**4. Mechanisms of repair**

Several molecular processes exist in cells that repair damage caused from PAH exposure, adduct formation, and ROS production. If the damage is repaired by these mechanisms no further consequences should result. However, if the lesions escape repair and survive to the next round of DNA replication faulty translesion bypass can occur causing mutagenesis and carcinogenesis. The most common mechanisms of repair used by cells exposed to PAHs are: nucleotide excision repair (NER), base excision repair (BER), non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs), homologous recombinational repair (HRR) and transcription coupled repair (TCR). On the other hand, some studies indicate that DNA damage induced by PAHs is preferentially repaired by NER or BER (Braithwaite, et al., 1998), and to a lesser extent by HRR (Meschini, et al., 2010).

#### **4.1 Nucleotide excision repair (NER)**

In this process, cells recognize damaged DNA regions based on their abnormal structure or chemistry, then excise and replace them. This pathway is complex, requiring more than 20 different proteins. NER is considered the main pathway for removal of bulky DNA adducts (Braithwaite, et al., 1998). There are two distinct forms of NER: GG-NER (Global Genomic-NER) and TC-NER (Transcription Coupled-NER). The first corrects damage in transcriptionally silent areas of the genome, while the second repairs lesions on the actively transcribed strand of the DNA. In GG-NER, the XPC (Xeroderma Pigmentosum Complementation Group-C)/hHR23B (Rad23 homolog B) protein complex is responsible for

the initial detection of damaged DNA. On the other hand, TC-NER does not require XPC, however the stalled RNA Polymerase complex is displaced in order to allow the NER proteins to access the damaged DNA. After this process, TC-NER and GG- NER proceed in identical ways. XPA and RPA (Replication Protein-A) then bind at the location of injury and further aid in damage detection. Subsequently, the XPB and XPD helicases unwind the DNA duplex in the surrounding area of the lesion. The endonucleases XPG and ERCC1 (Excision Repair Cross-Complementing group-1)/XPF then cleave one strand of the DNA at positions 3' and 5' to the damage, respectively, generating a 30 base oligonucleotide containing the lesion. This oligonucleotide is displaced, permitting gap repair synthesis (performed by DNA Pol Delta/Epsilon, and other accessory proteins). Finally, DNA ligase seals the nick in the repaired strand (Fig. 6). Several studies demonstrate that certain polymorphisms in NER genes alter the efficiency of DNA repair (Shen, et al., 2006, Vodicka, et al., 2004). Four polymorphisms, XPA - 4G/A (rs1800975), ERCC1 C8092A (rs3212986), XPD Lys751Gln (rs1052559), and XPF Ser835Ser (rs1799801), are associated with a reduced capacity for DNA repair and an increased susceptibility to various cancers (Hu, et al., 2004, Monzo, et al., 2007). However, a recent study reports that significant opportunities exist for an interaction between the XPA-4 G/A polymorphism and PAH exposure on sperm DNA damage (Gu, et al., 2010). Although some PAHs lack "bay" and/or "fjord" regions, such as anthracene for example, this molecule also induces DNA damage, activating repair mechanisms, and has in fact has been shown to induce NER and MMR pathways (Desler, et al., 2009).These findings provide support for the importance of NER pathway in DNA damage induced by PAHs.

### **4.2 Base excision repair (BER)**

132 Selected Topics in DNA Repair

mediated (Santodonato, 1997). Nevertheless, the most important mechanism of carcinogenesis is a deficient DNA repair system in key genes involved in cell cycle control. Since chronic exposure to PAHs is related to a high rate of mutagenesis it is probable that damage to DNA is cumulative. Several studies have associated chronic occupational PAH exposure to multiple types of cancer including cancer of the bladder, lung, kidney, liver, and breast (Boffetta, et al., 1997, Dickey, et al., 1997, Karami, et al., 2011, Shen, et al., 2003).

Fig. 5. Structures and interaction of two common adducts: (A) BPDE-dG adduct and (B)

Several molecular processes exist in cells that repair damage caused from PAH exposure, adduct formation, and ROS production. If the damage is repaired by these mechanisms no further consequences should result. However, if the lesions escape repair and survive to the next round of DNA replication faulty translesion bypass can occur causing mutagenesis and carcinogenesis. The most common mechanisms of repair used by cells exposed to PAHs are: nucleotide excision repair (NER), base excision repair (BER), non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs), homologous recombinational repair (HRR) and transcription coupled repair (TCR). On the other hand, some studies indicate that DNA damage induced by PAHs is preferentially repaired by NER or BER (Braithwaite, et al.,

In this process, cells recognize damaged DNA regions based on their abnormal structure or chemistry, then excise and replace them. This pathway is complex, requiring more than 20 different proteins. NER is considered the main pathway for removal of bulky DNA adducts (Braithwaite, et al., 1998). There are two distinct forms of NER: GG-NER (Global Genomic-NER) and TC-NER (Transcription Coupled-NER). The first corrects damage in transcriptionally silent areas of the genome, while the second repairs lesions on the actively transcribed strand of the DNA. In GG-NER, the XPC (Xeroderma Pigmentosum Complementation Group-C)/hHR23B (Rad23 homolog B) protein complex is responsible for

BPDE-dA adduct.

**4. Mechanisms of repair** 

1998), and to a lesser extent by HRR (Meschini, et al., 2010).

**4.1 Nucleotide excision repair (NER)** 

BER involves the combined activity of some specific proteins that recognize and excise DNA damage, replacing the damaged moiety with normal nucleotides. PAH adducts are repaired by this mechanism. BER consists of three important steps: first, removal of the incorrect base by an appropriate DNA N-glycosylase to create an AP site (apurinic/apyrimidinic site); second, cutting off the damaged DNA strand by AP endonuclease upstream of the AP site, therefore creating a 3'-OH terminus adjacent to the AP site and finally, extension of the 3'- OH terminus by DNA polymerase, accompanied by excision of the AP site. Several enzymes are required to complete these three steps. In humans, there are at least six different glycosylases that bind specifically to a target base and hydrolyze the N-glycosylic bond generating the AP or abasic site. Next, the AP site is processed by the APE1 system (AP Endonuclease-1, or HAP1/REF1/APEX), which cuts the phosphodiester backbone adjacent to the 5' end of the AP site, resulting in a 3' hydroxyl group and a transient 5' dRP (abasic deoxyribose phosphate). The removal of the dRP is accomplished by DNA Pol Beta (polymerase beta) activity, which adds one nucleotide to the 3' end of the nick and removes the dRP moiety through the action of an AP lyase (Bennett, et al., 1997). DNA Pol Beta also interacts with XRCC1. DNA Pol Beta is therefore crucial for the inclusion of different components of BER at sites of DNA damage and promoting repair efficiency (Fig. 6). The BER pathway deals with smaller damage to individual bases, such as oxidation, methylation, depurination, and deamination. If the adducts are left unrepaired, they may cause permanent mutations (Boysen & Hecht, 2003). If these mutations are situated at critical sites, including tumor suppressor genes, DNA repair-related genes or oncogenes, they may lead to cellular transformation and the development of tumors. A recent study demonstrates that BER plays an important role in DNA repair in cells exposed to PAHs. Chinese hamster ovary cells (CHO) deficient in the BER pathway were found to be more

DNA Damage Caused by Polycyclic Aromatic Hydrocarbons: Mechanisms and Markers 135

Fig. 6. Common mechanisms of DNA repair from PAH exposure and adduct formation:

nucleotide excision repair (NER) and base excision repair (BER).

sensitive to damage induced by DBPDE, as measured by frequency of chromosomal aberrations (Meschini, et al., 2010). Polymorphisms in proteins of the BER pathway are associated with an increase in DNA damage. One of these proteins, XRCC1, was discovered to have four functional polymorphisms: T-77C, Arg194Trp, Arg280His and Arg399Gln, associated with alteration in repair capacity of DNA damage induced by PAH adducts (Ji, et al., 2010).

#### **4.3 Homologous recombination (HR)**

Homologous recombination (HR) is a DNA metabolic process found in all forms of life that provides high-fidelity, template-dependent repair or tolerance of complex DNA damage including DNA gaps, DNA double-stranded breaks (DSBs), and DNA interstrand crosslinks (ICLs). The primary function of HR is to search for homology and DNA strand invasion through the Rad51-ssDNA presynaptic filament by positioning the invading 3′-end on a template duplex DNA to initiate repair synthesis. This mechanism participates in DNA repair induced by three specific PAHs: 1-nitrosopyrene (1-NOP), N-acetoxy-2 acetylaminofluorene (N-AcO-AAF), and 4-nitroquinoline 1-oxide (4-NQO). These PAHs were compared for their ability to cause intrachromosomal homologous recombination between two identical genes stably integrated into a genome of a mouse cell. In each case a dose-dependent increase in recombination frequency with differences in the efficiency of each compound was observed (Bhattacharyya, et al., 1989). Polymorphisms in the proteins related to the HR seem to be associated with a protective effect against PAH exposure.

One of these proteins, XRCC3, participates in homologous recombination repair of DNA double strand breaks and cross-links. This factor is a member of a family of Rad-51-related proteins. According to Shen, et al. (2003), it plays a protective role against bladder cancer for the XRCC3 codon 241 polymorphism, a higher risk in smokers. Considering that tobacco smoke has a high concentration of PAHs, this finding suggests that HR is related to some extent in the repair of damaged DNA induced by PAH exposure. Moreover, in an *in vitro* study using CHO cells deficient in the HR pathway, these cells were more sensitive to PAH exposure suggesting that this mechanism plays an important role in DNA repair (Meschini, et al., 2010).

#### **4.4 Translesion synthesis (TLS)**

To avoid cell death that may occur as a result of arrested DNA replication at unrepaired lesions cells have a mechanism, referred to as translesion synthesis (TLS), which allows them to overcome replication blockage from DNA damage. Once the lesion is generated in the DNA, the replication machine stalls, followed by either lesion repair or bypass by specialized polymerases (DNA polymerase IV or V, from the Y Polymerase family). Polymerase switching is mediated by, among other factors, the protein PCNA. TLS polymerases often have low fidelity; however, they are highly efficient, inserting the correct nucleotides at specific sites of damage. This process has been studied using the TLS performed by *Sulfolobus solfataricus* DNA polymerase Dpo4. The analysis of an oligonucleotide primer-extended and its dA−PAH adducts, using a liquid chromatography (LC)−mass spectrometry (MS)/MS, revealed this process in-depth including other proteins that may be involved (Zang, et al., 2006). The structure and nature, among other properties, of TLS polymerases is related to their fidelity and efficiency (Rechkoblit, et al., 2002). For a complete review of this process and the role of PAH adducts we recommend a review of Eoff et al. (2010).

sensitive to damage induced by DBPDE, as measured by frequency of chromosomal aberrations (Meschini, et al., 2010). Polymorphisms in proteins of the BER pathway are associated with an increase in DNA damage. One of these proteins, XRCC1, was discovered to have four functional polymorphisms: T-77C, Arg194Trp, Arg280His and Arg399Gln, associated with alteration in repair capacity of DNA damage induced by PAH adducts (Ji, et

Homologous recombination (HR) is a DNA metabolic process found in all forms of life that provides high-fidelity, template-dependent repair or tolerance of complex DNA damage including DNA gaps, DNA double-stranded breaks (DSBs), and DNA interstrand crosslinks (ICLs). The primary function of HR is to search for homology and DNA strand invasion through the Rad51-ssDNA presynaptic filament by positioning the invading 3′-end on a template duplex DNA to initiate repair synthesis. This mechanism participates in DNA repair induced by three specific PAHs: 1-nitrosopyrene (1-NOP), N-acetoxy-2 acetylaminofluorene (N-AcO-AAF), and 4-nitroquinoline 1-oxide (4-NQO). These PAHs were compared for their ability to cause intrachromosomal homologous recombination between two identical genes stably integrated into a genome of a mouse cell. In each case a dose-dependent increase in recombination frequency with differences in the efficiency of each compound was observed (Bhattacharyya, et al., 1989). Polymorphisms in the proteins related to the HR seem to be associated with a protective effect against PAH exposure. One of these proteins, XRCC3, participates in homologous recombination repair of DNA double strand breaks and cross-links. This factor is a member of a family of Rad-51-related proteins. According to Shen, et al. (2003), it plays a protective role against bladder cancer for the XRCC3 codon 241 polymorphism, a higher risk in smokers. Considering that tobacco smoke has a high concentration of PAHs, this finding suggests that HR is related to some extent in the repair of damaged DNA induced by PAH exposure. Moreover, in an *in vitro* study using CHO cells deficient in the HR pathway, these cells were more sensitive to PAH exposure suggesting that this mechanism plays an important role in DNA repair (Meschini,

To avoid cell death that may occur as a result of arrested DNA replication at unrepaired lesions cells have a mechanism, referred to as translesion synthesis (TLS), which allows them to overcome replication blockage from DNA damage. Once the lesion is generated in the DNA, the replication machine stalls, followed by either lesion repair or bypass by specialized polymerases (DNA polymerase IV or V, from the Y Polymerase family). Polymerase switching is mediated by, among other factors, the protein PCNA. TLS polymerases often have low fidelity; however, they are highly efficient, inserting the correct nucleotides at specific sites of damage. This process has been studied using the TLS performed by *Sulfolobus solfataricus* DNA polymerase Dpo4. The analysis of an oligonucleotide primer-extended and its dA−PAH adducts, using a liquid chromatography (LC)−mass spectrometry (MS)/MS, revealed this process in-depth including other proteins that may be involved (Zang, et al., 2006). The structure and nature, among other properties, of TLS polymerases is related to their fidelity and efficiency (Rechkoblit, et al., 2002). For a complete review of this process and the role of PAH adducts we recommend a review of

al., 2010).

et al., 2010).

Eoff et al. (2010).

**4.4 Translesion synthesis (TLS)** 

**4.3 Homologous recombination (HR)** 

Fig. 6. Common mechanisms of DNA repair from PAH exposure and adduct formation: nucleotide excision repair (NER) and base excision repair (BER).

DNA Damage Caused by Polycyclic Aromatic Hydrocarbons: Mechanisms and Markers 137

including scavenging oxidative radicals (Bonner, et al., 2005). In previous studies, the role of green tea polyphenols has been associated with protective effects against tumor induction in mice to which PAHs were topically applied (Wang, et al., 1989). Resveratrol (Leung, et al., 2009) and Genistain (Leung, et al., 2009) reduced DNA oxidative damage and adduct

> Sensitive methods and multiple biological

High sensitive to stable

High sensitivity and

High sensitivity to low concentrations, detection of different

Immunoassays Very high sensitivity Hard to obtain and

PAH molecules Indicators of exposure Some compounds

Genotyping Susceptibility markers Few studies on

These studies offer novel alternatives for the prevention of further DNA damage caused by PAH exposure. Additionally, mechanisms of damage and repair are revealed through understanding the interactions and pathways involved when these molecules come into

Both methods of detection and prevention of DNA damage are candidates for becoming new molecular alternatives for therapy and diagnosis of PAH-exposed individuals, and new

Worldwide, the population is, to some extent, exposed to PAHs. Of primary concern are the carcinogenic, teratogenic and mutagenic properties exhibited by some PAHs. The

Changes in gene expression of some genes are related with PAH exposure

Table 2. Methods to detect DNA damage caused by PAH exposure.

**Advantages Disadvantages Examples** 

1-hydroxypyrene

DNA adducts

Pyrene

CYP1A1 polymorphisms

CYP1B1 expression

Unstable metabolites

It may not be possible to detect unstable adducts

Suitable just for isotopes

Interference due endogen adducts

validate antibodies

are volatile

populations

Expensive and highly skilled techniques

formation induced by 7,12-dimethylbenz[*a*]anthracene in MCF-10A cells.

sources

adducts

selectivity

structures

**Target molecule or experimental technique** 

Metabolites as a result of exposure detected by chromatography

Radiolabeled compounds

32P-Postlabeling

Molecular biology

methods

tools in environmental biomonitoring.

Mass spectrometry

assay

**Strategies for detection of PAH exposure** 

**Metabolites detection and quantitation** 

> **Adduct detection**

**Primary compound detection** 

**Polymorphism analysis** 

> **Gene expression**

contact.

**6. Conclusions** 
