**2.2 Alkylating DNA damage**

238 Selected Topics in DNA Repair

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

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

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-7,8-

drugs will also be referred.

maintain genomic stability.

**2.1 Oxidative DNA damage** 

**2. DNA damage and genomic stability** 

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

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., 2010).

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 addressed.
