**2.1 Phase I metabolism of PAHs**

126 Selected Topics in DNA Repair

damage can also result. In this chapter, we review the mechanisms of damage caused by exposure to PAHs, factors involved in repairing the damage, and the important role of

Once PAHs enter the body they are metabolized in a number of organs (including liver, kidney, lungs), excreted in bile, urine or breast milk and stored to a limited degree in adipose tissue. The principal routes of exposure are: inhalation, ingestion, and dermal contact. The lipophilicity of PAHs enables them to readily penetrate cellular membranes (Yu, 2005). Subsequent metabolism renders them more water-soluble making them easier for the body to remove. However, PAHs can also be converted to more toxic or carcinogenic

Fig. 1. Structures of some polycyclic aromatic hydrocarbons.

biomarkers.

metabolites.

**2. Metabolism of PAHs** 

There are three main pathways for activation of PAHs: the formation of a PAH radical cation in a metabolic oxidation process involving cytochrome P450 peroxidase, the formation of PAH-o-quinones by dihydrodiol dehydrogenase-catalyzed oxidation and finally the creation of dihydrodiol epoxides, catalyzed by cytochrome P450 enzymes (Guengerich, 2000). The most common mechanism of metabolic activation of PAHs, such as benzo[a]pyrene (B[a]P), is via the formation of bay-region dihydrodiol epoxides e.g. benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), via CYP450 and epoxide hydrolase (EH) (Fig. 2). The most important enzymes in the metabolism of PAHs are CYPs 1A1, 1A2, 1B1 and 3A4. CYP1A1 is highly inducible by PAHs such as B[a]P and some polyhalogenated hydrocarbons. Recombinant human CYP1A1 metabolizes compounds such as B[a]P, 2 acetylaminofluorene and 7,8-diol,7-12-dimethylbenz[a]anthracene (Kim, et al., 1998). CYP1A2 and CYP1B2 are also inducible by the exposure to PAHs. In fact, these enzymes share the same mechanism with which PAH molecules interact with, the aryl hydrocarbon receptor (AhR). The AhR is present in the cytoplasm as a complex with other proteins such as heat shock protein 90 (Hsp90), p23 and AhR-interacting protein. After forming a complex with PAHs, the Hsp90 is released and the AhR-PAH complex translocates to the nucleus (Fig 3). Here, it creates a heterodimer with a ARNT (Ah Receptor Nuclear Translocator) and

<sup>1</sup> CAS (Chemical Abstracts Service) registry number

DNA Damage Caused by Polycyclic Aromatic Hydrocarbons: Mechanisms and Markers 129

corresponding diol epoxides of dibenzo[*a,l*]pyrene and benzo[*a*]pyrene as substrates for these enzymes (Sundberg, et al., 2002). Activity of GSTs is related to their glutathione redox status and genotoxic damage, at least in placenta tissue, therefore this status can be used as biomarker of PAH exposure (Obolenskaya, et al., 2010). On the other hand, polymorphisms of phase II metabolism are associated with carcinogenesis and with DNA damage. For instance, there is an important association between GSTM1 gene polymorphism and the DNA adduct levels (Binkova, et al., 2007). GSTs are also important for quenching and detoxifying ROS and

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

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).

bound to bacterial DNA polymerases (Hsu, et al., 2005, Ling, et al., 2004).

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

their derivatives (Bonner, et al., 2005).

cycle control and DNA repair (Castorena-Torres, et al., 2008).

**3. Mechanisms of damage** 

**3.1 Adduct formation** 

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 involves both CYP enzymes and epoxide hydrolase.

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

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

#### **2.2 Phase II metabolism of PAHs**

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 corresponding diol epoxides of dibenzo[*a,l*]pyrene and benzo[*a*]pyrene as substrates for these enzymes (Sundberg, et al., 2002). Activity of GSTs is related to their glutathione redox status and genotoxic damage, at least in placenta tissue, therefore this status can be used as biomarker of PAH exposure (Obolenskaya, et al., 2010). On the other hand, polymorphisms of phase II metabolism are associated with carcinogenesis and with DNA damage. For instance, there is an important association between GSTM1 gene polymorphism and the DNA adduct levels (Binkova, et al., 2007). GSTs are also important for quenching and detoxifying ROS and their derivatives (Bonner, et al., 2005).
