**Abstract**

Aflatoxins are a class of carcinogenic mycotoxins, products of *Aspergillus* fungi, which are known contaminants in a large portion of the world's food supply. Aflatoxin B1 (AFB1) is the most potent toxin, which has been strongly linked to the development of hepatocellular carcinoma (HCC), especially given coinfection with hepatitis B virus (HBV). AFB1 is catalyzed by cytochrome P450 (CYP450) into aflatoxin B1-8,9-exo-epoxide to form DNA adducts, which leads to carcinogenesis by disrupting DNA repair. 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 to HCC carry G-to-T transversion of p53 gene. When the p53 gene is mutated, it shows a "gain of oncogenic function." In addition, epigenetic alterations may potentially be beneficial for the treatment of HCC, because the epigenetic changes are reversible. This chapter will provide important information on the carcinogenicity of AFB1, including DNA damage checkpoint response and epigenetic alteration.

**Keywords:** aflatoxins, hepatocellular carcinoma, DNA adducts, carcinogenicity

## **1. Introduction**

Aflatoxins were reported to be potent liver carcinogens for laboratory animals since the 1960s [1]. The International Agency for Research on Cancer (IARC) has identified aflatoxins as one of the most harmful human carcinogens [2]. There are four main kinds of aflatoxins, B1, B2, G1, and G2, which are classified based on UV-induced fluorescence color and chromatography retention time [3].

Among these aflatoxins, aflatoxin B1 (AFB1), which is synthesized by *Aspergillus* fungi, is the most common carcinogenic and can be found in foods, such as corn, peanuts, cereals, rice, etc. [4, 5]. AFB1 is chemically stable and resistant to various thermal processes such as boiling, autoclaving, cooking, and fermentation [6]. AFB1 is catalyzed by cytochrome P450 into aflatoxin B1-8,9-exo-epoxide to form DNA adducts, which leads to carcinogenesis by disrupting DNA repair [7]. Although most countries introduce strict regulations for the maximum permitted concentrations of aflatoxin in food, excess of AFB1-DNA adducts has remained in normal and tumorous tissues of individuals with hepatocellular carcinoma (HCC) [8, 9]. HCC is ranked as the second leading cause of death from cancer globally, with 700,000 annual deaths recorded worldwide in 2012 [10, 11]. There are about 4.5 billion people in the world that are exposed to AFB1 and may develop HCC [12]. The risks of AFB1 to induce HCC are dependent on populations and areas, e.g., urban populations are generally exposed to lower levels of aflatoxins than rural populations. Moreover, AFB1 exposure is altered by strong seasonal variation that is correlated with increased food availability. In addition, hepatitis B virus (HBV) infection is associated with AFB1 exposure [12, 13].

Some studies reported that the detection of urinary AFB1-N7-guanine adduct, a good biomarker for AFB1 exposures and dietary AFB1 intake, has a significant association with HCC [13, 14]. The AFB1-DNA adduct is formed by the reactive intermediate of AFB1, AFB1-8,9-epoxide (AFB1-E), and binds to the DNA of hepatocytes. On the one hand, AFB1 is supposed to cause tumorigenesis by promoting the formation of DNA adducts, resulting in aberrant gene expression and genetic mutations, such as tumor suppressor gene p53, in targeted liver cells [15]. On the other hand, AFB1-developed HCC rat model presents various epigenetic (DNA methylation, histone modification, and noncoding RNA) alterations in hepatocytes [16, 17]. Therefore, this chapter focuses on the potential mechanisms of AFB1 induced development of HCC, including DNA damage checkpoint response and epigenetic alteration.

## **2. AFB1-DNA adduct formation**

AFB1 is mainly absorbed in the small intestine after ingestion and transported from blood to the liver [18]. It passes through hepatocytes by nonionic diffusion, which is not dependent on metabolic energy status [19]. Although AFB1 cannot directly bind to DNA, it is metabolized into reactive epoxide named AFB1-E by cytochrome P450 (CYP450) in the liver [7]. AFB1-E can rapidly conjugate with N7 of guanine residues, resulting in the formation of highly mutagenic AFB1- E-deoxyguanosine or AFB1-formamidopyrimidine (AFB1-FaPy-dG) adducts [20]. In fact, AFB1-E can spontaneously and irreversibly conjugate with guanine residues to form 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-E-N7-dG) adducts, leading to the occurrence of DNA damage and mutations [21, 22]. AFB1-E-N7-dG adduct may undergo imidazole ring opening or depurination spontaneously to yield AFB1-FaPy-dG, which has been found in many hepatocellular carcinoma cases [23]. Although both AFB1-FaPy and AFB1-E-N7-dG adducts have similar structures, they bind DNA in a different manner, which results in the differences in the lethality, mutagenicity, and repair capacity of these metabolites. In fact, enzymes involved in DNA repair pathways have higher affinity with AFB1- E-N7-dG than AFB1-FaPy analog. AFB1-FaPy-dG adduct block the replication more effectively than AFB1-E-N7-dG adduct, e.g., AFB1-FaPy-dG adducts induce at least six times more G-to-T transversion than AFB1-E-N7-dG [20]. Therefore, AFB1-FaPy-dG is recognized as the major adduct and the most lethal aflatoxininduced replication block in vivo.

AFB1 is bioactivated by the predominant enzyme cytochrome P450 in human liver microsomes. Specifically, CYP1A2 is the major enzyme for AFB1-E-dG formation and DNA damage at the low dietary intake of AFB1, while CYP3A4 is the major enzyme at higher doses of exposure [24, 25]. In general, CYP1A2 contributes to 95% of AFB1-DNA adduct formation [26]. AFB1 exposure significantly affected the expression of almost 200 genes and exhibited a fivefold decrease in histone transcripts, which is revealed by microarray studies [27].

In addition, the formation of urinary AFB1-E-N7-guanine excretion and levels of AFB1-serum albumin adducts are highly associated to AFB1 intake [28]. Accordingly, AFB1-E-N7-dG adduct is closely related with the incidence of liver tumors [29, 30]. Then, AFB1-E-N7-dG adduct is rapidly removed from DNA and excreted solely in urine [30]. Actually, people who excreted AFB1-E-N7-dG adduct has 9.1 times more risk of developing HCC than the ones with no adducts [31]. These findings indicate that AFB1-DNA adducts have strong effects on HCC development [26, 27].

**143**

*The Carcinogenicity of Aflatoxin B1*

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

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 [32]. On the one hand, AFB1 increases ROS generation, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione reductase (GR); on the other hand, AFB1 decreases glutathione (GSH) while increasing nitric oxide, fragmented DNA-conjugated dienes, caspase-3, superoxide anion radicals, lipid hydroperoxides, and malondialdehyde (MDA) [11, 33]. It suggests that AFB1 induced hepatocarcinogenesis is partially due to the increase of oxidative stress

In addition, the lipid peroxidation contributes to the progression of hepatocarcinogenesis by enhancing the susceptibility to mutation and provokes aldehyde-DNA adducts at p53 mutational hotspot codon 249 [35]. To prevent the further formation of ROS/RNS, p53 modulates the transcription of antioxidant genes; however, the extensive DNA damage promotes apoptosis by activating the prooxidant genes. The elevated oxidative stress is well correlated with the induction of AFB1-induced apoptosis of splenic lymphocytes [36]. Therefore, TP53 mutation is considered as a reliable biomarker for evaluating the extent of AFB1 exposure [37]. Vital antioxidant enzymes alleviate oxidative damage through removing free radicals, e.g., selenium, as a cofactor, and promote cell survival by activating the antioxidant system in response to AFB1-induced mitochondrial damage [38]. The dietary intake of AFB1 is associated with the amount of pathological lesions and apoptosis rate of hepatocytes [39]. Therefore, AFB1 causes instability between antioxidant system activity and ROS production, resulting in excessive reactive species that lead to the apoptosis of hepatocytes [11]. It has been reported that AFB1-treatment increases 8-hydroxy-2′-deoxyguanosine (8-OHdG), which was enormously found in tissues exposed to AFB1 [32]. The 8-OHdG adduct, which is generated from the binding between the hydroxyl radicals and guanine residues of DNA, is considered as a valuable bio-

In polluted food and water, especially in the regions that are warm and have high humidity, AFB1 coexists with microcystin-LR (MC-LR), a hepatotoxic toxin produced by *Cyanobacteria*. DNA damage is enhanced when cells are exposed to both AFB1 and MC-LR, compared to the exposure to AFB1 or MC-LR, alone, because exposure to both AFB1 and MC-LR further increases the release of ROS. In this case, AFB1 induces genotoxicity through increasing oxidative stress and inhibiting the activity of DNA base excision repair genes [41]. Thus, induction of oxidative DNA damage is responsible for AFB1-induced hepatocarcinogenesis besides the

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].

marker for the measurement of oxidative DNA damage [40].

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

elevated number of AFB1-DNA adducts.

**3. AFB1-induced oxidative stress**

biomarkers and DNA damage [34].
