**4. AFB1-induced DNA damage checkpoint response**

DNA damage checkpoint response is critical for the genomic integrity and the survival of the organism in response to AFB1-induced genotoxicity, especially in the DNA that is actively replicating [42]. Mechanically, AFB1 activates critical proteins in response to DNA damage, such as ataxia telangiectasia and Rad3-related (ATR), ataxia telangiectasia mutated (ATM), Chk2 (serine/threonine protein kinase Chk2 or checkpoint kinase 2), DNA repair enzymes, and p53 [43]. In this context, AFB1-induced DNA double-strand breaks trigger the activation of ATM, which is one of the earliest activated kinases in response to double-strand breaks [44, 45]. Notably, the ATR/Chk1 pathway is not activated in response to AFB1-induced DNA double-strand breaks [26].

Cell cycle-dependent regulation plays an important role in the modulation of DNA repair, such as checkpoint activation, which slows down the cell cycle progression to facilitate DNA repair [46]. In this way, temporary DNA lesions are restrained to become inheritable mutations [47]. AFB1 induces the mutation of ATM kinase, which results in the defectiveness of cell cycle checkpoints [48]. On the one hand, the damaged cells with activated checkpoints can be eliminated by apoptosis if the DNA lesions are severe; on the other hand, damaged cell can continue the cell cycle progression upon checkpoint termination if the DNA lesions have been repaired [49]. Actually, AFB1 decreased the rate of DNA double-strand break repair and apoptosis, resulting in elevated risk of cancer [50].

Since ATM plays a critical role in the activation of cell cycle checkpoints [51], the damaged DNA activates ATM kinase to trigger the DNA repair signaling pathways. One of the substrates of ATM kinase is the structural maintenance of chromosome 1 (SMC1) protein, which plays key roles in regulating DNA replication forks and DNA repair in response to the damage [52, 53]. The ATM signaling inhibits the cell cycle progression from G1 to S (the G1/S checkpoint) or G2 to mitosis (the G2/M checkpoint) by activating p53 and inhibiting cyclin-dependent kinases [45, 54]. Ultimately, ATM is involved in the regulation of G2/M checkpoint, which has a crucial role in the maintenance of genomic integrity [50, 55]. In addition, ATM activates Chk2 by promoting its phosphorylation in response to DNA damage [56]. Subsequently, phosphorylated Chk2 (pChk2) induces G2/M cell cycle checkpoint by activating p53 signal pathway [57, 58]. Indeed, AFB1 activates ATM-Chk2-p53 axis, leading to G2/M phase arrest via cdc25-cyclin B/ cdc2 route [59]. In this signaling pathway, Mdm2 is an E3 ubiquitin ligase that facilitates the ubiquitination of p53 and proteasome-mediated degradation [60, 61]. Thus, ATM-Chk2-p53 axis plays a critical role in AFB1-induced DNA damage response.

#### **5. AFB1 exposure and genetic mutation**

More than 50% of HCC patients are from the regions where the daily food is contaminated by excess amount of AFB1 [62]. In these cases, the AFB1 exposure is associated partly with the genetic mutations [20]. For example, high frequent mutation of p53 gene at codon 249 with G-to-T transversion has been found in HCC patients who are exposed to excess dietary aflatoxin [30].

AFB1 preferentially induces G-to-T transversion in exon 7 of p53 tumor suppressor gene, so as to the frequency of G-to-T transversion of p53 significantly associated with AFB1-DNA adducts [63]. Thus, HCC patients who have AFB1-DNA adducts also have AGG-to-AGT mutation at codon 249 of p53 gene [64]. In addition, AFB1 also promotes G-to-T transversion of ras genes, which can enhance the malignant transformation of normal cells. AFB1-FaPy-dG adduct that is a highly persistent DNA lesion in the liver is associated with the lethality and mutagenicity potential [65]. Indeed, the metabolically activated AFB1-E and its cationic metabolite, AFB1-E-N7-dG, are highly related with the genotoxicity of AFB1 [66]. Additionally, there are at least 80% of HCC patients who have chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) [67]. Although AFB1 exposure slows down the replication of HBV, AFB1-mediated DNA damage and p53 induction are not associated with virus infection [68].

#### **6. AFB1 and epigenetic alteration**

Epigenetic alterations are heritable phenotype changes that do not include alteration of the DNA sequences [17]. AFB1 exposure induces various kinds of

**145**

*The Carcinogenicity of Aflatoxin B1*

to the development of HCC.

cancer, such as HCC [76].

**7. Conclusion**

etic changes are reversible.

*DOI: http://dx.doi.org/10.5772/intechopen.88353*

tion machinery associated with HCC development.

tion and proliferation of liver tumor cells [74, 80, 81].

epigenetic alterations, such as DNA methylation and histone modifications, leading

the CpG site of DNA, where a cytosine (C) is found next to a guanine (G) separated by a single phosphate (p) [69]. AFB1-induced change of DNA methylation is considered as one of the major mechanisms that are associated with AFB1-induced hepatocarcinogenesis [70]. The global hypomethylation of DNA has been identified as one of the most common biomarkers in human cancer [71]. It can lead to the silencing of critical tumor suppressor genes, such as p53 [72]. AFB1 exposure interferes DNA methylation patterns, resulting in an interruption of the methyla-

DNA methylation is an enzymatic process that a methyl group is conjugated into

A nucleosome is composed of a segment of DNA, which is wrapped around an octamer of histone protein cores composed of two copies of each of the protein histones H2A, H2B, H3, and H4 [73, 74]. Thousands of nucleosome units highly compact into a structure called chromatin [17, 75]. Several translational modifications occur at the N-terminal domains of the histones, e.g., acetylation, methylation, phosphorylation, and ubiquitination, which regulate important cellular processes, including DNA repair, chromatin structure, gene expression, and DNA replication [74]. AFB1 exposure can alter the pattern of modifications in histones, resulting in an increase or repression of genes, facilitating the development of

The most common modifications in histones regulated by AFB1 are methylation and acetylation [74]. Methylation is catalyzed by enzymes called histone methyltransferases, which add the methyl group into the lysine of the histones H3 and H4, occurring as three different forms, including mono-, di-, or trimethylation. Additionally, histone demethylase can remove the methyl group. AFB1-induced histone methylation causes conformational changes of chromatin, leading to gene silencing [77, 78]. In contrast to methylation, acetylation leads to a relaxation of chromatin, resulting in the activation of the gene transcription. Histone acetyltransferases, which catalyze the addition of the acetyl group, and the histone deacetylase (HDAC) enzymes, which remove the acetyl group, are the major enzymes involved in this dynamic process [73, 79]. The increased expression of HDACs causes the hypoacetylation of histone H3, leading to the repression of the silencing of specific tumor suppressor genes, such as p21, inducing the differentia-

Aflatoxin B1 (AFB1), which is synthesized by *Aspergillus* fungi, is the most common carcinogenic type of aflatoxins and can be found in human diet, such as corn, peanuts, cereals, rice, etc. Although AFB1 cannot directly conjugate with DNA, its metabolite, AFB1-E, can form carcinogenic AFB1-E-N7-dG and AFB1-FaPy-dG adducts spontaneously and irreversibly. Besides the generation of AFB1-DNA adducts, AFB1-induced DNA damage is also caused by the production of excessive ROS, leading to the oxidation of DNA bases. The majority of AFB1-related HCC carry G-to-T transversion of p53 gene. When the p53 protein is mutated, it shows a "gain of oncogenic function." In addition, epigenetic alterations may potentially be beneficial for the treatment of HCC, because the epigen-

#### *The Carcinogenicity of Aflatoxin B1 DOI: http://dx.doi.org/10.5772/intechopen.88353*

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

apoptosis, resulting in elevated risk of cancer [50].

**5. AFB1 exposure and genetic mutation**

patients who are exposed to excess dietary aflatoxin [30].

induction are not associated with virus infection [68].

**6. AFB1 and epigenetic alteration**

Cell cycle-dependent regulation plays an important role in the modulation of DNA repair, such as checkpoint activation, which slows down the cell cycle progression to facilitate DNA repair [46]. In this way, temporary DNA lesions are restrained to become inheritable mutations [47]. AFB1 induces the mutation of ATM kinase, which results in the defectiveness of cell cycle checkpoints [48]. On the one hand, the damaged cells with activated checkpoints can be eliminated by apoptosis if the DNA lesions are severe; on the other hand, damaged cell can continue the cell cycle progression upon checkpoint termination if the DNA lesions have been repaired [49]. Actually, AFB1 decreased the rate of DNA double-strand break repair and

Since ATM plays a critical role in the activation of cell cycle checkpoints [51], the damaged DNA activates ATM kinase to trigger the DNA repair signaling pathways. One of the substrates of ATM kinase is the structural maintenance of chromosome 1 (SMC1) protein, which plays key roles in regulating DNA replication forks and DNA repair in response to the damage [52, 53]. The ATM signaling inhibits the cell cycle progression from G1 to S (the G1/S checkpoint) or G2 to mitosis (the G2/M checkpoint) by activating p53 and inhibiting cyclin-dependent kinases [45, 54]. Ultimately, ATM is involved in the regulation of G2/M checkpoint, which has a crucial role in the maintenance of genomic integrity [50, 55]. In addition, ATM activates Chk2 by promoting its phosphorylation in response to DNA damage [56]. Subsequently, phosphorylated Chk2 (pChk2) induces G2/M cell cycle checkpoint by activating p53 signal pathway [57, 58]. Indeed, AFB1 activates ATM-Chk2-p53 axis, leading to G2/M phase arrest via cdc25-cyclin B/ cdc2 route [59]. In this signaling pathway, Mdm2 is an E3 ubiquitin ligase that facilitates the ubiquitination of p53 and proteasome-mediated degradation [60, 61]. Thus, ATM-

Chk2-p53 axis plays a critical role in AFB1-induced DNA damage response.

More than 50% of HCC patients are from the regions where the daily food is contaminated by excess amount of AFB1 [62]. In these cases, the AFB1 exposure is associated partly with the genetic mutations [20]. For example, high frequent mutation of p53 gene at codon 249 with G-to-T transversion has been found in HCC

AFB1 preferentially induces G-to-T transversion in exon 7 of p53 tumor suppressor gene, so as to the frequency of G-to-T transversion of p53 significantly associated with AFB1-DNA adducts [63]. Thus, HCC patients who have AFB1-DNA adducts also have AGG-to-AGT mutation at codon 249 of p53 gene [64]. In addition, AFB1 also promotes G-to-T transversion of ras genes, which can enhance the malignant transformation of normal cells. AFB1-FaPy-dG adduct that is a highly persistent DNA lesion in the liver is associated with the lethality and mutagenicity potential [65]. Indeed, the metabolically activated AFB1-E and its cationic metabolite, AFB1-E-N7-dG, are highly related with the genotoxicity of AFB1 [66]. Additionally, there are at least 80% of HCC patients who have chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) [67]. Although AFB1 exposure slows down the replication of HBV, AFB1-mediated DNA damage and p53

Epigenetic alterations are heritable phenotype changes that do not include alteration of the DNA sequences [17]. AFB1 exposure induces various kinds of

**144**

epigenetic alterations, such as DNA methylation and histone modifications, leading to the development of HCC.

DNA methylation is an enzymatic process that a methyl group is conjugated into the CpG site of DNA, where a cytosine (C) is found next to a guanine (G) separated by a single phosphate (p) [69]. AFB1-induced change of DNA methylation is considered as one of the major mechanisms that are associated with AFB1-induced hepatocarcinogenesis [70]. The global hypomethylation of DNA has been identified as one of the most common biomarkers in human cancer [71]. It can lead to the silencing of critical tumor suppressor genes, such as p53 [72]. AFB1 exposure interferes DNA methylation patterns, resulting in an interruption of the methylation machinery associated with HCC development.

A nucleosome is composed of a segment of DNA, which is wrapped around an octamer of histone protein cores composed of two copies of each of the protein histones H2A, H2B, H3, and H4 [73, 74]. Thousands of nucleosome units highly compact into a structure called chromatin [17, 75]. Several translational modifications occur at the N-terminal domains of the histones, e.g., acetylation, methylation, phosphorylation, and ubiquitination, which regulate important cellular processes, including DNA repair, chromatin structure, gene expression, and DNA replication [74]. AFB1 exposure can alter the pattern of modifications in histones, resulting in an increase or repression of genes, facilitating the development of cancer, such as HCC [76].

The most common modifications in histones regulated by AFB1 are methylation and acetylation [74]. Methylation is catalyzed by enzymes called histone methyltransferases, which add the methyl group into the lysine of the histones H3 and H4, occurring as three different forms, including mono-, di-, or trimethylation. Additionally, histone demethylase can remove the methyl group. AFB1-induced histone methylation causes conformational changes of chromatin, leading to gene silencing [77, 78]. In contrast to methylation, acetylation leads to a relaxation of chromatin, resulting in the activation of the gene transcription. Histone acetyltransferases, which catalyze the addition of the acetyl group, and the histone deacetylase (HDAC) enzymes, which remove the acetyl group, are the major enzymes involved in this dynamic process [73, 79]. The increased expression of HDACs causes the hypoacetylation of histone H3, leading to the repression of the silencing of specific tumor suppressor genes, such as p21, inducing the differentiation and proliferation of liver tumor cells [74, 80, 81].
