**DNA Damage Protection and Induction of Repair by Dietary Phytochemicals and Cancer Prevention: What Do We Know?**

Alice A. Ramos1, Cristóvão F. Lima2 and Cristina Pereira-Wilson1 *1CBMA – Molecular and Environmental Biology Centre 2CITAB – Centre for the Research and Technology of Agro-Environmental and Biological Sciences Department of Biology, School of Sciences, University of Minho, Campus de Gualtar, Braga, Portugal* 

#### **1. Introduction**

236 Selected Topics in DNA Repair

Zhang, C., Guo, H., Zhang, J., Guo, G., Schumaker, K.S., and Guo, Y. (2010). Arabidopsis

Zhang, Y., and Schroeder, D.F. (2010). Effect of overexpression of Arabidopsis damaged

DNA-binding protein 1A on de-etiolated 1. Planta 231, 337-348.

Plant Cell 22, 2353-2369.

cockayne syndrome A-like proteins 1A and 1B form a complex with CULLIN4 and damage DNA binding protein 1A and regulate the response to UV irradiation.

> DNA damage accumulates in cells over time as a result of exposure to a variety of exogenous and endogenous agents. These damages, if not repaired properly, can generate mutations in somatic or germline cells, which are involved in the pathogenesis of many diseases, such as cancer. To maintain genomic integrity generation after generation, organisms possess multiple mechanisms such as inhibition of carcinogen uptake into the cells, induction of detoxification enzymes, increased cellular defenses that prevent DNA damage, enhancement of DNA repair, increased anti-inflammatory activity, inhibition of cell proliferation, and modulation of apoptosis through effects on signal transduction pathways (de Kok *et al.*, 2008); (Pan *et al.*, 2008). In the last decades, several dietary constituents have been shown to modulate all these processes. Epidemiological studies as well as laboratory data suggest that consumption of fruits and vegetables is associated with a reduced risk of developing a wide range of cancers. It has been estimated that 30- 40% of all tumours can be prevented with a correct lifestyle and diet, in particular colon cancer (Rajamanickam and Agarwal, 2008). Multiple mechanisms have been proposed to explain the chemopreventive effects of phytochemicals. Protection of DNA from damage and modulation of DNA repair assume an important role on prevention of mutations and consequently of the carcinogenic process. The comet assay or single cell gel electrophoresis (SCGE) assay is a rapid, sensitive and relatively simple method for assessing DNA damage and its repair in individual cells. The standard comet assay measures DNA breaks and alkali-labile sites that are converted to strand breaks. With its widespread use, several modifications on the comet assay have been made that allow the quantification of other types of DNA damage as well as DNA repair rates. With the inclusion of an extra step on the comet assay by using specific DNA repair enzymes, different base lesions can be identified by the introduction of breaks at sites of base damage. In this regard, Endonuclease III, FPG and AlkA have been used to detect oxidized pyrimidines, modified purines and alkylpurines bases, respectively. With these

DNA Damage Protection and Induction of Repair

**2.2 Alkylating DNA damage** 

2010).

addressed.

**3. Mechanisms of DNA repair** 

transversions through mispairing in replication.

antioxidant have been found in tumor cells (Maynard et al., 2009).

by Dietary Phytochemicals and Cancer Prevention: What Do We Know? 239

dihydroguanine) is one of the most abundant forms of DNA oxidation and can cause G to T

Tumor cells are characterized by high levels of ROS. Some studies showed that human tumor cells have an increase of levels of 8-oxoG relative to normal cells. Also, low levels of

The removal of oxidative lesions by cellular repair processes is essential for maintaining genome integrity and survival limiting mutagenesis. However, there are a number of studies indicating that oxidative DNA damage could not account entirely by itself for tumor development, since elevated levels of 8-oxoGua have been shown not to reflect reflect increased cancer rates. In fact, oxidative damage is the most studied DNA damage, however, alkylating DNA damage is not less important. Human exposure to alkylating agents can arise from diet (e.g. presence of heterocyclic amines on food), environment (e.g. exposure to cigarette smoke and fuel combustion), or produced endogenously (e.g.

Alkylating agents can cause a wide spectrum of DNA adducts, including N-alkylated adducts, such as N7-methylguanine (N7MeG), N3-methyladenine (N3MeA) and N3 methylguanine (N3MeG), and O-alkylated adducts, such as O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT). N-alkylated adducts correspond to more than 80% of alkylated bases and exhibit different stabilities. For example, N7MeG (correspond aprox. 70%) is the most stable N-methylation adduct in vitro with 80hr of half-life. N3MeA and N3MeG are less abundant with 9 and 2%, respectively, of total methylation adducts. O6MeG vary from 0.3% (for methyl methanesulfonate) to 8% (for methylnitrosourea) of the total DNA methyl adducts and it is stable in the absence of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). O4MeT is produced in very low amounts (<0.4% of the total DNA methyl adducts) and its mutagenicity and cytotoxicity are unclear. In general, Oalkylations are highly mutagenic and genotoxic, whereas N-alkylations are cytotoxic, but less mutagenic (Drablos *et al.*, 2004; Kondo *et al.*, 2010). O6MeG is the major pre-mutagenic, pre-carcinogenic and pre-cytotoxic DNA lesion induced by methylating agents (Wirtz et al.,

Prevention of DNA damage and modulation of DNA repair by dietary phytochemicals phytochemicals is the main focus of this review but first a brief overview of DNA repair mechanisms will be presented. Effects on chemoprevention of colon cancer will be

Maintainance of genomic integrity is complex due to great diversity of damage that can occur in DNA. In contrast to other biomolecules, DNA cannot be replaced, only repaired. To avoid the deleterious consequences of damage accumulation, cells have a variety of DNA repair pathways, each recognize and repair specific types of DNA damage. Base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), non-homologous end-joining repair (NHEJ) and direct damage reversal repair are some of the most important pathways used by cells to repair oxidative and alkylating DNA damage (Table I). These cellular repair pathways are not completely independent from one another. Some studies have show physical interactions between some

nitrosation of amides and amines mediated by enteric bacteria) (Wirtz et al., 2010).

modifications the comet assay can be used to estimate oxidative and alkylating DNA damage. Cells' DNA repair capacity can also be measured by using modified protocols of the comet assay, such as following the repair kinetics by the cellular repair assay, or in vitro by using cellular substrates with specific damages to measure base excision repair (BER) and nucleotide excision repair (NER) (Collins *et al.*, 2001b; Collins, 2004). These different modifications of the comet assay have been successfully used to evaluate the chemopreventive potential of several phytochemicals present in our diet. In this review, we will show evidence that dietary agents can protect DNA from oxidative and alkylating agents as well modulate DNA repair in eukaryotic cells, by using the comet assay. Data from different experimental systems, including primary cell cultures, human cell lines, animal models and human biomonitoring studies, will be discussed in order to provide an overview of effects of dietary phytochemicals on DNA damage and repair with particular emphasis on colon cancer chemoprevention. Recently, we have shown that dietary phytochemicals such as quercetin, rutin, rosmarinic acid, luteolin and others not only protect DNA damage but also stimulate DNA repair in liver and colon cell lines (Lima *et al.*, 2006; Ramos *et al.*, 2008; Ramos *et al.*, 2010b; Ramos *et al.*, 2010a). These effects may contribute to their anti-carcinogenic effects. Effects of phytochemicals through DNA repair modulation and their interaction with alkylating agents used as chemotherapeutic drugs will also be referred.

### **2. DNA damage and genomic stability**

Cells of all organisms are under continual attack from reactive oxygen species (ROS) and alkylanting species generated by environmental pollutants, drugs, radiation, cigarette smoke and endogenous metabolism. Theses endogenous and exogenous agents can induce harmful effects if the cell's defense mechanisms are not enough to maintain cellular redox homeostasis. Protection and repair of DNA damage represent two important mechanisms to maintain genomic stability.

#### **2.1 Oxidative DNA damage**

Endogenous and exogenous agents can induce disruption of the cellular redox homeostasis in favor of oxidant state. This imbalance is described as oxidative stress. As a consequence, different types of molecules such as proteins, lipids and nucleic acids, can be damaged, resulting in severe metabolic dysfunction (including lipid peroxidation, protein oxidation, membrane disruption and DNA damage) (Aherne *et al.*, 2007). Oxidative stress has been involved in the development of several pathologies such as certain cancers, once it may lead to mutations that activate oncogenes or inactivate tumor suppressor genes (Allen and Tresini, 2000; Maynard *et al.*, 2009). In tumor cells, oxidative stress can act as a selective factor in favour of these cells, through induction of DNA damage that may generate more mutations; activation of growth-promoting transcription factors and modulation of genes involved in apoptosis and proliferation (Allen and Tresini, 2000; Karihtala and Soini, 2007). A significant consequence of oxidative stress is DNA damage, which may result in genomic instability. It has been estimated that around 104 lesions are induced in a mammalian cell genome every day (Hegde *et al.*, 2008). There are several types of oxidative DNA damage, such as oxidized bases, abasic sites (also called apurinic/apyrimidinic (AP)) and DNA strand breaks. Hydroxylation of guanine at C-8 position, 8-oxoGua (8-Oxo-7,8dihydroguanine) is one of the most abundant forms of DNA oxidation and can cause G to T transversions through mispairing in replication.

Tumor cells are characterized by high levels of ROS. Some studies showed that human tumor cells have an increase of levels of 8-oxoG relative to normal cells. Also, low levels of antioxidant have been found in tumor cells (Maynard et al., 2009).
