzo(a)pyrene [B(a)P]-induced DNA damage **(Gajecka et al., 1999)**. In rats, using Comet assay, the genotoxicity of p-dimethylaminoazobenzene (DAB), a hepatocarcinogen, was found to be decreased by AA administration. Besides, vitamin A, vitamin E and combination of these three vitamins were also found be effective against the toxicity **(Velanganni et al., 2007)**. A significant increase in the levels of protein oxidation, DNA strand breaks, and DNA-protein cross-links was observed in blood, liver, and kidney of rats exposed to arsenic (100 ppm in drinking water) for 30 days. Co-administration of AA and vitamin E in the form of -tocopherol to arsenic-exposed rats showed a substantial reduction in the levels of arsenic-induced oxidative products of protein and DNA **(Kadirvel et al., 2007)**. For anti-cancer drugs there are inconclusive results. AA was protective against epirubicin- and adriamycin-induced genotoxicity in cancer patients **(Mousseau et al., 2005; Shimpo et al., 1991)**. However, there was no evidence of a protective effect of AA against the damage caused by bleomycin **(Anderson & Phillips, 1999)**. Moreover, results were also inconclusive when oestrogenic compounds were co-incubated with AA (0.5 and 1 mM) in isolated lymphocytes showing no common pattern in the responses **(Anderson et al., 2003)**.

Nitrosamines (NOCs) can be formed endogenously from nitrate and nitrite and secondary amines under certain conditions such as strongly acidic pHs of the human stomach **(Jakszyn and Gonzalez, 2006; Bofetta et al., 2008; Tricker, 1997)**. Humans are exposed to a wide range of NOCs from diet (cured meat products, fried food, smoked preserved foods, foods subjected to drying, pickled and salty preserved foods), tobacco smoking, work place and

AA is able to suppress ROS efficiently *in vivo*; thus, reducing DNA damage to tumor suppressor genes which might explain its anticancer properties **(Crott et al., 1999)**. *In vitro*, AA acts in conjunction with vitamin E, present in lipid membranes, to quench free radicals

In the Comet assay, evidence of protection was seen against the effects of H2O2 when AA was present at low concentrations (up to 1 mM); by contrast, there was exacerbation at higher doses (>5 mM) **(Harréus et al., 2005; Anderson et al., 1994; Anderson and Phillips, 1999)**. After 2–4 h after intake, AA provided significant protection to the DNA of isolated lymphocytes when challenged with H2O2 **(Panayiotidis and Collins, 1997)**. Besides, AA was found to be protective against H2O2-induced DNA damage (DNA strand breaks and oxidized purines/pyrimidines) in human hepatoma cells (HepG2 cells) **(Arranz et al., 2007a, Arranz et al., 2007b)**. In intervention studies, supplementation of 100 mg/day to 50–59 yearold men led to a decrease in oxidative base damage and enhanced resistance against oxidative damage **(Duthie et al., 1996)**. In a long-term study, the antioxidant effect of AA was studied by measuring oxidative DNA damage and DNA repair in blood cells with the Comet assay. Male smokers were given AA (2 × 250 mg) daily in the form of plain or slow release tablets combined with plain release vitamin E (2 × 91 mg), or placebo for 4 weeks. The results of this study suggested that long-term AA supplementation at a high dose, i.e. 500 mg, together with vitamin E in moderate dose, i.e. 182 mg, decreased the steady-state level of oxidative DNA damage in lymphocytes of smokers **(Møller et al., 2004)**. In a study performed on gastric epithelial cells SGC-7901, both AA and SS were found to be protective against Helicobacter pylori-induced oxidative stress and genotoxicity **(Shi and Zheng,** 

AA was also tested for its protective effects against the genotoxicity of several toxic chemicals, drugs and metals. Using peripheral blood lymphocytes, AA as well as vitamin E were found to be protective against benzo(a)pyrene [B(a)P]-induced DNA damage **(Gajecka et al., 1999)**. In rats, using Comet assay, the genotoxicity of p-dimethylaminoazobenzene (DAB), a hepatocarcinogen, was found to be decreased by AA administration. Besides, vitamin A, vitamin E and combination of these three vitamins were also found be effective against the toxicity **(Velanganni et al., 2007)**. A significant increase in the levels of protein oxidation, DNA strand breaks, and DNA-protein cross-links was observed in blood, liver, and kidney of rats exposed to arsenic (100 ppm in drinking water) for 30 days. Co-administration of AA and vitamin E in the form of -tocopherol to arsenic-exposed rats showed a substantial reduction in the levels of arsenic-induced oxidative products of protein and DNA **(Kadirvel et al., 2007)**. For anti-cancer drugs there are inconclusive results. AA was protective against epirubicin- and adriamycin-induced genotoxicity in cancer patients **(Mousseau et al., 2005; Shimpo et al., 1991)**. However, there was no evidence of a protective effect of AA against the damage caused by bleomycin **(Anderson & Phillips, 1999)**. Moreover, results were also inconclusive when oestrogenic compounds were co-incubated with AA (0.5 and 1 mM) in isolated lymphocytes

Nitrosamines (NOCs) can be formed endogenously from nitrate and nitrite and secondary amines under certain conditions such as strongly acidic pHs of the human stomach **(Jakszyn and Gonzalez, 2006; Bofetta et al., 2008; Tricker, 1997)**. Humans are exposed to a wide range of NOCs from diet (cured meat products, fried food, smoked preserved foods, foods subjected to drying, pickled and salty preserved foods), tobacco smoking, work place and

showing no common pattern in the responses **(Anderson et al., 2003)**.

and prevent lipid peroxidation **(Niki et al., 1995)**.

**2006)**.

#### drinking water **(Bartsch and Spiegelhalder, 1996; Bofetta et al., 2008; Jakszyn & Gonzalez, 2006; Tricker, 1997)**.

In several studies, AA was found to be protective against NOC-induced genotoxicity using Comet assay. In a study by **Robichová et al. (2004)**, the researchers used three cell lines (HepG2, V79 and VH10) to determine the genotoxic effect of N-Nitrosomorpholine (NMOR). NMOR was found to induce DNA damage in a dose-dependent manner but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by Comet assay confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pretreated with vitamin A, vitamin E and AA the researchers found a significant decrease of % tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E**.** AA did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. In a study by **Arranz et al. (2007)**, HepG2 cells were simultaneously treated with AA and the genotoxic effects of the N-nitrosamines, namely, N-nitrosodimethylamine (NDMA), Nnitrosopyrrolidine (NPYR), N-nitrosodibutylamine (NDBA) or N-nitrosopiperidine (NPIP) were reduced in a dose-dependent manner. At concentrations of 1-5 M AA, the protective effect was higher towards NPYR-induced oxidative DNA damage (78-79%) than against NDMA (39-55%), NDBA (12-14%) and NPIP (3-55%), in presence of Fpg enzyme. However, a concentration of 10 M AA led to a maximum reduction in NDBA (94%), NPYR (81%), NPIP (80%) and NDMA (61%)-induced oxidative DNA damage, in presence of Fpg enzyme. The greatest protective effect of AA (10 M) was higher towards NDBA-induced oxidative DNA damage. The authors concluded that one feasible mechanism by which AA exerted its protective effect could be that it might interact with the enzyme systems catalyzing the metabolic activation of the N-nitrosamines, blocking the production of genotoxic intermediates.

In our previous studies performed using Comet assay, we have shown that AA was highly protective in HepG2 cells against the genotoxicity of both nitrite and three important NOC, namely NDMA, Nitrosodiethylamine (NDEA) and NMOR **(Erkekoglu et al., 2010c)**. Nitrite was added as 20 µM, NDMA as 10 mM, NDEA as 10 mM and NMOR as 3 mM to the medium for 30 min with or without AA (10 M). When compared to untreated cells, nitrite (p>0.05), NDMA (p<0.05), NDEA (p<0.05), and NMOR (p<0.05) raised the tail intensity up to 1.18-, 3.79-, 4.24-, and 4.16-fold, respectively. AA was able to reduce the tail intensity caused by nitrite, NDMA, NDEA, and NMOR to 34%, 59%, 44%, and 44%, respectively, and these reductions were statistically significant when compared to each individual toxic compound applied group (all, p<0.05). Besides, nitrite, NDMA, NDEA, and NMOR increased the tail moment up to 1.94, 6.04, 6.05, and 5.70, respectively. AA (10 μM) enabled a reduction of 27%, 30%, 23%, and 22% in the tail moment in nitrite, NDMA, NDEA, and NMOR-treated cells, respectively, and these reductions were statistically significant when compared to each individual toxic compound applied group (all, p<0.05) **(Erkekoglu et al., 2010c)**.

In an experiment performed on multiple organs of mice, the genotoxicity of endogenously formed N-nitrosamines from secondary amines and sodium nitrite was evaluated in, using Comet assay. Dimethylamine, proline, and morpholine were simultaneously with sodium

Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 429

nucleated cells (MNCs) following atrazine administration. In the animals administrated vitamin E along with atrazine**,** there was a significant decrease in percentage of micronuclei as compared to atrazine treated rats. The increase in frequency of micronuclei in liver cells and tail length of comets confirm genotoxicity induced by atrazine in blood and liver cells. In addition, the findings clearly demonstrated protective effect of vitamin E in attenuating atrazine-induced DNA damage **(Singh et al., 2008)**. In mouse retina, both vitamin E and AA were shown to markedly reduce the cell apoptosis, lipid peroxidation and DNA damage caused by the organophosphorus insecticide chlorpyrifos **(Yu et al, 2008)**. Vitamin E supplementation was also protective against pyrethroid (both cypermethrin and

Vitamin E was also shown to reduce the genotoxic effects of the anti-HIV drug stavudine (**Kaur & Singh**, **2007)** and the antibiotic, ciprofloxacin **(Gürbay et al., 2006)**. In a study performed on primary culture of rat astrocytes, the researchers incubated the cultured cells with various concentrations of ciprofloxacin, and DNA damage was monitored by Comet assay. The results showed a concentration-dependent induction of DNA damage by ciprofloxacin. Pretreatment of cells with Vitamin E for 4 h provided partial protection

Vitamin E was also found to be protective against the toxicity of anesthesics. In a study performed with sevoflurane on rabbits, vitamin E and SS were administered 15 days before the anesthesia treatment and blood samples were collected after 5 days of treatment with sevoflurane. Both vitamin E and SS administration prevented the sevoflurane induced

Several supplementation studies have also been performed both vitamin E and AA. Supplementation of the diet for 12 weeks with AA and vitamin E resulted in a significant decrease in the DNA damage in diabetic patients **(Sardaş et al., 2001)**. Vitamin E supplementation was also shown to reduce oxidative DNA damage in both hemodialysis and peritoneal dialysis patients **(Domenici et al., 2005)**. In another study performed on 26 healthy subjects, a daily drink including 1.8 mg vitamin E was administered for 26 days and blood samples were obtained. The DNA damage was measured in the lymphocytes subjected to oxidative stress and genotoxicity was found to be significantly lower (42%,

There are few protection studies with vitamin E against radiation toxicity using Comet assay. An *in vitro* study on dermal microvascular endothelial cells by the same research group, gamma- irradiated cells at 3 and 10 Gy, and 0.5 mM of pentoxifylline (PTX) and trolox (Tx, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-soluble derivative of vitamin E), were added either before (15 min) or after (30 min or 24 h) irradiation. ROS measured by the dichlorodihydrofluorescein diacetate assay, and DNA damage, assessed by the Comet and micronucleus assays, were measured at different times after exposure (0 - 21 days). The PTX/Tx treatment decreased the early and delayed peak of ROS production by a factor of 2.8 in 10 Gy-irradiated cells immediately after irradiation and the basal level by a factor of 2 in non-irradiated control cells. Moreover, the level of DNA strand breaks, as measured by the comet assay, was shown to be reduced by half immediately after irradiation when the PTX/Tx treatment was added 15 min before irradiation. However, unexpectedly, DNA strand breaks was decreased to a similar extent when the drugs were added 30 min after radiation exposure. This reduction

permethrin), induced lymphocyte DNA damage **(Gabbianelli et al., 2004)**.

against this effect **(Gürbay et al., 2006)**.

p<0.0001) **(Porrini et al., 2005)**.

genotoxicity in the lymphocytes **(Kaymak et al., 2004)**.

nitrite and the stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow were sampled 3 and 24 h after these compounds had been ingested. DNA damage was observed mainly in the liver following simultaneous oral ingestion of these compounds **(Ohsawa et al., 2003)**.
