**6.2.2 Whole foods**

Many whole foods and plant crude extracts have also been found to protect against alkylating damage. The water extract of *Salvia officinalis* prevent formation of aberrant crypt foci (ACFs, a pre-carcinogenic lesion) induced by AOM in rat colon and also protected DNA

DNA Damage Protection and Induction of Repair

poor correlation between activity and mRNA was found.

a specific type of damage.

**6.3.1 Polyphenols** 

**6.3.2 Carotenoids** 

**6.3.3 Whole foods** 

fibroblasts.

2007).

by Dietary Phytochemicals and Cancer Prevention: What Do We Know? 255

measures the ability of cells to rejoin strand breaks induced by a DNA damaging agent; while the "in vitro repair assay" measures the excision repair activity of a protein extract prepared from cells treated with dietary agents, incubating with a DNA substrate containing

Recently, we reported that luteolin and luteolin-7-glucoside increased rejoining of strand breaks after treatment with H2O2 in Caco-2 cells. Luteolin-7-glucoside also had a BER inductive effect increasing incision activity in Caco-2 cells (Ramos *et al.*, 2010a; Ramos *et al.*, 2010b). Quercetin also increased rejoining of strand breaks induced by *t*-BHP in HepG2 (Ramos et al., 2008). Moreover, dietary agents such as flavonoids, vitamins E and C had been reported as inducers of oxidative DNA damage repair activity (Davis and Milner,

An in vitro study using HeLa and Caco-2 cells reported that -cryptoxanthin increased rejoining of strand breaks induced by H2O2 and increase the repair of oxidised bases. However, increase of repair activity was not related with increase of hOGG1 or APE1expression (Lorenzo et al., 2009). The lack of correlation between activity and mRNA expression of OGG1 and APE1 has also been demonstrated in other studies (Collins *et al.*, 2003; Silva *et al.*, 2009). -carotene, lutein and lycopene also enhanced strand breaks rejoining in lymphocytes (Fillion *et al.*, 1998; Torbergsen and Collins, 2000). Paz-Elizur *et al.*, (2007) measured OGG1 activity and mRNA expression in 120 healthy individuals and a

Water extracts of Salvia species increased rejoining of strand breaks after treatment with H2O2 in Caco-2 cells. These water extracts also increased incision activity of a Caco-2 cell extract on a DNA substrate containing specific oxidative damage (8-oxoGua) (Ramos *et al.*, 2010a). Nakamura et al., (2000) reported that aqueous fractions of Fushimi sweet pepper increase repair against ultraviolet-induced cyclobutane pyrimidine dimers in human

Collins et al., (2003), in a human intervention study, showed that 3 weeks of a dietary supplementation with kiwifruit increased DNA repair capacity measured by "in vitro repair assay". Also Freese, (2006) showed that kiwifruit consumption increased DNA repair capacity in human lymphocytes. Brevik *et al.*, (2011) in a human biomonitoring study, reported that consumption of kiwifruits and antioxidant-rich plant products reduced DNA strand breaks in lymphocytes. Increase of BER activity was observed in the group that consumed antioxidant-rich plant products. However, a reduction of NER activity was observed in both groups. No explanations have been found for this decrease in NER pathway. Diet supplementation with cooked carrots, during 3 weeks, increased in vitro

High dietary folate intake has been associated with a decreased risk of cancer development, such as colorectal cancer. In vitro, rodent and human studies demonstrated that low folate intake increases uracil misincorporation leading to increase of DNA damage, chromosomal breakage and malignant transformation; modulates DNA repair by inhibiting thymidine

repair activity and strand break rejoining in lymphocytes (Astley *et al.*, 2004).

of colonocytes (unpublished observations). Using the same experimental model, Sengupta *et al.*, (2004 and 2003) reported that tomato and garlic prevent ACFs, induced by AOM in rat colon. Tomato also decreased incidence and progression of 9,10-dimethyl benzanthracene (DMBA)-induced mouse skin tumours (De and Das, 2001). Intake of beer reduced ACF formation and protected against DNA damage induced by AOM in the rat colon mucosa (Nozawa et al., 2004). de Lima *et al.*, (2005) evaluated the effect of aqueous extract of propolis on the formation of DMH-induced ACF and DNA damage in rat colon. Propolis had no effect on ACF formation, however, modulation of DMH-induced DNA damage in colon cells was observed. At lower concentrations (12, 34 and 108 mg/Kg bw/day) aqueous extract of propolis decrease the level of DNA damage. However, the highest concentration (336mg/Kg bw/day) induced DNA damage in rat colon. Dietary agents such as, ginseng, lemon grass and propolis, have been found to inhibit DMH- and AOM-induced colon carcinogenesis and DNA damage in animal model (Bazo et al., 2002; Suaeyun *et al.*, 1997; Volate et al., 2005). Consumption of onion, blueberries (Boateng et al., 2007), and garbanzo beans (Murillo *et al.*, 2004) also decreased the number of AOM-induced ACFs in rats and mice, respectively.

Some studies reported antigenotoxic effects of diet against alkylating DNA damage using cytogenetic assays (micronucleus assay) (Azevedo Bentes Monteiro Neto et al., 2011; Gurbuz *et al.*, 2009). Edenharder *et al.*, (1998) reported that sweet cherries, strawberries, cucumber, tomatoes, bananas, oranges, asparagus, yellow red peppers and specially spinach had a protective effect against clastogenicity of cyclosphosphamide (an alkylating drug) in mice.

Using comet assay some in vivo studies have shown the antigenotoxic effects of dietary agents, namely, artepillin C (a polyphenolic acid found in Brazilian green propolis and Baccharis dracunculifolia) (Azevedo Bentes Monteiro Neto et al., 2011), safranal, (a constituent of Crocus sativus L. stigmas) (Hosseinzadeh and Sadeghnia, 2007), orange juice (Franke *et al.*, 2005b) and vitamin C (Franke *et al.*, 2005a) against DNA damage induced by methyl methanesulfonate (MMS). Also lemongrass protected leukocytes from DNA damage induced by N-methyl-N-nitrosurea (MNU) (Bidinotto *et al.*, 2010). Data from application of comet assay in the assessment of genoprotective effects of diet against alkylating DNA damage is limited. First, AlkA is, as far as we know, the only repair enzyme used for detection of alkylating DNA damage by the comet assay (Collins *et al.*, 2001a). Alk A recognises 3-MeA in DNA, but its specificity is low, detecting other modified bases, some of which are also other alkylated bases. Furthermore, 3-MeA is not the most abundant alkylating damage and it does not represent the alkylating damage with more mutagenic/carcinogenic potential. Other alkylating lesions, like N7MeG, the most abundant lesion, and O6MeG, the most mutagenic, are until now undectectable by comet assay.

#### **6.3 Effect of diet on induction of DNA repair**

DNA damage can arrest cell cycle progression to allow DNA repair, preventing, therefore, replication of errors, or to induce apoptosis to eliminate cells severely damaged. Defective DNA repair is usually linked to human cancer development. Therefore, enhancement of DNA repair can be understood as a prevention strategy against cancer and an important molecular target for dietary phytochemicals (Davis and Milner, 2007). However, few studies have investigated whether DNA repair activity can be modified by diet in humans.

New modifications of the comet assay have been developed to assess effects of dietary agents on DNA repair ability (as described above). Briefly, the "cellular repair assay" measures the ability of cells to rejoin strand breaks induced by a DNA damaging agent; while the "in vitro repair assay" measures the excision repair activity of a protein extract prepared from cells treated with dietary agents, incubating with a DNA substrate containing a specific type of damage.
