**4.2.2 Vitamin E**

Vitamin E refers to a group of fat-soluble compounds that include both tocopherols and tocotrienols **(Brigelius- Flohé and Traber, 1999)**. Naturally occurring vitamin E exists in eight chemical forms (alpha-, beta-, gamma-, and delta-tocopherol and alpha-, beta-, gamma-, and delta-tocotrienol) that have varying levels of biological activity. Alpha- (or α-) tocopherol is the only form that is recognized to meet human requirements. γ-tocopherol is the most common in the North American diet **(Traber, 1998)**. γ-tocopherol can be found in corn oil, soybean oil, margarine and dressings **(Bieri and Evarts, 1974; Brigelius-Flohé & Traber, 1999)**. The most biologically active form of vitamin E, α-tocopherol, is the second most common form of vitamin E in the North American diet and perhaps the common form in European and Mediterranean diet. This variant of vitamin E can be found most abundantly in wheat germ oil, sunflower, and safflower oils **(Reboul et al., 2006)**. Serum concentrations of α-tocopherol depend on the liver, which takes up the nutrient after the various forms are absorbed from the small intestine. The liver preferentially resecretes only α-tocopherol *via* the hepatic α-tocopherol transfer protein **(Traber, 2006)**. As a result, blood and cellular concentrations of other forms of vitamin E are lower than those of α -tocopherol and have been the subjects of less research **(Sen et al., 2006; Dietrich et al., 2006)**.

Vitamin E is an important vitamin for preventing lipid peroxidation and it has many reported health effects and is recognized as the most important lipid-soluble, chain-breaking antioxidant in the body **(Fenech & Ferguson, 2001)**. This vitamin might have a protective role against chromosomal damage, DNA oxidation and DNA damage. Vitamin E has also been reported to play a regulatory role in cell signaling and gene expression. Epidemiological studies showed that high blood concentrations of vitamin E were associated with a decreased risk of certain cancers. This effect might emerge in part, by enhancing immune function **(Frank, 2005; Claycombe & Meydani, 2001, Salobir et al., 2010)**. Vitamin E might also block the formation of carcinogenic NOCs formed in the stomach from nitrite and secondary amines **(Weitberg and Corvese, 1997)**.

Vitamin E was shown to prevent the genotoxicity of several environmetal chemicals and several drugs. Nitrosamine toxicity was shown to be protected by vitamin E. Hepatocytes freshly isolated from rats fed with a common diet or a vitamin A- or vitamin Esupplemented diet were assayed for sensitivity to DNA breakage and cytogenetic changes induced by several carcinogens including NMOR. NMOR was the only agent that induced DNA breaks, chromosomal aberrations, and micronuclei. Both vitamin A and vitamin E were able to reduce these effects, and the protection by vitamin A was more pronounced **(Slamenová, 2001)**. On the other hand, vitamin E was also found to be protective against the genotoxic properties of one of the most commonly used herbicides**,** atrazine**,** in male rats. Atrazine caused a significant increase in tail length of comets from blood and liver cells compared to controls. Co-administration of vitamin E (100 mg/kg bw) along with atrazine resulted in decrease in tail length of comets as compared to the group treated with atrazine alone. Besides, micronucleus assay revealed a significant increase in the frequency of micro-

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

Vitamin E refers to a group of fat-soluble compounds that include both tocopherols and tocotrienols **(Brigelius- Flohé and Traber, 1999)**. Naturally occurring vitamin E exists in eight chemical forms (alpha-, beta-, gamma-, and delta-tocopherol and alpha-, beta-, gamma-, and delta-tocotrienol) that have varying levels of biological activity. Alpha- (or α-) tocopherol is the only form that is recognized to meet human requirements. γ-tocopherol is the most common in the North American diet **(Traber, 1998)**. γ-tocopherol can be found in corn oil, soybean oil, margarine and dressings **(Bieri and Evarts, 1974; Brigelius-Flohé & Traber, 1999)**. The most biologically active form of vitamin E, α-tocopherol, is the second most common form of vitamin E in the North American diet and perhaps the common form in European and Mediterranean diet. This variant of vitamin E can be found most abundantly in wheat germ oil, sunflower, and safflower oils **(Reboul et al., 2006)**. Serum concentrations of α-tocopherol depend on the liver, which takes up the nutrient after the various forms are absorbed from the small intestine. The liver preferentially resecretes only α-tocopherol *via* the hepatic α-tocopherol transfer protein **(Traber, 2006)**. As a result, blood and cellular concentrations of other forms of vitamin E are lower than those of α -tocopherol

and have been the subjects of less research **(Sen et al., 2006; Dietrich et al., 2006)**.

stomach from nitrite and secondary amines **(Weitberg and Corvese, 1997)**.

Vitamin E is an important vitamin for preventing lipid peroxidation and it has many reported health effects and is recognized as the most important lipid-soluble, chain-breaking antioxidant in the body **(Fenech & Ferguson, 2001)**. This vitamin might have a protective role against chromosomal damage, DNA oxidation and DNA damage. Vitamin E has also been reported to play a regulatory role in cell signaling and gene expression. Epidemiological studies showed that high blood concentrations of vitamin E were associated with a decreased risk of certain cancers. This effect might emerge in part, by enhancing immune function **(Frank, 2005; Claycombe & Meydani, 2001, Salobir et al., 2010)**. Vitamin E might also block the formation of carcinogenic NOCs formed in the

Vitamin E was shown to prevent the genotoxicity of several environmetal chemicals and several drugs. Nitrosamine toxicity was shown to be protected by vitamin E. Hepatocytes freshly isolated from rats fed with a common diet or a vitamin A- or vitamin Esupplemented diet were assayed for sensitivity to DNA breakage and cytogenetic changes induced by several carcinogens including NMOR. NMOR was the only agent that induced DNA breaks, chromosomal aberrations, and micronuclei. Both vitamin A and vitamin E were able to reduce these effects, and the protection by vitamin A was more pronounced **(Slamenová, 2001)**. On the other hand, vitamin E was also found to be protective against the genotoxic properties of one of the most commonly used herbicides**,** atrazine**,** in male rats. Atrazine caused a significant increase in tail length of comets from blood and liver cells compared to controls. Co-administration of vitamin E (100 mg/kg bw) along with atrazine resulted in decrease in tail length of comets as compared to the group treated with atrazine alone. Besides, micronucleus assay revealed a significant increase in the frequency of micro-

**(Ohsawa et al., 2003)**.

**4.2.2 Vitamin E** 

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

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

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

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

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

Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 431

evidence in literature for a cancer-protective effect of the vegetables of the family *Cruciferae* that includes broccoli, watercress, cabbage, kale, horseradish, radish, turnip, and garden cress **(Verhoeven et al., 1996; Hecht, 1999)**. This effect is attributed to ITCs, which occur naturally as thioglucoside conjugates (glucosinolates). They are hydrolysis products of glucosinolates and are generated through catalytic mediation of myrosinase, which is released upon processing (cutting or chewing) of cruciferous vegetables from a compartment separated from glucosinolates. Evidence exists for conversion of glucosinolates to ITCs in the gut. At least 120 different glucosinolates have been identified. ITCs have a common basic skeleton but differ in their terminal R group, which can be an alkyl, an alkenyl, an alkylthioalkyl, an aryl, a β-hydroxyalkyl, or an indolylmethyl group. The widely studied ITCs include phenethyl isothiocyanate (PEITC), benzyl isothiocyanate (BITC), indole-3-carbinol (I3C) and allyl isothiocyanate (AITC) **(Fahey et al., 2001; Arranz et** 

The most important biological property discovered about ITCs is their ability to inhibit carcinogenesis, induced by several chemicals including nitrosamines in the lung, stomach, colon, liver, esophagus, bladder and mammary glands in animal models **(Hecht, 1999; Zhang et al., 2003; Zhang and Talalay, 1994; Hecht et al., 1995; Munday et al., 2003)**. Two mechanisms can be suggested for the protective effect of ITCs against nitrosamine-induced

a. Blocking the production of genotoxic intermediates by inhibiting Phase I enzymes: PEITC was shown to reduce p-nitrophenol hdroxylase (CYP2E1), ethoxyresorufin O-deethylase (CYP1A1) and coumarin hdroxylase (CYP2A6) activities **(García et** 

b. Enhancement of detoxification pathways through the induction of Phase II enzymes

Furthermore, ITCs may have ROS scavenging capacity, alter cell proliferation, stimulate DNA-repair, and induce NAD(P)H: quinine oxidoreductase activity as also mentioned for AA before **(Gamet-Payrastre et al., 2000; Chaudière and Ferrari-Iliou et al., 1999; Surh,** 

ITCs were shown to be effective in the inhibition of lung tumorigenesis in mice and rats induced by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Because NNK is believed to play a significant role as a cause of lung cancer in smokers, PEITC is being developed as a chemopreventive agent, which is presently in Phase I a clinical trial in healthy smokers **(Hecht, 1996; Stoner et al., 1991)**. PEITC is a potent inhibitor of rat esophageal tumorigenesis induced by NBMA **(Stoner et al., 1991)**. A comparative study demonstrates that phenylpropyl isothiocyanate (PPITC) is even more potent, whereas BITC and 4-phenylbutyl isothiocyanate (PBITC) have little effect on tumorigenesis **(Wilkinson et al., 1995)**. However, phenylhdroxyl isothiocyanate (PHITC) enhances tumorigenesis in the same model **(Stoner et al., 1995)**. Mechanistic studies clearly show that PEITC inhibits the metabolic activation of NBMA in the rat esophagus, probably through inhibition of a cytochrome P450 (CYP450) enzyme **(Morse et al., 1997)**. Concomitant with this inhibition, inhibition of *O*6-methylguanine formation in rat esophageal DNA was observed. The inhibitory effects on tumorigenicity correlate with their inhibitory effects on *O*6-methylguanine formation **(Wilkinson et al., 1995; Stoner & Morse, 1997)**. Inhibition of

**al., 2006)**.

DNA damage:

**al., 2008)**.

**(Arranz et al., 2006)**.

**2002; Surh et al., 2001; Roomi et al., 1998)**.

was accompanied by a 2.2- and 3.6-fold higher yield in the micronuclei frequency observed on days 10 and 14 post-irradiation, respectively. These results suggest that oxidative stress and DNA damage induced in dermal microvascular endothelial cells by radiation can be modulated by early PTX/Tx treatment. These drugs acted not only as radical scavengers, but they were also responsible for the increased micronuclei frequency in 10 Gy-irradiated cells. Thus, these drugs may possibly interfere with DNA repair processes **(Laurent et al., 2006)**.

In another study, the effects of vitamin E supplementation were evaluated in cultured primary human normal fibroblasts exposed to UVA. Cells were incubated in medium containing α-tocopherol, α-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by Comet assay, immediately following irradiation or after different repair periods. The generation of H2O2 and superoxide ion was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. Pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photo-induction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and superoxide, compared to controls. The researchers indicated that in human normal fibroblasts, exogenously added vitamin E exerted a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks **(Nocentini et al., 2001)**.

In an *in vivo* study by **Konopacka at al. (1998),** the modifying effects of treatment with vitamin E, AA and vitamin A in the form of β-carotene on the clastogenic activity of gamma rays were investigated in mice. Damage *in vivo* was measured by the micronucleus assay in bone marrow polychromatic erythrocytes and exfoliated bladder cells. The vitamins were administered orally, either for five consecutive days before or immediately after irradiation with 2 Gy of gamma rays. The results showed that pretreatment with vitamin E (100-200 mg/kg/day) and -carotene (3-12 mg/kg/day) were effective in protecting against micronucleus induction by gamma rays. AA depending on its concentration enhanced the radiation effect (400 mg/kg/day), or reduced the number of micro-nucleated polychromatic erythrocytes (50-100 mg/kg/day). Such effect was weekly observed in exfoliated bladder cells. The most effective protection in both tissues was noted when a mixture of these vitamins was used as a pretreatment. Administration of the all antioxidant vitamins to mice immediately after irradiation was also effective in reducing the radiation-induced micronucleus frequency. The data from the *in vitro* experiments based on the Comet assay show that the presence of the vitamins in culture medium influences the kinetic of repair of radiation-induced DNA damage in mouse leukocytes.

### **4.3 Prevention of genotoxicity by thiocyanates**

Human cancer can be prevented by changing the dietary habits **(Kelloff , 2000; Vallejo et al., 2002; Hecht, 1996; Milner , 2004; Davis & Milner, 2006)**. Studies show that antioxidantrich diets are associated with low risk of cancer and whole diet plays a more important role than the individual components. The protective effects of vegetables and fruits may be attributed to the combined effect of various phytochemicals, vitamins, fibers, and allium compounds rather than the effect of a single component **(Lee et al., 2003)**. There is powerful

was accompanied by a 2.2- and 3.6-fold higher yield in the micronuclei frequency observed on days 10 and 14 post-irradiation, respectively. These results suggest that oxidative stress and DNA damage induced in dermal microvascular endothelial cells by radiation can be modulated by early PTX/Tx treatment. These drugs acted not only as radical scavengers, but they were also responsible for the increased micronuclei frequency in 10 Gy-irradiated cells. Thus, these drugs may possibly interfere with DNA repair

In another study, the effects of vitamin E supplementation were evaluated in cultured primary human normal fibroblasts exposed to UVA. Cells were incubated in medium containing α-tocopherol, α-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by Comet assay, immediately following irradiation or after different repair periods. The generation of H2O2 and superoxide ion was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. Pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photo-induction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and superoxide, compared to controls. The researchers indicated that in human normal fibroblasts, exogenously added vitamin E exerted a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and

In an *in vivo* study by **Konopacka at al. (1998),** the modifying effects of treatment with vitamin E, AA and vitamin A in the form of β-carotene on the clastogenic activity of gamma rays were investigated in mice. Damage *in vivo* was measured by the micronucleus assay in bone marrow polychromatic erythrocytes and exfoliated bladder cells. The vitamins were administered orally, either for five consecutive days before or immediately after irradiation with 2 Gy of gamma rays. The results showed that pretreatment with vitamin E (100-200 mg/kg/day) and -carotene (3-12 mg/kg/day) were effective in protecting against micronucleus induction by gamma rays. AA depending on its concentration enhanced the radiation effect (400 mg/kg/day), or reduced the number of micro-nucleated polychromatic erythrocytes (50-100 mg/kg/day). Such effect was weekly observed in exfoliated bladder cells. The most effective protection in both tissues was noted when a mixture of these vitamins was used as a pretreatment. Administration of the all antioxidant vitamins to mice immediately after irradiation was also effective in reducing the radiation-induced micronucleus frequency. The data from the *in vitro* experiments based on the Comet assay show that the presence of the vitamins in culture medium influences the kinetic of repair of

Human cancer can be prevented by changing the dietary habits **(Kelloff , 2000; Vallejo et al., 2002; Hecht, 1996; Milner , 2004; Davis & Milner, 2006)**. Studies show that antioxidantrich diets are associated with low risk of cancer and whole diet plays a more important role than the individual components. The protective effects of vegetables and fruits may be attributed to the combined effect of various phytochemicals, vitamins, fibers, and allium compounds rather than the effect of a single component **(Lee et al., 2003)**. There is powerful

processes **(Laurent et al., 2006)**.

mutagenic risks **(Nocentini et al., 2001)**.

radiation-induced DNA damage in mouse leukocytes.

**4.3 Prevention of genotoxicity by thiocyanates**

evidence in literature for a cancer-protective effect of the vegetables of the family *Cruciferae* that includes broccoli, watercress, cabbage, kale, horseradish, radish, turnip, and garden cress **(Verhoeven et al., 1996; Hecht, 1999)**. This effect is attributed to ITCs, which occur naturally as thioglucoside conjugates (glucosinolates). They are hydrolysis products of glucosinolates and are generated through catalytic mediation of myrosinase, which is released upon processing (cutting or chewing) of cruciferous vegetables from a compartment separated from glucosinolates. Evidence exists for conversion of glucosinolates to ITCs in the gut. At least 120 different glucosinolates have been identified. ITCs have a common basic skeleton but differ in their terminal R group, which can be an alkyl, an alkenyl, an alkylthioalkyl, an aryl, a β-hydroxyalkyl, or an indolylmethyl group. The widely studied ITCs include phenethyl isothiocyanate (PEITC), benzyl isothiocyanate (BITC), indole-3-carbinol (I3C) and allyl isothiocyanate (AITC) **(Fahey et al., 2001; Arranz et al., 2006)**.

The most important biological property discovered about ITCs is their ability to inhibit carcinogenesis, induced by several chemicals including nitrosamines in the lung, stomach, colon, liver, esophagus, bladder and mammary glands in animal models **(Hecht, 1999; Zhang et al., 2003; Zhang and Talalay, 1994; Hecht et al., 1995; Munday et al., 2003)**. Two mechanisms can be suggested for the protective effect of ITCs against nitrosamine-induced DNA damage:


Furthermore, ITCs may have ROS scavenging capacity, alter cell proliferation, stimulate DNA-repair, and induce NAD(P)H: quinine oxidoreductase activity as also mentioned for AA before **(Gamet-Payrastre et al., 2000; Chaudière and Ferrari-Iliou et al., 1999; Surh, 2002; Surh et al., 2001; Roomi et al., 1998)**.

ITCs were shown to be effective in the inhibition of lung tumorigenesis in mice and rats induced by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Because NNK is believed to play a significant role as a cause of lung cancer in smokers, PEITC is being developed as a chemopreventive agent, which is presently in Phase I a clinical trial in healthy smokers **(Hecht, 1996; Stoner et al., 1991)**. PEITC is a potent inhibitor of rat esophageal tumorigenesis induced by NBMA **(Stoner et al., 1991)**. A comparative study demonstrates that phenylpropyl isothiocyanate (PPITC) is even more potent, whereas BITC and 4-phenylbutyl isothiocyanate (PBITC) have little effect on tumorigenesis **(Wilkinson et al., 1995)**. However, phenylhdroxyl isothiocyanate (PHITC) enhances tumorigenesis in the same model **(Stoner et al., 1995)**. Mechanistic studies clearly show that PEITC inhibits the metabolic activation of NBMA in the rat esophagus, probably through inhibition of a cytochrome P450 (CYP450) enzyme **(Morse et al., 1997)**. Concomitant with this inhibition, inhibition of *O*6-methylguanine formation in rat esophageal DNA was observed. The inhibitory effects on tumorigenicity correlate with their inhibitory effects on *O*6-methylguanine formation **(Wilkinson et al., 1995; Stoner & Morse, 1997)**. Inhibition of

Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 433

79%) and by PEITC (1 M, 67%) and I3C (1 M, 61%) towards NDMA (in presence of Fpg enzyme). However, in absence of Fpg enzyme, AITC (1 M, 72%) exerted the most drastic reduction towards NPYR-induced oxidative DNA damage, and PEITC (1 M, 55%) towards NDMA. These results indicated that ITCs protect human-derived cells against the DNA damaging effect of NPYR and NDMA, two carcinogenic compounds that occur in the environment. Another study performed by **García et al. (2008)** aimed to investigate the protective effect of ITCs alone or in combination with AA towards NDBA or NPIP-induced oxidative DNA damage in HepG2 cells by Comet assay. PEITC and I3C alone showed a weak protective effect towards NDBA (0.1 M, 26-27%, respectively) or NPIP (1 M, 26- 28%, respectively)-induced oxidative DNA damage. AITC alone did not attenuate the genotoxic effect provoked by NDBA or NPIP. In contrast, HepG2 cells simultaneously treated with PEITC, I3C and AITC in combination with AA showed a stronger inhibition of oxidative DNA-damage induced by NDBA (0.1 M, 67%, 42%, 32%, respectively) or NPIP (1 M, 50%, 73%, 63%, respectively) than ITCs alone. One feasible mechanism by which ITCs alone or in combination with AA exert their protective effects towards N-nitrosamineinduced oxidative DNA damage could be by the inhibition of their CYP450 dependent bioactivation. PEITC and I3C strongly inhibited the p-nitrophenol hydroxylation (CYP2E1) activity (0.1 M, 66-50%, respectively), while the coumarin hydroxylase (CYP2A6) activity was slightly reduced (0.1 M, 25-37%, respectively). However, the ethoxyresorufin Odeethylation (CYP1A1) activity was only inhibited by PEITC (1 M, 55%). The results indicated that PEITC and I3C alone or PEITC, I3C and AITC in combination with AA protect human-derived cells against the oxidative DNA damaging effects of NDBA and NPIP.

In our study performed on HepG2 cells, we tested AITC (0.5 µM) against the nitrite and nitrosamine toxicity. 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 AITC. When compared to untreated cells, nitrite, NDMA, NDEA and NMOR raised the tail intensity up to 17 %, 279 %, 324 % and 288 %, respectively (all, p<0.05). AITC was able to reduce the tail intensity caused by nitrite 36 %, by NDMA 36 %, by NDEA 49 % and by NMOR 32 %, respectively. These reductions were statistically significant when compared to each individual toxic compound applied group (all, p<0.05). Besides, when compared to untreated cells, nitrite, NDMA, NDEA and NMOR raised the tail intensity up to 94%, 126%, 157% and 207%, respectively (all, p<0.05). AITC was able to reduce the tail moment caused by nitrite 16 %, by NDMA 32 %, by NDEA 41 % and by NMOR 19 %, respectively and these reductions were statistically significant when compared to each individual toxic compound applied

The protective effect of antioxidants is universally accepted. However, as also seen in AA, the mode of action of antioxidants particularly with dual behavior (prooxidant and antioxidant) remain unclear and more research must be conducted on these compounds. For instance, the elucidation of how antioxidant properties operate *in vitro* can provide a better understanding of the *in vivo* situation. On the other hand, Comet assay can be an important tool for the determining of the genotoxic effect of several environmental chemicals, as well

group **(Erkekoglu & Baydar, 2010d)**.

as the antioxidant properties of several compounds.

**5. Conclusion** 

*N*′- nitrosonornicotine (NNN) tumorigenicity in the rat esophagus by PEITC also appears to be due to inhibition of its metabolic activation **(Stoner et al., 1998)**.

The antimutagenic properties of ITCs have been reported towards NDMA and NPYRinduced oxidative stress before. In studies performed by **Knasmüller et al. (1996, 2003)**  using PEITC as a chemopreventive agent, the researchers observed a reduction in NDMAand NPYR-induced DNA damage in *Escherichia coli* K-12 and a considerable reduction in NDMA-induced micronuclei in HepG2 cells. The results of several studies demonstrated that ITCs exhibited strong antimutagenic effects against NDMA and NPYR in a dose dependent manner. In a study by **Smerák at al. (2009)**, the researchers investigated the effect of PEITC on the mutagenic activity of indirect-acting mutagens and carcinogens like aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) using the *Ames* bacterial mutagenicity test, the Comet assay, an *in vivo* micronucleus test, and direct-acting mutagen and carcinogen N-nitroso-N-methylurea (MNU). In the Ames test, the antimutagenic activity of PEITC was studied in the concentration range 0.3-300 g/plate. PEITC at concentrations of 0.3, 3 and 30 g/plate reduced dose-dependently mutagenicity of AFB1 and IQ in both *Salmonella typhimurium* TA98 and TA100 strains. In the case of the direct mutagen MNU, the antimutagenic effect of PEITC was detected only at concentration of 30 g/plate in the strain TA100. The PEITC concentration 300 g/plate was toxic in the Ames test. The 24 h pre-treatment of HepG2 cells with PEITC at concentration 0.15 g/ml resulted in a significant decrease of DNA breaks induced by MNU at concentrations 0.25 and 0.5 mM. Although a trend towards reduced strand break level were determined also at PEITC concentrations 0.035 and 0.07 g/ml**,** it did not reach the statistical significance. No effect, however, of PEITC on IQ-induced DNA breaks was observed. Chemopreventive effect of PEITC was revealed also *in vivo*. Pretreatment of mice with PEITC concentrations of 25 and 12.5 mg/kg bw administered to mice in three daily doses resulted in reduction of micronucleus formation in mice exposed to all three mutagens under study, with statistically significant effect at concentration of 25 mg/kg. Results of this study indicated that the strong PEITC antimutagenic properties may have an important role in the prevention of carcinogenesis and other chronic degenerative diseases that share some common pathogenetic mechanisms. In a recent study by **Tang et al. (2011),** PEITC was shown to induce a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G2/M phase of DU 145 human prostate cells. Besides, PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G2/M phase arrest was mediated by the increase of p53 and Wee1 and it reduced the level of M-phase inducer phosphatase 3 (CDC25C) protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results of this study also demonstrated that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. The researchers concluded that PEITC might exhibit anticancer activity and become a potent agent for human prostate cancer cells in the future.

There are a few studies on ITCs against nitrosamine-induced genotoxicity in literature. In a study by **Arranz et al. (2006)**, the protective effect of three ITCs was tested. ITC were highly protective against NPYR-induced oxidative DNA damage than against NDMA. The greatest protective effect towards NPYR-induced oxidative DNA damage was shown by I3C (1 M, 79%) and by PEITC (1 M, 67%) and I3C (1 M, 61%) towards NDMA (in presence of Fpg enzyme). However, in absence of Fpg enzyme, AITC (1 M, 72%) exerted the most drastic reduction towards NPYR-induced oxidative DNA damage, and PEITC (1 M, 55%) towards NDMA. These results indicated that ITCs protect human-derived cells against the DNA damaging effect of NPYR and NDMA, two carcinogenic compounds that occur in the environment. Another study performed by **García et al. (2008)** aimed to investigate the

protective effect of ITCs alone or in combination with AA towards NDBA or NPIP-induced oxidative DNA damage in HepG2 cells by Comet assay. PEITC and I3C alone showed a weak protective effect towards NDBA (0.1 M, 26-27%, respectively) or NPIP (1 M, 26- 28%, respectively)-induced oxidative DNA damage. AITC alone did not attenuate the genotoxic effect provoked by NDBA or NPIP. In contrast, HepG2 cells simultaneously treated with PEITC, I3C and AITC in combination with AA showed a stronger inhibition of oxidative DNA-damage induced by NDBA (0.1 M, 67%, 42%, 32%, respectively) or NPIP (1 M, 50%, 73%, 63%, respectively) than ITCs alone. One feasible mechanism by which ITCs alone or in combination with AA exert their protective effects towards N-nitrosamineinduced oxidative DNA damage could be by the inhibition of their CYP450 dependent bioactivation. PEITC and I3C strongly inhibited the p-nitrophenol hydroxylation (CYP2E1) activity (0.1 M, 66-50%, respectively), while the coumarin hydroxylase (CYP2A6) activity was slightly reduced (0.1 M, 25-37%, respectively). However, the ethoxyresorufin Odeethylation (CYP1A1) activity was only inhibited by PEITC (1 M, 55%). The results indicated that PEITC and I3C alone or PEITC, I3C and AITC in combination with AA protect human-derived cells against the oxidative DNA damaging effects of NDBA and NPIP.

In our study performed on HepG2 cells, we tested AITC (0.5 µM) against the nitrite and nitrosamine toxicity. 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 AITC. When compared to untreated cells, nitrite, NDMA, NDEA and NMOR raised the tail intensity up to 17 %, 279 %, 324 % and 288 %, respectively (all, p<0.05). AITC was able to reduce the tail intensity caused by nitrite 36 %, by NDMA 36 %, by NDEA 49 % and by NMOR 32 %, respectively. These reductions were statistically significant when compared to each individual toxic compound applied group (all, p<0.05). Besides, when compared to untreated cells, nitrite, NDMA, NDEA and NMOR raised the tail intensity up to 94%, 126%, 157% and 207%, respectively (all, p<0.05). AITC was able to reduce the tail moment caused by nitrite 16 %, by NDMA 32 %, by NDEA 41 % and by NMOR 19 %, respectively and these reductions were statistically significant when compared to each individual toxic compound applied group **(Erkekoglu & Baydar, 2010d)**.
