**6.1 Effect of diet on prevention of oxidative DNA damage**

Prevention of DNA damage is one of the cellular mechanisms that may prevent cancer. Diet plays an important role in regulating DNA damage for instance by modulating the antioxidant/oxidant balance. The protective effect observed in many studies could be due, in part, to the presence of phenolic and/or non-phenolic constituents that have ability to act as antioxidant by free radical scavenging and chelating metal ions (Anderson *et al.*, 2000); (Ross and Kasum, 2002). They can also act as indirect antioxidant by increasing levels of antioxidants such as glutathione (GSH) and/or by increasing the activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD) (Alia *et al.*, 2005; Lima *et al.*, 2005; Lima *et al.*, 2006; Frei and Higdon, 2003). Phytochemicals can also modulate phase I (activating) enzymes and phase II (detoxifying) enzymes involved in xenobiotic metabolism (Chen and Kong, 2004; Ferguson *et al.*, 2004; Moon *et al.*, 2006; Ross and Kasum, 2002).

Phytochemicals are bioactive compounds present in plants where they are produced as secondary metabolites to protect themselves from several pathogenic agents. Their consumption by humans confers protection against some diseases. Most bioactive phytochemicals belong to one of 5 groups: polyphenols, carotenoids, alkaloids, nitrogencontaining compounds, and organosulfur compounds. Polyphenols and carotenoids include several hundreds of compounds and are the most studied groups. Polyphenols may be further classified into 4 groups, according with the number of phenol rings that they contain: Flavonois, phenolic acids, stilbenes, and lignans. The flavonoids may themselves be classified into the follow subgroups: flavonols, flavones, isoflavones, flavanones, flavanols and anthocyanidins (Fig.2).

### **6.1.1 Polyphenols**

248 Selected Topics in DNA Repair

SBs and AP sites) to the condition treated with enzyme gives a value that correspond to the damage recognized by the enzyme and is usually referred as 'netenzyme-sensitive sites'.

Beside effects on protection against DNA damage, comet assay was also developed to assess DNA repair ability of the cells and effects of diet on DNA damage repair rates. For that, two

1. The "cellular repair assay" that measures the ability of cells to rejoin strand breaks induced by a DNA damaging agent. In this assay two different treatment regimens can be used: (A) Pretreatment of cells with dietary agent followed by exposure to DNA damaging agent, and cells allowed to recover in fresh culture medium at 37ºC. At different time points, samples are taken for analysis with the standard comet assay. Thus, we evaluate the effect of preincubation with test extract/compound on the ability of cells to rejoin SBs; and (B) treatment with DNA damaging agent treatment is done before cells are incubated with the test extract/compounds. In this case the aim is to test effects on nonenzymatic repair. Inclusion of lesion-specific enzymes allow to assess

2. The "in vitro repair assay" was developed by (Collins *et al.*, 2001b) to measure excision repair activity of cell extracts, such as lymphocytes collected in dietary intervention studies or from cells treated with dietary agents. These cell extracts are used as an extra step (similar to the use of lesion-specific enzymes) in DNA substrates containing a specific damage. The first application of this assay was the measurement of repair rates of oxidized bases, called "in vitro BER assay". In this case substrate cells are treated with the photosensitiser Ro 19-8022 (Roche) plus visible light to induce 8-oxoGua, and then cells are embedded in agarose on a slide. After lysis, substrate nucleoids are incubated with a cell extract (e.g. cells incubated with dietary agent) that have enzymes that recognize 8-oxoGua and cut at the place of the lesion. This assay allows to measure the activity of the repair enzyme 8-oxoguanine DNA glycosylase OGG1present in cell extract. A modification of the "in vitro repair assay" was introduced by Langie et al., (2006) to assess nucleotide excision repair (NER), the "in vitro NER assay". In this version cells were treated with benzo(a)pyrene diolepoxide and bulky adducts were formed. In 2009, Gaivao *et al.*, (2009) exposed cells to UVC irradiation inducing cyclobutane pyrimidine dimers and 6-4 photoproducts. Irradiated cells are embedded in agarose on a slide, and lysed to expose the DNA, which is then incubated with the cell extract. Incision at damage sites is detected using the alkaline comet assay and indicates repair ability. In these assays, higher DNA intensity in the tail indicates higher DNA repair activity of the cell extracts. Cell extracts of cells incubated with vehicle are

The format of comet assay most used is 2 gels per slide. To large numbers of samples new formats have been developed, such as 12 gels per slide, that have several advantages: 1) reduces the number of cells required for each gel (approx. 200 cells/gel); 2) allows different conditions in the same slide (different genotoxic chemicals; enzymes or cell extracts); 3) requires low volume of solutions; 4) increases the number of samples processed at one time. This new format has been used in human biomonitoring studies with a great number of

repair ability of others damages beyond strand breaks and AP sites.

**5.3 DNA repair assays** 

different methodologies are frequently used:

used as control (basal) repair abilities.

**5.4 Other modifications** 

Numerous studies have shown the antioxidant and DNA protective properties of polyphenols. Quercetin is a flavonoid found in a variety of foods including apples, onions, wine and tea. Several studies, including our own showed the protective effects of quercetin against oxidative DNA damage in HepG2 cells (Lima et al., 2006; Ramos et al., 2008), Caco-2 cells (Aherne and O'Brien, 1999), HMB-2 cells (Horvathova *et al.*, 2005), human macrophage (U-937 cells) (Kanupriya et al., 2006; Moon et al., 2006); human lymphocytes (healthy volunteers) (Wilms *et al.*, 2007), and murine leukemia L1210 cells (Horvathova *et al.*, 2003). Wilms *et al.*, (2005) reported the protective effects of quercetin against the formation of oxidative DNA damage and bulky DNA adducts in human lymphocytes, induced by H2O2 and benzo(a)pyrene (B(a)P, respectively. In the same study, results obtained in an in vivo experiments showed that four weeks of quercetin-rich blueberry/apple juice mixture

DNA Damage Protection and Induction of Repair

hydrogen peroxide.

**6.1.2 Carotenoids** 

(Panayiotidis and Collins, 1997).

by Dietary Phytochemicals and Cancer Prevention: What Do We Know? 251

Naringin, a citrus grapefruit flavonone, showed antigenotoxic properties in V79 cells (Jagetia *et al.*, 2007). 4-Coumaric acid, a phenolic acid present in many foods and drinks, such as wine, tea, berries, apples, spinach and cereals reduced basal oxidative DNA damage in rat colonic mucosa when evaluated by comet assay (Guglielmi *et al.*, 2003). Resveratrol, found in substantial amounts in several types of beverages including red wine and fruits, showed genoprotective effects under conditions of oxidative stress induced by H2O2 in C6 glioma cells (Quincozes-Santos et al., 2007). Apigenin, a flavonoid widely distributed in many herbs, fruits, and vegetables, has shown chemopreventive activities in several biological systems. Jeyabal *et al.*, (2005) reported the protective role of apigenin against the oxidative stress caused by N-nitrosodiethylamine and phenobarbital in Wistar albino rats. Rusak et al., (2010), showed genoprotective effects of apigenin and other flavonoids (luteolin and kaempferol) in human peripheral lymphocytes against oxidative damage induced by

Tomato is one of the main sources of lycopene, a carotenoid with antioxidant properties. Pretreatment of rat hepatocytes with lycopene (1.86, 9.31 and 18.62 µM) in culture, showed a significant decrease in the levels of TBARS and DNA damage induced by gamma-radiation. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as, the levels of GSH, vitamins A, E, C, increased significantly when hepatocytes were pretreated with the carotenoid (Srinivasan et al., 2007). At physiological concentrations (0.3-10 µM) lycopene and beta-carotene also protect Chinese hamster lung fibroblasts against DNA damage induced by 3-morpholinosydnonimine (SIN-1) (Muzandu et al., 2006). In a human intervention study, healthy volunteers were submitted to a supplementation with lycopene during 8 weeks. Consumption of lycopene decreased oxidative DNA damage in lymphocytes and urinary 8-hydroxy deoxoguanosine levels when compared with the basal levels (Devaraj et al., 2008). Also, Zhao *et al.*, (2006) evaluated the protective effect of lycopene and others carotenoids against DNA damage in humans. Here, thirty-seven healthy, nonsmoking postmenopausal women consumed for 56 days a daily dose of mixed carotenoids (lycopene, lutein and beta-carotene; 4 mg each), 12 mg of a single carotenoid (lycopene, lutein or beta-carotene), or placebo. At the end the lymphocytes were isolated and DNA damage measured by comet assay. The results showed that all groups that consumed carotenoids had significantly lower endogenous DNA damage than that found on baseline measurements. No differences were found in placebo group. Lutein also decreased DNA damage induced by cisplatin in mice when evaluated by comet assay and increased GSH levels when compared with a negative control group (Serpeloni *et al.*, 2010). Lorenzo *et al.*, (2009) reported that β-cryptoxanthin at low concentrations, close to those found in plasma, protects Caco-2 and HeLa cells from oxidative DNA damage induced by H2O2 or by visible light in the presence of a photosensitizer. Consumption of βcarotene decreased the number of strand breaks induced by H2O2 in lymphocytes

Pre-treatment with astaxanthin, a red carotenoid used as a dietary additive, at 12.5, 25 and 50 mg/kg/day for 5 days before cyclophosphamide treatment resulted in the amelioration of antioxidant defenses (glutathione and superoxide dismutase) in the liver and decreased DNA damage evaluated by standard comet assay and using specific-enzymes (FPG and EndoIII) in bone marrow cells and peripheral blood lymphocytes isolated from mice. This

Fig. 2. Classification of phytochemicals and the main natural compounds in each group.

consumption led to a decrease of oxidative DNA damage and BPDE-DNA adduct levels by 41% and 11%, respectively, although the results were not statistically significant. Increases in the total antioxidant capacity of plasma and in plasma quercetin content were observed. Others flavonoids, such as luteolin (Cai *et al.*, 1997; Horvathova et al., 2005; Lima et al., 2006; Min and Ebeler, 2008; Ramos *et al.*, 2010b), rutin (Moon et al., 2006; Ramos et al., 2008), genistein (Cai et al., 1997), catechin and epicatechin (Kanupriya et al., 2006), showed antigenotoxic effects against oxidative DNA damage in several cell models.

In an ex vivo study, lymphocytes from three healthy subjects were pre-incubated with several dietary antioxidants. Quercetin and caffeic acid ( a phenolic acid) protected against H2O2 induced DNA damage, however in this study no effect was observed when cells were treated with catechin, epicatechin, catechin gallate and epicatechin gallate (Szeto and Benzie, 2002).

Besides quercetin and rutin, we reported that ursolic acid (a triterpenoid) protect against DNA damage induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells (Ramos et al., 2008). Other reports showed that ursolic acid and/or luteolin protect DNA from damage induced by H2O2, t-BHP or AZT (3\_-azido-3\_-dideoxythymidine) in several cell lines, such as leukemic cells, HEI-OC1 auditory cells and PC12 cells (Horvathova et al., 2003; Horvathova *et al.*, 2004; Ovesna *et al.*, 2006; Yu et al., 2009; Noroozi *et al.*, 1998; Silva *et al.*, 2008). Rosmarinic acid (RA), reduced the frequency of micronuclei and the extent of DNA damage induced by doxorubicin in V79 cells (Furtado *et al.*, 2009). Caffeic acid (3,4-dihydroxy cinnamic acid), also a dietary phenolic compound, showed a photoprotective effect (Devipriya *et al.*, 2008; Benkovic et al., 2009). Human blood lymphocytes irradiated with UVB (280-320) after pretreatment with caffeic acid exhibited lower levels of lipid peroxidation markers such as thiobarbituric acid reactive substance (TBARS) and lipid hydroperoxide (LPH) and also a decrease of UVBinduced DNA damage (Prasad *et al.*, 2009).

Naringin, a citrus grapefruit flavonone, showed antigenotoxic properties in V79 cells (Jagetia *et al.*, 2007). 4-Coumaric acid, a phenolic acid present in many foods and drinks, such as wine, tea, berries, apples, spinach and cereals reduced basal oxidative DNA damage in rat colonic mucosa when evaluated by comet assay (Guglielmi *et al.*, 2003). Resveratrol, found in substantial amounts in several types of beverages including red wine and fruits, showed genoprotective effects under conditions of oxidative stress induced by H2O2 in C6 glioma cells (Quincozes-Santos et al., 2007). Apigenin, a flavonoid widely distributed in many herbs, fruits, and vegetables, has shown chemopreventive activities in several biological systems. Jeyabal *et al.*, (2005) reported the protective role of apigenin against the oxidative stress caused by N-nitrosodiethylamine and phenobarbital in Wistar albino rats. Rusak et al., (2010), showed genoprotective effects of apigenin and other flavonoids (luteolin and kaempferol) in human peripheral lymphocytes against oxidative damage induced by hydrogen peroxide.
