**3. Mechanisms of damage**

128 Selected Topics in DNA Repair

afterwards binds to DNA via the xenobiotic response element (XRE) situated in the promoter region of CYP1A and CYP1B genes (Shimada, et al., 2002)). CYP3A4 and CYP3A5 are known to activate PAHs present in cigarette smoke, such that increased protein levels and activity of these enzymes in cells exposed to smoke have been detected (Piipari, et al., 2000)). Furthermore, genetic variants of CYPs are associated with risk of carcinogenesis. Some polymorphisms of CYP1A1 are associated with adduct formation and mutagenesis in several populations and species (Ichiba, et al., 1994, Rojas, et al., 2000, Shields, et al., 1992). This fact is related to the metabolizing rate and the subsequent repair mechanism associated with DNA damage. Polycyclic aromatic hydrocarbons are activated by a pathway that

Other phase I enzymes related to PAHs metabolism are the aldo-keto reductases. These enzymes oxidize polycyclic aromatic (PAH) trans-dihydrodiols to reactive and redox-active o-quinones *in vitro* (Quinn & Penning, 2006). Specifically, AKR1A1, and members of the AKR1C dihydrodiol/hydroxysteroid dehydrogenase subfamily, AKR1C1-AKR1C4 are involved in metabolic activation of PAH trans-dihydrodiol. Production of o-quinone metabolites by these enzymes has been shown *in vitro* and in cell lines to amplify ROS and oxidative damage to DNA bases to form the highly mutagenic lesion 8-oxo-dGuo and

Fig. 2. Mechanism of activation of BaP by cytochrome P450 (CYP) and epoxide hydrolase

Phase II metabolism includes conjugation of metabolites from phase I with small molecules catalyzed by specific enzymes such as sulfotransferases (SULTs), UDP-glucuronyl transferases (UGTs) or glutathione S-transferases (GSTs). SULTs have been shown to activate some metabolites of PAHs such as 7,12-dimethylbenz[a]anthracene and its methyl-hydroxylated derivatives, in different tissues (Chou, et al., 1998). Polymorphisms of SULT1A1 have been associated with PAH-DNA adduct levels (Tang, et al., 2003). Glucuronidation is also a main pathway for PAH detoxification metabolism. Like sulfation, glucuronidation produces polar conjugates that are readily excreted. Oxygenated benzo[*a*]pyrene derivatives are common substrates of UDP-glucuronyltransferase (Bansal, et al., 1981), the resulting metabolite, 1 hydroxypyrene glucuronide, and the parental 1-hydroxypyrene are used as biomarkers of PAH exposure (Strickland, et al., 1994). Finally, GSTs are also involved in conjugation of PAH derivatives. The importance of this activity has been demonstrated *in vitro* using the

involves both CYP enzymes and epoxide hydrolase.

render damaged and carcinogenic DNA (Quinn, et al., 2008).

(EH).

**2.2 Phase II metabolism of PAHs** 

PAHs undergo metabolic activation to diol-epoxides as we discussed before, which bind covalently to DNA. Afterwards, they form adducts or induce oxidative stress that provokes mutations. If DNA repair mechanisms are afflicted by the adduct formation rate the result is an accumulation of mutations in DNA that may induce carcinogenesis. Several studies indicate that the number of adducts formed is related to the degree of PAH exposure. However, is also important to consider the effect of life stage of the organism at exposure to PAHs (Bolognesi, et al., 1991), as well as concentration and genetic profiles of PAHs, among other factors. PAH exposure induces several molecular and cellular responses that modify the endogenous environment. Exposure to PAHs induces genes involved in apoptosis, cell cycle control and DNA repair (Castorena-Torres, et al., 2008).

### **3.1 Adduct formation**

When PAHs are metabolized reactive diol epoxide enantiomers are generated. These enantiomers form DNA adducts with different structures, motifs and biological activities. DNA adducts of diverse conformations are excised by DNA repair enzymes at different rates. PAH diol epoxides (PAHDEs) bind covalently to exocyclic amino groups of guanine and adenine, forming stable adducts within DNA (Lin, et al., 2001). Futhermore, there are correlations between DNA adduct levels and mutagenesis. The structure of some PAHDEs forms a region called "Fjord", which some studies indicate is a region that is highly involved with high tumorigenicity. These molecules are mostly non-planar, reactive, and bind preferentially to adenine nucleotides. On the other hand, PAHDES with a "bay" region are planar, less reactive and bind to guanine nucleotides (Fig. 4). Geacintov and colleagues (1997) have described several structural motifs by nuclear magnetic resonance analysis. These structural types are divided into: (a) minor groove, when the PAH is partially accessible to the solvent; (b) classical intercalation, when the PAH is protected from the environment and forms a "sandwich structure" and (c) base-displaced intercalation, when PAHs substitute the healthy base (Buterin, et al., 2000, Geacintov, et al., 1997).

Molecular studies have revealed that adducts in DNA block polymerase replication activity, contributing to increased DNA damage by reducing repair activity (Hsu, et al., 2005). An example of adduct formation between adenine or guanine and benzopyrene diol epoxide (BPDE) is shown in Figure 5. Interestingly, some compounds present in food are capable of preventing adducts such as ellagic acid (EA) by the formation of adducts previous to DNA binding (Lagerqvist, et al., 2011). The presence of adducts have been evaluated in marine and aquatic species as an indicator of environmental occurrence of PAHs. Some studies have revealed, by X-ray crystallography, structures of PAH-adducted oligonucleotides bound to bacterial DNA polymerases (Hsu, et al., 2005, Ling, et al., 2004).

DNA Damage Caused by Polycyclic Aromatic Hydrocarbons: Mechanisms and Markers 131

It has been reported that BaP derivatives have the capacity to enter redox cycles and induce the production of reactive oxygen species (ROS), thereby causing oxidative stress (An, et al., 2011). BaP radical-cations are precursors for 6-OH-BaP. Auto-oxidation of this derivative may result in the formation of BaP quinones such as 6, 12-, 1,6- and 3,6-BaP dione (Briede, et al., 2004). These metabolites can undergo redox-cycling to their corresponding BaP diols and produce superoxide reactive oxygen species which are then converted to hydroxyl radicals by the Haber-Weiss reaction (Lesko & Lorentzen, 1985). Free radicals react with guanine and cause DNA damage, including the production of 7-hydro-8-oxo-20-deoxyguanosine (8-oxodG) (Chatgilialoglu & O'Neill, 2001). The OGG1 gene codes for a DNA glycosylase involved in base excision repair of 8-oxo-dG that arises from ROS. When this system fails there is an increase in mutation rate (Bonner, et al., 2005). Balance between generation of ROS species and scavenging of these molecules is fundamental in repairing DNA damage. If the rate of ROS generation is greater than their removal it is likely that more DNA damage will result. PAHs may absorb light energy in UVR (280–400 nm) region and may induce DNA damage by production of ROS. For example, chrysene, induces apoptosis and DNA damage in human keratinocytes by generating ROS in response to UVB radiation (Ali, et al., 2011).

The failure of repair mechanisms and constant exposure to PAHs induce mutagenesis in cells. These mutations are present in multiple genes including those that participate in cell survival. In particular, p53 mutations are associated with risk of carcinogenesis in PAHexposed individuals. Since the p53 protein is a transcription factor that regulates cell proliferation, differentiation, apoptosis, and DNA repair, mutations induced in this important protein could lead to severe damage in cells and genes. Some studies have associated p53 mutations to PAH exposure (Mordukhovich, et al., 2010, Yoon, et al., 2003). Another common target of mutagenesis is the ras gene (Ross & Nesnow, 1999). A study by Gray et al. (2001) revealed that exposure to BaP in mice increases mutation of the K-ras gene.

Forming adducts in DNA repair-related genes is not the only mechanism by which PAHs induce carcinogenesis. An additional danger amounts from their resemblance to steroid hormones allowing PAHs the ability to activate estrogen receptors and metabolism. The ability of several PAHs to displace natural estrogens and occupy ER binding sites, at least to some extent, implies a potential mechanism of action in endocrine tissues that is ER-

Fig. 4. Difference between bay and fjord regions in two PAH conformations.

**3.2 Oxidative stress** 

**3.3 Mutations** 

**3.4 Carcinogenesis** 

Fig. 3. Aryl hydrocarbon receptor (AhR) pathway activated by BaP induces expression of *cyp1A1* and *cyp1B1*.

Fig. 4. Difference between bay and fjord regions in two PAH conformations.
