**2.4 AFB1-associated DNA adducts and cellular targets**

 AFB1-associated DNA adducts have been characterized *in vitro* and isolated from organisms that were exposed *in vivo*. DNA exposed to synthesized AFBO reacts predominately with the N<sup>7</sup> group of guanine bases forming 8,9-dihydro-8-(N7 -guanyl)-9-hydroxyaflatoxin B1 (AFB1-N<sup>7</sup> -Gua), as identified by mass spectrometry analysis. In the presence of hydroxyl ions (base), N7 -guanine DNA adduct is unstable and decays into an apurinic site and a AFB1 formamidopyrimidine (Fapy, **Figure 2**) DNA adduct; for review, see [68]. It is unclear whether both apurinic sites and AFB1-Fapy DNA adducts are equally generated; based on mutations generated by DNA lesions constructed *in vitro*, it has been suggested that AFB1-Fapy DNA adducts are the primary source of genetic mutations [69, 70], especially G to T transversion mutations that are found in AFB1-associated liver cancer [71, 72]. The AFB1-Fapy adduct is stable and can be present in two anomer forms; the alpha and the beta forms. While the beta form is highly mutagenic in *Escherichia coli* [69], the alpha form can stabilize the duplex helix and interfere with DNA replication [70]. In the rat liver, the half-life for AFB1-N<sup>7</sup> -Gua is 7.5 h, while that for AFB1-Fapy is at least 24 h [68]. While the AFB1-N7 -Gua is unstable, the accumulation of AFB1-Fapy in the rat liver may also result from differential repair of the two types of DNA adducts.

AFB1 exposure also generates oxidative stress (ROS) in exposed cultured cells *in vitro* and in the liver and lung *in vivo* [73]. Multiple factors may contribute to

**Figure 2.**  *Intercalation of the AFB1-Fapy-dG in duplex DNA. The adduct is in lighter tone; adapted from Ref. [121].* 

AFB1-associated oxidative stress including cytochrome P450 activity that involves iron-catalyzed reactions and Kupffer cells [68]. Oxidative stress generates hydroxyl radicals that form 8′-hydroxy-2′-deoxyguanosine (8oxodG) DNA damage. AFB1 exposure increases the 8OH-dG in the livers of ducks [74] and rats [75] and cultured woodchuck hepatocytes [68].

Interestingly, Niranjan et al. [76] observed that in rats, AFB1 bound to mitochondria l DNA exceeded the amount that was bound to the nuclear DNA and persisted for a longer period of time [76]. Furthermore, the persistence of mitochondrial DNA adducts correlated with a longer delay in expression of mitochondrial proteins, compared to that of nuclear-encoded proteins. The authors speculated that the persistence of AFB1 in the mitochondria may result from the lack of NER in the mitochondria. These studies support the notion that mitochondria are a prime target for acute effects of AFB1 exposure, and oxidative stress associated with AFB1-exposure could be indirect due to damage to mitochondria and the generation of superoxide.

To further elucidate the pathological consequences of AFB1-associated mitochondrial DNA lesions, Liu and Wang [77] measured AFB1-associated mitochondrial damage in primary broiler hepatocytes by monitoring mitochondrial membrane potential (MMP), ROS generation, apoptosis, and nuclear factor erythroid 2-like factor 2 (Nrf2)-related signal pathway. They observed mitochondrial ROS generation, decreased MMP and induced apoptosis. The increase in apoptotic cells correlated with an increase expression of caspase-9 and caspase-3. They concluded that AFB1 exposure results in a disruption of mitochondrial functions, generating more ROS, and consequently inducing apoptosis while triggering the Nrf2 signaling pathway [77].

## **2.5 Epigenetic changes associated with AFB1-associated damage**

While genetic instability associated with AFB1 have been described, less well known are epigenetic changes. Epigenetic changes are inheritable changes that result in phenotypic changes without affecting the DNA sequence. Epigenetic changes can result from DNA methylation (hypermethylation) or demethylation (hypomethylation), histone modifications, and changes in microRNA (miRNA) expression [78]. AFB1-associated epigenetic changes have been observed in cell cultures, animal studies, and human tumors (**Table 1**). Hypomethylation has been observed to increase the expression of oncogenes and repetitive sequences, while hypermethylation may decrease expression of DNA repair and tumor suppressor genes (**Table 1**). Zhang et al. [79] observed global hypomethylation in AFB1 associated cancers, where particular genomic repetitive elements, such as LINE-1 elements, were hypomethylated; correlating with increased retro transposition and genetic instability [80]. Hypomethylation also correlated with increased expression of the oncogene c-MET, which is associated with accelerated liver cancer progression and poor prognosis [81]. Hyper-methylated genes include the DNA repair gene methylguanine methyl transferase (MGMT) and p16, which have a negative effect on DNA repair and apoptosis [82]. Thus, methylation patterns may possibly serve as biomarker that can indicate increased risk for HCC [83–85].

Additional biomarkers that indicate AFB1 exposure include alterations in miRNA expression. miRNAs are small noncoding RNAs that are generally 19–25 nucleotides in length and regulate gene express at the post-transcriptional level. They are important factors in regulating HCC development in mammalian organisms [87, 88], and a list of miRNAs that correlate with AFB1 exposure is shown in **Table 1**. This comprises a partial group of total miRNAs that have been associated with HCC. Several miRNAs upregulated after rats or liver cell lines are exposed to


### *Cellular Responses to Aflatoxin-Associated DNA Adducts DOI: http://dx.doi.org/10.5772/intechopen.81763*

**Table 1.** 

*Epigenetic changes associated with AFB1 exposure.* 

AFB1 may be protective by downregulating cell proliferation, while upregulated miRNAs found in AFB1-associated HCC may promote tumor size or carcinogenesis. While an individual microRNA may target multiple genes, the expression of individual microRNA can be influenced by multiple transcriptional and epigenetic factors, as well as by genomic changes. These factors include CpG methylation, c-Met signaling, and gene copy number.

Among HCC tumor cells associated with AFB1 exposure, upregulation of several miRNAs, such as miR-429 and miR-24 [86], are associated with larger tumor size [83]. In human bronchial epithelial cells that express CYP2A13 (P50- B-2a13 CELLS), AFB1 exposure induces malignant transformation of immortalized cells [89]. Among transformed cells, one downregulated miRNA was miR-138-1, observed to inhibit proliferation, colony formation, and transformation of P50- B-2a13 CELLS [89]. This miRNA preferentially inhibits 3-phosphoinositide dependent protein kinase-1 (PDK1), which lowers the expression of the P13K/PDK/Akt pathway [89]. These studies indicate that changes in miRNA expression in AFB1 associated HCC may promote carcinogenesis.

HBV infection also upregulates the expression of miRNAs in hepatocytes and may promote HBV-associated HCC. The expression of miR106b-25 is upregulated in HCC patients in general, and in HCC patients infected with HBV [90]. Hep 3B cells transformed with an HBx expression plasmid also express higher levels of miR106b-25, compared to cells that do not express HBx. The miR106b-25 is a member of a cluster of miRNAs in MCM7 that downregulate the expression of several tumor suppressors, including p21, E2F, BIM, and pTEN [91]. Thus, HBV infection may not only interfere with DNA repair mechanisms but also epigenetically silence tumor suppressor genes and accelerate HCC progression.

### **2.6 Mutagenic signatures associated with AFB1-associated DNA adducts**

 Mutation signatures are useful biomarkers to determine AFB1 exposure and HCC progression. AFB1 is known to induce mutations in *E. coli*, *Saccharomyces cerevisiae* (budding yeast), and in mammalian cells. AFB1 was one of the original carcinogens published in the Ames assay [92, 93]. While G to T transversions are considered associated with chronic AFB1 exposure in humans [19, 20, 68, 71], in *E. coli*, carcinogen-induced transversion mutations require over-expression of expression of MucAB, which encodes the polV error-prone polymerase [94]. In budding yeast expressing either human CYP1A2 or CYP1A1, AFB1 has been shown to increase mutation frequencies at a *CAN1*, *LYS2*, and *URA3*; however the mutagenic signature of AFB1 in yeast has yet to be identified [95, 96]. The mutagenicity of AFB1 in yeast, however, is low compared to many alkylating agents, such as ethyl methane sulfonate (EMS) [95].

 While AFB1 is well-known to cause G to T transversion mutation in mammalian cells, other nucleotide substitutions occur, some of which are in the vicinity of the AFB1-DNA adduct. Investigators have used two approaches to determine the DNA sequence context of AFB1-associated mutations; one technique utilizes PCR (QPCR) and ligation-mediated PCR (LMPCR), and the second technique utilizes whole genome sequencing. Using the first technique, Denissenko et al. [97] mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide. In a second experiment, Denissenko et al. [97] mapped total AFB adducts in hepatocytes exposed to either AFB1 activated by rat liver microsomes or AFB1 activated by human liver microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40–400 μM AFB1, were 0.07–0.74 adducts per kilobase (kb). *In vitro* modification with 1–4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03–0.58 lesions per kb. The adduct patterns obtained with the epoxide

#### *Cellular Responses to Aflatoxin-Associated DNA Adducts DOI: http://dx.doi.org/10.5772/intechopen.81763*

 and the different microsomal systems were virtually identical indicating that AFB1 adducts share similar sequence-specificity whether occurring *in vitro* and *in vivo*.

 With the advent of next generation sequencing (NGS) [98], investigators have studied the entire genome and determine whether particular mutation signatures. Huang et al. [99] determine whole genome sequencing data to determine the position of >40,000 mutations in two human cell lines, and in liver tumors from wild type mice and a transgenic mouse carrying the hepatitis B surface antigen. The mutational signature from all four experimental systems was remarkably similar and compared well with experimental mutational signatures derived from sequenced HCCs form Qidong County in China, an area of high AFB1 exposure [100]. The Catalog of Somatic Mutations in Cancer (COSMIC) mutational signature 24 [101], previously associated with AFB1-associated liver cancer, was confirmed and also shown to be present in a high proportion (16%) in HCC from Hong Kong, but in 1% or less from HCC from Japan or North America. The COSMIC mutation signature 24 indicates guanine damage with a very strong transcriptional strand bias for C>A mutations. Additional studies being performed by multiple research groups [25, 102] confirm the presence of signature 24 in human HCC tumors and in tumors induced by AFB1 in mice. In addition to signature 24, investigators have also noted the presence of transition mutations that might also occur in the context of oxidative stress. It has not been determined which of these minor mutation classes drive HCC.

#### **2.7 AFB1 is a potent recombinagen**

 In budding yeast expressing CYP1A2, AFB1 is potent recombinagen but a poor mutagen [95]. Exposure to AFB1 stimulates homologous recombination between sister chromatids (sister chromatid exchange or SCE), chromosome homologs, and repeated sequences located on non-homologous chromosomes. Using a recombination assay involving truncated fragments of *his3* [103] positioned on non-homologous chromosomes, Sengstag et al. [95] showed that homologous recombination could be stimulated 50-fold in contrast to a less than 10-fold stimulation of mutations. AFB1 concentrations as low as 5 μM were shown to be effective at stimulating the formation of reciprocal translocations, and the karyotypes were confirmed by pulse field gel electrophoresis [103]. AFB1 is also a recombinagen in human and Chinese hamster ovary (CHO) cells and can increase the frequencies of SCE [104–107]. It is unclear whether the same AFB1-associated DNA lesions can stimulate both mutations and recombination. For example, it could be possible that particular lesions that stall DNA replication and generate breaks generate more recombination events while other lesions that can be bypassed by DNA polymerases generate more mutagenic events. These studies thus demonstrate that the genotoxicity of AFB1 extends beyond making mutations and involves stimulating chromosomal rearrangements in model eukaryotic organisms and in humans.

#### **2.8 Repair of AFB1-associated DNA damage**

Considering the genotoxicity of AFB1-associated DNA adducts and possible hindrance of DNA replication, it is important to identify which DNA repair pathways and which replication bypass mechanisms are used to tolerate the most persistent AFB1-associated DNA adducts. There are several pathways that are involved in repairing AFB1-associated DNA damage. Among these repair pathways are nucleotide excision repair NER, BER, and recombinational repair; for a general review see [108]. Post-replication repair pathways to bypass DNA adducts involve (1) either errorprone or error-free DNA polymerases, or (2) template switch mechanisms. The later

mechanism involves DNA recombination mechanisms, which are utilized in tolerating UV-induced DNA damage and alkylated DNA bases. While in some organisms there are preferred pathways, a general theme in DNA repair is that organisms have evolved redundant DNA repair mechanisms. A prediction of redundant DNA repair pathways is that eliminating genes in two or more repair pathways should effectively lead to a synergistic decrease in AFB1 resistance, while eliminating genes in the same pathway should confer no greater sensitivity than the most sensitive mutant.

 Nucleotide excision repair (NER) involves the recognition of the DNA adduct, the opening of the helix at the DNA damage site, the excision of the DNA adduct and the re-synthesis of DNA using the non-damaged DNA strand as a template. In general, 12–13 nucleotides are excised in prokaryotes (for review see [109]) while 24–32 nucleotides are excised in eukaryotes. Global genome repair (GGR) can occur on either the transcribed or non-transcribed strand. Transcription-coupled repair (TCR) does discriminate and preferentially repairs the transcribed strand. The mechanistic difference between the two pathways is how the DNA adduct is recognized; in GGR specific proteins recognize the DNA helical distortion while in TCR, the RNA polymerase stalled complex is recognized; for general review see [110]. In eukaryotes and prokaryotes, both mechanisms are used. While the mechanism is widely conserved among eukaryotes, the mechanism differs between prokaryotes and eukaryotes in the amount of DNA that is excised.

 NER is likely to be the predominant mechanism for the repair of AFB1 associated DNA damage in many eukaryotic and prokaryotic organisms [68]. The AFB1-N7 -guanine adduct is fairly unstable while the AFB1-Fapy DNA adduct can insert between the base pairs of the DNA double helix [111]. UvrABC from *E. coli*  can effectively excise both DNA adducts, although the AFB1-Fapy adduct appears to be more chemically stable [68, 112]. The excision of the DNA adducts does not depend on the SOS response; thus, basal levels of the DNA repair enzymes appear to be adequate in repairing the DNA lesions. In *E. coli*, both AFB1-N7 -Gua and AFB1-Fapy adduct appear to be excised at a similar rate. One explanation is that the UvrABC complex does not rely on helix distortion to repair the DNA adduct, but rather size and structure of the aromatic rings [68, 112].

Other insights from model organism yeast revealed that the NER genes are required to excise AFB1-associated DNA adducts [96, 113, 114]. *RAD14* (XPA) and *RAD1-RAD10* (XPF-ERCC1) are required for AFB1 resistance. Failure to repair the DNA adducts in a *rad4* (XPC) haploid mutant results in S phase arrest, supporting the notion that particular AFB1-associated DNA adducts interfere with DNA replication [113, 114]. In addition, in *rad4* mutants the level of AFB1-N7 -Gua DNA adducts was reported to increase three fold [114]. These studies support the notion that the yeast NER pathway recognizes and repairs AFB1-N<sup>7</sup> -Gua DNA adducts.

In mammalian cells, the NER pathway preferential repairs AFB1-N<sup>7</sup> -Gua DNA adducts but still participates in the repair of AFB1-Fapy DNA adducts [115, 116]. In XPA human fibroblast cells, the loss of AFB1-N7 -Gua DNA is much slower and the accumulation of the AFB1-Fapy DNA adducts is greater compared to wild type cells [116]. XPA<sup>−</sup>/<sup>−</sup> deficient mice are also more susceptible to AFB1-associated tumorigenesis compared to wild-type mice [117]. Since the accumulation of DNA adducts correlate with the increased carcinogenicity of the DNA adducts [71, 72, 118], it is likely that the burden of AFB1-associated DNA adducts increases the frequencies of carcinogen-associated mutations in the XPA deficient mice.

The second major pathway to repair DNA involves the BER pathway (for review, see Fortini and Dogliotti [119]). As in NER, the DNA damage base is excised and new DNA is synthesized using the undamaged DNA as template for repair (**Figure 3**). In BER, the modified DNA base is recognized and excised by a specific enzyme that generally referred to as a glycosylase. Subsequently, a apurinic endonuclease (APE1)

*Cellular Responses to Aflatoxin-Associated DNA Adducts DOI: http://dx.doi.org/10.5772/intechopen.81763* 

**Figure 3.** 

*BER (left) and NER (right) mechanisms to repair the AFB1-Fapy adduct. Both mechanisms involve incision and excision of the damage base, followed by unscheduled DNA synthesis.* 

generates a 3′OH for primer recognition and new DNA synthesis. In mammalian cells, polymerase β synthesizes new DNA across the gap and removes the deoxyribose residue, and XRCC1/Ligase III cooperate to seal the nick An alternative pathway that does not involve APE1, employs endonuclease VIII like-1 (NEIL1). Following excision of the damaged base by a βδ excision mechanism, the 3′ phosphate is excised by polynucleotide kinase (PNK) to yield a 3′OH for primer recognition and new DNA synthesis. For long patch repair, DNA polymerase δ/PCNA/RFC synthesizes across the gap, the displaced oligonucleotide is excised by FEN1, and the nick is sealed by Ligase I [119]. Poly(ADP-ribose) polymerase PARP1 generally protects the single-strand gap from being subjected to further cleavage or from serving as a substrate for recombinational repair proteins although additional pathways have been proposed [120].

Interestingly, while BER mechanisms have been thought to play a minor role for DNA repair of some AFB1-associated DNA adducts in yeast, BER mechanisms for AFB1-associated DNA adducts can occur in mammalian cells. In budding yeast, the *apn1/apn2* haploid double mutant is no more AFB1 sensitive than the haploid wild type [96]. However, AFB1-associated mutagenesis is lower in the *apn1/apn2* haploid double mutant compared to wild type [96], suggesting that either Apn1 or Apn2 still function in processing the AFB1-associated adducts for post-replication repair. One interpretation of these results is that there is redundancy in both NER and BER mechanisms for conferring AFB1 resistance, while another interpretation is that budding yeast lack the BER enzymes, such as NEIL1, which may actively participate in the repair of AFB1-associated DNA adducts.

In mice, the NEIL1 gene has been isolated and knock-out of the gene leads to higher levels of AFB1-associated DNA adducts and AFB1-associated HCC [121]. The NEIL1 enzyme recognizes and excises AFB1-Fapy-dG adducts in "bubble" DNA structures, such as the one described by Brown et al. [70, 111]. One idea is that AFB1-Fapy-dG adducts may stably intercalate in the helix and be recognized by NEIL1-dependent BER pathway but not by the NER pathway; the repair pathway may thus depend on the DNA sequence context of the AFB1-Fapy-dG adduct. Knock-out of NEIL1 in mice leads to an increase of AFB1-associated tumors and

 an accumulation of Fapy-adducts [121]. Vartanian et al. [121] assert that the AFB1-associated carcinogenicity in *Neil1*<sup>−</sup>/<sup>−</sup> mice is as high if not higher than that observed in *Xpa*<sup>−</sup>/<sup>−</sup> mice, noting that both the size and number of tumors are greater in the *Neil1*<sup>−</sup>/<sup>−</sup> mice compared to the *Xpa*<sup>−</sup>/<sup>−</sup> mice. However, the investigators indicate that spontaneous tumors arise at a much higher frequency in *Xpa*<sup>−</sup>/<sup>−</sup> mice, so that the increase in AFB1-associated tumors were measured until the mice were 11 months in age and not when the mice were 15 months in age.

AFB1 exposure is also associated with oxidative stress, as evident by the accumulation of 8-oxodG lesions. It is particularly interesting whether 8-oxodG accumulates in particular DNA repair mutants and contributes to genotoxicity and the etiology of liver cancer. The contribution of 8-oxodG to overall AFB1-associated genotoxicity is unclear; *Ogg1− /Ogg1−* transgenic mice do not exhibit more AFB1-associated lung tumors than those that are wild type, but do exhibit increased weight loss and mortality [122]. However, *Ogg1*<sup>−</sup>/<sup>−</sup> null mice succumb to other cancers after being exposed to oxidizing agents and carcinogens [123]. These studies suggest that AFB1-associated 8-oxodG lesions are not the causative lesions in liver or lung cancer.

 The third major pathway in cells that function in AFB1-associated DNA damage is recombination repair. Knocking out *RAD51* in either *rad14* or *rad4* cells leads to a synergistic increase in AFB1 sensitivity in yeast [96, 113]. There are two different explanations. One explanation is that some AFB1-associated DNA lesions that accumulate in *rad4* cells are converted into single or double-strand breaks and require recombinational repair. A single double-strand break has previously been shown to be lethal in strains defective in homologous recombination [124]. An alternative explanation is that cells require *RAD51* to bypass the DNA lesions and accumulate stalled replication forks. Studies have shown that *RAD51* is required for DNA damage-associated SCE [125], which likely occur by replication bypass mechanisms. This second reason is also supported by the notion that *rad4* cells tend to arrest in a small budded stage upon entry into the cell cycle.

#### **2.9 DNA damage tolerance and AFB1-associated DNA damage**

 DNA damage tolerance pathways allow cellular replication mechanisms to bypass blocking DNA adducts, such as the AFB1-Fapy DNA adduct, resulting in persistence of the DNA adduct in the divided cells. These mechanisms are divided into error-free mechanisms where the original "correct" base is opposite the modified base and error-prone mechanisms where an "incorrect" base is inserted opposite the damaged base, thereby generating mutations. The insertion of the "incorrect" base is generally accomplished by substituting a "high fidelity" polymerase with a lower fidelity polymerase that also has lower processivity. The polymerase switch mechanism is accomplished by a series of ubiquitination reactions on PCNA, which is the processivity factor for DNA polymerase on the DNA template; for review, see [126, 127]. The first ubiquitination reaction of PCNA is a monoubiquitination reaction catalyzed by Rad18/Rad6. Subsequent polyubiquitination of PCNA by Rad5/Ubc13/Mms2 is required for error-free by-pass mechanisms, which includes template-switch mechanisms. Both *RAD18/RAD6* and *RAD5* genes are well conserved in eukaryotes.

The function of replication bypass in conferring AFB1 resistance has been validated in model organisms. In budding yeast, *RAD18*, *RAD5*, *REV1*, and *REV7/ REV* are required for AFB1 resistance [96]. These genes are also required for AFB1 associated mutagenesis [96]. These results indicate that all three translesion polymerases are required for AFB1 resistance, while it is unclear which gene is required for replication bypass of individual AFB1-associated DNA adduct.

While there are only three translesion DNA polymerases in budding yeast, in humans, there are at least 11 translesion polymerases, forming the majority of the 15 DNA template-dependent DNA polymerases [128]. Both the AFB1-N7-Guanine and

*Cellular Responses to Aflatoxin-Associated DNA Adducts DOI: http://dx.doi.org/10.5772/intechopen.81763* 

the AFB1-Fapy DNA adducts can be bypassed by translesion polymerases [129, 130]. Of the translesion polymerases, DNA polymerase ζ, also referred to as Rev3L the Rev3 homolog in humans, limits chromosomal damage and promotes cell survival following AFB1 exposure [131]. The authors suggest that Rev3 is required for progression through S phase since mouse embryonic fibroblasts, derived from *Rev3L−/−* knock down mice, arrest in S/G2 after AFB1 exposure [131]. These cells also exhibit an increase in gamma-H2AX foci, micronuclei, and chromosomal aberrations; the kinetics of micronuclei formation support a replication-dependent mechanism that results in the accumulation of unrepaired DSBs in. The Rev3 requirement for DNA replication of an AFB1-associated DNA adduct was also demonstrated for a single lesion present on a replicating plasmid in HEK239 cells [131]. Considering the number of mammalian translesion polymerases [128], the Rev3 requirement for replication bypass may reflect the efficiency by which AFB1-associated DNA adducts block other polymerases or Rev3's ability to minimize detrimental chromosomal damage [132].

#### **2.10 Template-switch mechanisms as an alternative mechanism for tolerating DNA damage**

Exposure to AFB1 stimulates SCE in multiple organisms. One possible mechanism is that in post-replication repair, processing of AFB1-associated DNA damage generates apurinic sites and/or subsequent DNA single-strand gaps, which initiate SCE by serving as substrates for DNA recombination proteins. Template switch mechanisms are another mechanism (**Figure 4**) that avoid the necessity of using error-prone polymerase for replication bypass. In support of the role of template switching in AFB1-associated SCE, studies have been performed in budding yeast indicating that *rad51* null mutants, deficient in DNA damage-associated SCE [125], exhibit higher frequencies of AFB1-associated mutations [96, 133]. In addition, Rad51 foci appear as cells enter S phase [114] and not in G2, suggesting that the appearance of Rad51 foci are replication-dependent and not associated with double- or single-strand breaks after replication. However, it is possible that multiple mechanisms are involved.

AFB1-associated SCE are also observed in human and mammalian cells. SCEs have been detected in human lymphocytes, Chinese hamster V79 cells, rat and mouse hepatocyte cell lines [104–107]. It has not yet been determined whether mammalian cells defective in homologous recombination exhibit more AFB1 associated mutations. Nonetheless, it is interesting that polymorphisms of XRCC3 [62–64], which functions in homologous recombination, are a risk factor for HCC.

#### **2.11 Tissue specificity of DNA damage repair of AFB1-associated DNA adducts**

Since AFB1-associated DNA adducts are found in different tissues, the question can be asked whether there are tissue-specific differences in repair mechanisms. Mudler et al. [134] addressed the question whether oxidative damage caused by AFB1, 8-oxodG, was repaired more efficiently in the mouse lung compared to the mouse liver. They exposed mice to a low chronic amount of AFB1 (0.2 or 1.0 ppm AFB1) and then assayed for the amount of dGTP incorporation. Interestingly, they found that although Ogg1 was present in both the lung and the liver, there was a lower repair efficiency in the liver after exposure to 1.0 ppm AFB1. The lower efficiency of the repair in the liver did not correspond to AFB1-associated cytotoxic effects, and they speculated that the differences could result from AFB1 directly inhibiting Ogg1 [134].

Bedard et al. [135] asked the question whether AFB1-N<sup>7</sup> -Gua and AFB1-Fapy DNA adducts were repaired more efficiently in the mouse liver or lung. They also compared the efficiency of repair in the rat liver and the mouse liver. After exposing mice to 50 mg/kg AFB1, extracts were obtained from the various tissues and used to determine the repair of plasmid DNA AFB1-N7 -guanine or AFB1-Fapy adducts as

#### **Figure 4.**

*DNA damage tolerance mechanisms used to bypass a AFB1-Fapy DNA adduct blocking the leading strand polymerase on a growing replication fork. Error-free (left) bypass uses a template switch mechanism while errorprone (right) bypass uses a low fidelity DNA polymerase, resulting in the insertion of an A opposite the DNA adduct.* 

substrates. Mouse liver extracts repaired AFB1-N7 -guanine and AFB1-Fapy adducts 5- and 30-fold more effectively, respectively, than did extracts from the mouse lung. Mouse liver extracts also repaired the adducts 6-fold and 4-fold more effectively, respectively, than did liver extracts from rats. They conclude that there is a tissuespecific induction in repair in the mouse liver that renders the mouse liver more resistant to AFB1-associated carcinogenesis. However, further studies are needed to determine which NER and BER enzymes are preferentially induced in the liver.

#### **2.12 AFB1-induction of DNA repair and protective mechanisms**

The redundancy in repair mechanisms for DNA adducts in yeast and in mammalian organisms provokes the question of which genes are transcriptionally induced after the exposure of AFB1. Two complementary studies have been performed using budding yeast and several studies have been performed in mammalian cells. While studies in yeast utilized microarrays, more recent studies in mammalian cells have used RNAseq and NGS technology. The common genes that are induced have provided clues into which pathways are shared among eukaryotic organisms.

 Keller-Seitz et al. [113] determined which budding yeast genes were induced after exposure to AFB1. Essentially, an exponentially grown culture was concentrated to 4 × 10e8 cells/ml and then exposed to 25 μM AFB1 in phosphate buffer (pH 7.5). After RNA was extracted, cDNA was synthesized and labeled for analysis on microarrays. Fourteen DNA repair genes were upregulated more than two-fold, with *RAD51*  being upregulated more than seven-fold. Among NER genes, *RAD16*, *RAD3*, and *RAD1* were AFB1-inducible. The upregulation of selected genes was verified by RT-PCR. Additional genes that were induced included those involved in mismatch repair and DNA synthesis, while genes participating in NHEJ were downregulated.

 A similar study was done by Guo et al. [136], except AFB1-inducible genes were identified in actively growing cultures. Similar to the Keller-Seitz study [113], *RAD51* was upregulated over seven-fold. However, additional genes involved in

#### *Cellular Responses to Aflatoxin-Associated DNA Adducts DOI: http://dx.doi.org/10.5772/intechopen.81763*

 regulating dNTP levels were also upregulated including *DUN1*, which encodes a DNA damage-signaling kinase, and *RNR2* and *RNR4*, which are subunits of ribonucleotide reductase. Although the functional significance of the AFB1-associated inducibility is unknown, there is good overlap with a cluster of genes identified as DNA damage-inducible but not generally stress-inducible [137]; DNA damageinducible genes from multiple studies include *DUN1*, *RAD51*, *RNR2*, and *RNR4*. In contrast to the previous study by Keller-Seitz, NER genes were not upregulated. In both studies, the DNA damage-inducibility of *RAD51* is *MEC1*-dependent; *MEC1* is the ATM/ATR orthologue of yeast. The functional significance of the upregulation was illustrated by showing that the recombination deficiency exhibited by *mec1* mutants could be partially suppressed by over-expression of *RAD51* [113]. Thus, upregulation of particular DNA repair genes could enhance AFB1 genotoxic effects.

Additional genes that were upregulated in both studies included genes involved in cell cycle control, protein transport, DNA metabolism, and ion homeostasis [113, 136]. Although the functional significance of the upregulation of each of these genes is unknown, many of these genes are involved cell cycle regulation. Interestingly, genes involved in histone biosynthesis were downregulated, reflecting a delay in S phase [136]. The delay in S phase may result from the stability of the AFB1-Fapy DNA adduct during the exposure time.

 Identification of AFB1-inducible genes in mammalian cells revealed broader classes of upregulated genes, compared to the yeast studies, reflecting the hepatic cell's ability to metabolize and neutralize xenobiotic agents. Merrick et al. [138] performed RNA seq analysis on liver cells after the rat was injected with AFB1. In brief, RNA was obtained from male rats exposed 1 ppm AFB1 in feed for 90 days, and RNA seq analysis was performed using the appropriate number of unexposed rats as controls. 1026 differentially induced transcripts were identified. Genes upregulated more than five-fold relevant to hepatocellular proliferation include follistatin (442 fold), Aldh3a1 (302-fold), Mybl2 (21-fold), Mybl1 (6-fold), and Sox9 (6-fold). Genes upregulated and involving the E2f1 transcription factor included Cdk1, Mdm2, Ect2, Mad2L1, and Nuf2. Of those genes that were upregulated, of particular interest are those involved in DNA damage tolerance and repair. A two to four-fold increase was observed for Mgmt, Top2a, Rad51, Rad18, Xrcc6, Mnd1, and Tynns [138]. These studies indicate that chronic AFB1 exposure in animals can also induce DNA repair genes that are involved in cell cycle regulation and DNA replication bypass.

#### **2.13 Signal transduction and checkpoint activation**

Both studies in yeast and in mammalian cells indicate that AFB1 triggers a checkpoint response that delays cell cycle progression so that DNA damage can be repaired. The mechanism by which the AFB1 DNA adducts are sensed is unknown. However, it is likely that DNA replication stress triggers S phase delay that is associated with Rad53 (Chk2 orthologue) phosphorylation [133]. In budding yeast, exposure to 50 μM AFB1 is sufficient to delay S phase [133, 135]. The Rad53 phosphorylation is dependent on MEC1, the ATM/ATR orthologue. Fasullo et al. [133] observed that the downstream effector of Rad53, DUN1, was required for both AFB1-associated mutation and AFB1 associated recombination. However, the substrates for the signaling cascade that affect AFB1-associated recombination and mutation are unknown. One possibility is that Rad55 phosphorylation is important in triggering AFB1-associated recombination.

In mammalian cells, the DNA damage response to AFB1-associated DNA adducts has been addressed by only a few studies. After exposure to AFB1, HepG2 cells exhibit 53BP1 foci and H2AX foci but not Chk1 or Chk2 activation [139]. However, other studies [140] in other cell lines suggest a robust stimulation of the checkpoint response. In human bronchial epithelial cells (BEAS-2B) expressing CYP2A13 and exposed to

low concentrations of AFB1, AFB1-DNA adducts and 8oxodG significantly increased, along with phosphorylation of ATR and BRCA1. In addition, Mre11, Rad50 and Rad51 were significantly increased. These studies suggest that similar to yeast, checkpoint activation leads to higher expression of DNA recombination genes in3BEAS-2B cells.

### **3. Conclusions**

Liver cancer is the third leading cause of cancer deaths, and unfortunately the incidence of liver cancer is increasing in the USA. Environmental and lifestyle factors include AFB1 exposure and infection with HCV and HBV viruses. AFB1 is a potent liver carcinogen because it is a potent genotoxin and AFB1 exposure is correlated to signature mutations found in HCC. Liver injury and inflammation set the stage for regenerative cell proliferation that enhances AFB1-associated genetic instability. As liver cancer progresses, multiple genetic mutations and epigenetic changes accumulate that eventually accelerate an irreversible path toward malignancy and poor prognosis.

Nonetheless, cellular defense mechanisms have evolved to diminish the AFB1 genotoxicity and repair or tolerate AFB1 DNA adducts so that mutations and chromosomal instability are avoided. First, there are multiple pathways to repair AFB1-associated DNA adducts. These include BER repair involving NEIL1 and NER pathways that excise AFB1-associated DNA adducts. However, it is still unclear which pathway is favored in humans and whether they are redundant. Second, there are common repair and checkpoint pathways that are upregulated in both model organisms and in mammalian organisms; these include ATR signaling pathways and recombinational repair pathways. These pathways may suppress chromosomal instability by error-free mechanisms by which DNA adducts can be bypassed by the DNA replication machinery. One error free mechanism involves recombination-mediated template switch mechanisms. Supporting this idea, RAD51 expression is enhanced in yeast and particular polymorphisms XRCC3, a RAD51 paralogue, may be risk factors for HCC. Nonetheless the DNA repair process can be thwarted by HBV virus, where Hepatitis B virus may directly interfere with NER and perpetuate the replication of cells containing damaged DNA.

 The studies presented in this chapter point to future directions in elucidating repair mechanisms of AFB1-associated DNA damage and genetic susceptibility to AFB1-associated cancer. The advent of NGS technology has made it possible to profile the yeast and mammalian genomes for AFB1 resistance which will facilitate identifying the most prominent AFB1 resistant genes. This will facilitate epidemiological studies in determining potential gene polymorphisms that may pose the greatest risk for HCC. NGS technology can facilitate characterizing the DNA sequence contexts where AFB1-associated mutations occur. With the advent of NGS it may be possible to determine the temporal and sequence contexts by with AFB1-associated mutations occur. With the accumulation of genetic information, new biomarkers may be available to aid clinicians and epidemiologists to detect individuals most of risk for HCC and to take appropriate prophylactic actions at earl signs of HCC progression.

#### **Acknowledgements**

The author was supported by NIH grants R21ES015954, F33ES021133, and R15E023685.

## **Conflict of interest**

The author declares no conflict of interest.

*Cellular Responses to Aflatoxin-Associated DNA Adducts DOI: http://dx.doi.org/10.5772/intechopen.81763* 
