**3. AFB1-induced oxidative stress**

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

epigenetic alteration.

**2. AFB1-DNA adduct formation**

induced replication block in vivo.

scripts, which is revealed by microarray studies [27].

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

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 aflatoxin-

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 tran-

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

**142**

development [26, 27].

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 biomarkers and DNA damage [34].

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

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 elevated number of AFB1-DNA adducts.
