**3. The molecular mechanisms of aflatoxin-induced hepatocarcinoma**

As described earlier, the main chronic toxification of aflatoxins is chronic liver damage and induced tumorigenesis of hepatocarcinoma. AFB1 has been proved as an I-type chemical carcinogen. Mechanisms of AFB1-induced hepatocarcinoma mainly involve in DNA damage and repair, the inactivation of tumor suppressor genes and the activation of oncogenes from genic mutations, abnormal immunoreaction, and inheritance alterations.

#### **3.1. Aflatoxin-induced DNA damage**

**Figure 4.** The third stage of aflatoxin biosynthesis. The third stage of aflatoxin biosynthesis, including the 13th (R13) to 18th reaction (R18) of biosynthesis, refers from versicolorin B (VB) to the formation of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). *Abbreviations*. VBD, versicolorin B (VB) desaturase; DMSTSS, demethylsterigmatocystin (DMST) synthase system; OMTI, *O*-methyltransferase I; OMTII, *O*-methyltransferase II; OAE, OrdA enzyme; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide-adenine dinucleotide

phosphate (reduced form). *Noted:* adapted from Yabe and Nakajima [7].

118 Liver Research and Clinical Management

Increasing evidence has shown that the carcinogenicity of aflatoxins results from aflatoxininduced DNA damage, including the formation of DNA adducts, DNA single strand breaks (SSBs) or double strand breaks (DSBs), chromosomal aberration damage (CAD), unscheduled DNA synthesis (USDS), abnormal chromatid exchange (ACE), the formation of micronuclei and macronuclei, and oxidation DNA damage. Of these DNA damages, AFB1-DNA adducts are the most common damage types and consist of 8,9-dihydro-8-(N<sup>7</sup> -guanyl)-9-hydroxy–AFB1 adduct (AFB1-GA) and ring-opened formamidopyrimidine AFB1 adduct (AFB1-FAPYA). The formation of AFB1-GA begins from AFB1 covalent binding to DNA and its product 8,9-epoxide-AFB1 (AFBE) by CYP450 [12, 13]. This adduct can automatically not only give rise to AFB1-FAYPA, which is accumulated using a time-dependence and nonenzyme pathway, but also be transferred into AFP1, AFM1, AFQ1, and other products by metabolic enzymes.

mutation was further proved to locate in the GC-rich regions via the plasmid system identifying mutational target enzyme and named as hot-spot regions for aflatoxin-induced mutations [29–31]. Results from quantitative analyses based on the *in vitro* cell model, which was transfected by pS189 (a shuttle vector having mutative targets), also showed that more than 90% of mutative spectra caused by aflatoxins was GC to TA (about 50% of mutations) and GG to TC transversion (about 30% of mutations) [32]. It has been proved that the accumulation of these transversions will result in the mutations of some important genes such as TP53 and Ras and

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121

Studies *in vivo* and *in vitro* have examined the abnormality of tumor suppressor genes by aflatoxin exposure (**Table 2**). Among these known genes, the abnormality of TP53 induced by aflatoxins has been proved to be an important molecule change [34, 35]. In high aflatoxinexposure areas, the mutations of TP53 gene, especially hot-spot mutation at codon 249, are present among more than 40% of patients with AFB1-related hepatocarcinoma, whereas this kind of mutation is very rare among cases with null or low AFB1 exposure [14, 36, 37]. Therefore, the mutation at codon 249 of TP53 gene has been defined as a molecular symbol for hepatocarcinoma caused by AFB1 exposure. Results from clinical sample and experimental studies further display that consistent exposure of aflatoxins may result in the accumulation of TP53 mutant protein and abnormal DNA damage repair, apoptosis, and immunoreaction [38]. Other genes such as bcl2, p27, p16, and p21 are found to produce different expression or abnormal structural change under the conditions of aflatoxin expression (**Table 2**). Taken together, inactivation of tumor suppressor genes from mutation and increasing mutant

In the past decades, the abnormality of oncogenes induced by aflatoxins has mainly been focused on c-myc and ras genes, involving in the activation, expression, and mutation of proto-oncogenes (**Table 3**). For example, Tashiro et al. investigated the effects of AFB1 exposure on oncogenes based on rat model with AFB1-induced hepatomas and found that the expression of both c-myc and c-Ha-ras was upregulated in all the tumors [65]. They also observed c-Ha-ras amplification and rearrangement [65]. In Fischer rat models with AFB1 and AFG1-induced liver tumors, Sinha et al. observed that aflatoxins can induce activation of N-ras and spot mutation of G to A at codon 12 of Ki-ras [66]. This type of activation and mutation will increase in the tissues with liver cancer than those with noncancers [66–69]. Results from *in vitro* studies have further proved that aflatoxins can induce gene mutations of oncogenes [70]. Together, these data suggest that aflatoxins may activate proto-oncogenes by

The interaction of aflatoxins and hepatitis B virus (HBV) has been proved in the carcinogenesis of hepatocarcinoma by molecular epidemiological and clinicopathological studies and sys-

**3.3. The abnormality of tumor suppressor genes induced by aflatoxins**

expression may be a crucial step of malignant transformation for liver cells.

inducing gene mutations and promote the carcinogenesis of hepatocarcinoma.

**3.5. The interaction of aflatoxins and hepatitis B virus promoting** 

**hepatocarcinogenesis**

**3.4. The abnormality of oncogenes induced by aflatoxins**

promote hepatocarcinogenesis [31, 33].

Additionally, AFB1 also induces oxidation DNA damage such as 8-oxodeoxyguanosine (8-oxyG). These damages induced by aflatoxins, if not timely repaired, can cause subsequent repair-resistant adducts and depurination or lead to error-prone DNA repair resulting in DSBs, SSBs, USDs, CAD, ACE, and frame shift mutations. Interestingly, the accumulation of DNA damages is positively associated with the time and the levels of aflatoxin exposure and modifies the risk of hepatocarcinoma through regulating the expression of some genes such as a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) [14], X-ray repair complementing 4 (XRCC4) [15], microRNA-4651 [16], and so on (**Table 1**). For example, Huang et al. [14] investigated the association between AFB1-DNA adducts via a hospitalbased case control study and found increasing AFB1-DNA adducts negatively correlated with ADAMTS5 expression. It is known that ADAMTS5 may act as a tumor suppressor gene via decreasing vascular endothelial growth factor (VEGF) expression and inhibiting tumor angiogenesis and metastasis [17]. The downregulation of XRCC4 by increasing AFB1-DNA adducts decreases repair capacity for SSBs and DSBs and increases risk of tumor suppressor gene TP53 mutation and tumors [15, 18–22]. These genes progress the tumorigenesis and progression of hepatocarcinoma via regulating DNA repair capacity and angiogenesis. Although AFB1- DNA adducts are mainly produced in liver cells, they are also found in the immune cells and may regulate the immune function. Thus, DNA damage may be an important molecular event and may play a crucial role in the carcinogenesis of hepatocarcinoma caused by aflatoxins.

#### **3.2. The mutagenesis of aflatoxins**

Aflatoxin-induced DNA adducts can produce depurination, DSBs, the substitution of DNA bases, and frame shift mutations. In the past decades, the *in vivo* and *in vitro* studies have shown that the mutagenesis of aflatoxins can induce the mutation from GC to TA. As previously shown, mispairing of the aflatoxin-DNA adducts can cause both transition and transversion mutations [25–27]. In an *in vitro* non-sense analysis, Foster et al. found that the action form of AFB1 (namely AFBE) can induce more than 90% of GC to TA mutation [28]. This


**Table 1.** The change of gene expression related to DNA damage induced by aflatoxins.

mutation was further proved to locate in the GC-rich regions via the plasmid system identifying mutational target enzyme and named as hot-spot regions for aflatoxin-induced mutations [29–31]. Results from quantitative analyses based on the *in vitro* cell model, which was transfected by pS189 (a shuttle vector having mutative targets), also showed that more than 90% of mutative spectra caused by aflatoxins was GC to TA (about 50% of mutations) and GG to TC transversion (about 30% of mutations) [32]. It has been proved that the accumulation of these transversions will result in the mutations of some important genes such as TP53 and Ras and promote hepatocarcinogenesis [31, 33].

#### **3.3. The abnormality of tumor suppressor genes induced by aflatoxins**

are the most common damage types and consist of 8,9-dihydro-8-(N<sup>7</sup>

120 Liver Research and Clinical Management

**3.2. The mutagenesis of aflatoxins**

adduct (AFB1-GA) and ring-opened formamidopyrimidine AFB1 adduct (AFB1-FAPYA). The formation of AFB1-GA begins from AFB1 covalent binding to DNA and its product 8,9-epoxide-AFB1 (AFBE) by CYP450 [12, 13]. This adduct can automatically not only give rise to AFB1-FAYPA, which is accumulated using a time-dependence and nonenzyme pathway, but also be transferred into AFP1, AFM1, AFQ1, and other products by metabolic enzymes.

Additionally, AFB1 also induces oxidation DNA damage such as 8-oxodeoxyguanosine (8-oxyG). These damages induced by aflatoxins, if not timely repaired, can cause subsequent repair-resistant adducts and depurination or lead to error-prone DNA repair resulting in DSBs, SSBs, USDs, CAD, ACE, and frame shift mutations. Interestingly, the accumulation of DNA damages is positively associated with the time and the levels of aflatoxin exposure and modifies the risk of hepatocarcinoma through regulating the expression of some genes such as a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) [14], X-ray repair complementing 4 (XRCC4) [15], microRNA-4651 [16], and so on (**Table 1**). For example, Huang et al. [14] investigated the association between AFB1-DNA adducts via a hospitalbased case control study and found increasing AFB1-DNA adducts negatively correlated with ADAMTS5 expression. It is known that ADAMTS5 may act as a tumor suppressor gene via decreasing vascular endothelial growth factor (VEGF) expression and inhibiting tumor angiogenesis and metastasis [17]. The downregulation of XRCC4 by increasing AFB1-DNA adducts decreases repair capacity for SSBs and DSBs and increases risk of tumor suppressor gene TP53 mutation and tumors [15, 18–22]. These genes progress the tumorigenesis and progression of hepatocarcinoma via regulating DNA repair capacity and angiogenesis. Although AFB1- DNA adducts are mainly produced in liver cells, they are also found in the immune cells and may regulate the immune function. Thus, DNA damage may be an important molecular event and may play a crucial role in the carcinogenesis of hepatocarcinoma caused by aflatoxins.

Aflatoxin-induced DNA adducts can produce depurination, DSBs, the substitution of DNA bases, and frame shift mutations. In the past decades, the *in vivo* and *in vitro* studies have shown that the mutagenesis of aflatoxins can induce the mutation from GC to TA. As previously shown, mispairing of the aflatoxin-DNA adducts can cause both transition and transversion mutations [25–27]. In an *in vitro* non-sense analysis, Foster et al. found that the action form of AFB1 (namely AFBE) can induce more than 90% of GC to TA mutation [28]. This

**Gene Expression change Role of change in the hepatocarcinoma carcinogenesis Ref** ADAMTS5 Down Angiogenesis, metastasis, prognosis [14] XRCC4 Down Low DNA repair capacity, gene mutation [15] MicroRNA-4651 Up Angiogenesis, metastasis, prognosis [16] MicroRNA-24 Up Angiogenesis, metastasis, prognosis [23] MicroRNA-429 Up Angiogenesis, metastasis, prognosis [24]

**Table 1.** The change of gene expression related to DNA damage induced by aflatoxins.


Studies *in vivo* and *in vitro* have examined the abnormality of tumor suppressor genes by aflatoxin exposure (**Table 2**). Among these known genes, the abnormality of TP53 induced by aflatoxins has been proved to be an important molecule change [34, 35]. In high aflatoxinexposure areas, the mutations of TP53 gene, especially hot-spot mutation at codon 249, are present among more than 40% of patients with AFB1-related hepatocarcinoma, whereas this kind of mutation is very rare among cases with null or low AFB1 exposure [14, 36, 37]. Therefore, the mutation at codon 249 of TP53 gene has been defined as a molecular symbol for hepatocarcinoma caused by AFB1 exposure. Results from clinical sample and experimental studies further display that consistent exposure of aflatoxins may result in the accumulation of TP53 mutant protein and abnormal DNA damage repair, apoptosis, and immunoreaction [38]. Other genes such as bcl2, p27, p16, and p21 are found to produce different expression or abnormal structural change under the conditions of aflatoxin expression (**Table 2**). Taken together, inactivation of tumor suppressor genes from mutation and increasing mutant expression may be a crucial step of malignant transformation for liver cells.

#### **3.4. The abnormality of oncogenes induced by aflatoxins**

In the past decades, the abnormality of oncogenes induced by aflatoxins has mainly been focused on c-myc and ras genes, involving in the activation, expression, and mutation of proto-oncogenes (**Table 3**). For example, Tashiro et al. investigated the effects of AFB1 exposure on oncogenes based on rat model with AFB1-induced hepatomas and found that the expression of both c-myc and c-Ha-ras was upregulated in all the tumors [65]. They also observed c-Ha-ras amplification and rearrangement [65]. In Fischer rat models with AFB1 and AFG1-induced liver tumors, Sinha et al. observed that aflatoxins can induce activation of N-ras and spot mutation of G to A at codon 12 of Ki-ras [66]. This type of activation and mutation will increase in the tissues with liver cancer than those with noncancers [66–69]. Results from *in vitro* studies have further proved that aflatoxins can induce gene mutations of oncogenes [70]. Together, these data suggest that aflatoxins may activate proto-oncogenes by inducing gene mutations and promote the carcinogenesis of hepatocarcinoma.

#### **3.5. The interaction of aflatoxins and hepatitis B virus promoting hepatocarcinogenesis**

The interaction of aflatoxins and hepatitis B virus (HBV) has been proved in the carcinogenesis of hepatocarcinoma by molecular epidemiological and clinicopathological studies and sys-


tematically reviewed by several studies [73–75]. In brief, the first clinicopathological evidence of aflatoxins interacting with HBV was provided by Yeh et al. [76]. Through a case-control study design conducted in Guangxi Area, they found that these HBV-positive individuals with high AFB1 exposure consumption featured 10-times the mortality rate compared with those with low exposure consumption. Results from multivariable interactive analyses have further convinced that AFB1 multiplicatively interacted with HBV status for promoting hepatocarcinoma risk [77–80]. For example, Williams et al. reported that the risk of developing hepatocarcinoma was 6.37 for aflatoxin exposure, 11.3 for HBV infection, and 73.0 for the combination of aflatoxin and HBV [77]. The following several molecular epidemiological studies with large-size samples from areas with high aflatoxin exposure and high HBV infection in China showed remarkably multiplicative effect for hepatocarcinoma risk (multiplicative inter-

**Gene Study design Change Significance Ref** N-ras HCCs (n = 36) Mutation at codon 61 Carcinogenesis [51] c-myc Mice model with HNP Expression ↑, amplification, rearrangement Carcinogenesis [65] c-Ha-ras Mice model with HNP Expression ↑, amplification, rearrangement Carcinogenesis [65] Ki-ras Mice model with HNP Activation Carcinogenesis [69] N-ras Mice model with HNP Activation Carcinogenesis [66] Ki-ras Mice model with HNP Mutation at codon 12 Carcinogenesis [66] N-ras Mice model with HCC Activation Carcinogenesis [67] Ki-ras Mice model with HCC Activation Carcinogenesis [67] c-Ha-ras Mice model with HNP Mutation at codon 61: 40–60% Carcinogenesis [71, 72]

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This interaction of two hepatocarcinogenic causes has been proved in the transgenic mice models with overexpressing HBV large envelope polypeptide [81]. Results from this study exhibited that animals will produce more rapid and extensive hepatic dysplasia and hepatocarcinoma under the conditions with aflatoxin consumption [81]. Similar findings have also

The aflatoxins interacting with HBV infection promoting hepatocarcinoma development mechanically involve in the following aspects. First, HBV infection directly or indirectly increases the sensitivity of hepatocytes on the toxification of aflatoxins. Evidence from observation studies have displayed that HBV-positive carriers have more amount of aflatoxin adducts than those with negative HBV status, although they are from the same high aflatoxin exposure area [85, 86]. The active product of aflatoxin AFBE is found to significantly increase the risk of viral DNA integrating into damaged DNA strand [87]. This promotes malignant transformation of damaged hepatocytes by aflatoxins. Second, HBV

action: 63.2 (both positive) > 1.9 (AFB1 positive) × 9.5 (HBV positive) [78–80].

*Abbreviations.* HNP, hepatic neoplasms; HCC, hepatocarcinoma.

**Table 3.** The change information of oncogenes induced by aflatoxins.

shown in the studies based on woodchuck and duck models [82–84].

**Table 2.** The change information of tumor suppressor genes induced by aflatoxins in hepatic cells and hepatocarcinoma cells.


**Table 3.** The change information of oncogenes induced by aflatoxins.

**Gene Study design Change Significance Ref** TP53 Mice model with HNP Expression ↑ DNA damage ↑ [39] bcl2 Mice model with HNP Expression ↓ DNA damage ↑ [39] p27 Hepatocytes *in vitro* Expression ↓ DNA damage ↑ [40] p21 Hepatocytes *in vitro* Expression ↓ DNA damage ↑ [40] TP53 HCCs (n = 223) Expression ↑, multiplot mutation Carcinogenesis [41] TP53 HCCs (n = 124) Mutation at codon 249: 60% Carcinogenesis [42] H2AX HCC cells *in vitro* Phosphorylation Carcinogenesis [43] BP1 HCC cells *in vitro* Phosphorylation Carcinogenesis [43] TP53 HCCs (n = 52) Mutation at codon 249: 50% Carcinogenesis [44] p16 HCCs (n = 40) Methylation Carcinogenesis [45] p53 HCCs (n = 40) Multiplot mutation Carcinogenesis [45]

Mutation at codon 249: 36%, protein

TP53 Mice model with HNP Multiplot mutation Carcinogenesis [48]

TP53 Atcc-Ccl13 *in vitro* Mutation at codon 249 Carcinogenesis [50] TP53 HCCs (n = 36) Mutation at codon 249 Carcinogenesis [51]

TP53 HCCs (n = 60) Mutation at codon 249: 69% Carcinogenesis [58, 59] TP53 Hepatocytes *in vitro* Multiplot mutation Carcinogenesis [60]

TP53 HCCs (n = 15) Mutation at codon 249 and 254 Carcinogenesis [62] TP53 HCC cells *in vitro* AFB1-induced Mutation at codon 249 Carcinogenesis [63] TP53 HCCs (n = 18) Mutation at codon 249: 53% Carcinogenesis [64]

**Table 2.** The change information of tumor suppressor genes induced by aflatoxins in hepatic cells and hepatocarcinoma cells.

promoting IGF-II expression

accumulation: 50%

protein increasing

TP53 HCCs (n = 110) Mutation at codon 249: 69% DNA damage,

TP53 HCC cells *in vitro* AFB1-induced mutation at codon 249

TP53 Mice model Mutation at codon 249 and 346, mutant

*Abbreviations.* HNP, hepatic neoplasms; HCC, hepatocarcinoma.

Multiplot mutation at CpG Carcinogenesis [46]

Carcinogenesis [47]

Carcinogenesis [49]

Carcinogenesis [52–57]

[61]

carcinogenesis

p53 AFB1-induced mutation *in vitro*

122 Liver Research and Clinical Management

TP53 HCCs (n = 64) plus a meta-analysis

tematically reviewed by several studies [73–75]. In brief, the first clinicopathological evidence of aflatoxins interacting with HBV was provided by Yeh et al. [76]. Through a case-control study design conducted in Guangxi Area, they found that these HBV-positive individuals with high AFB1 exposure consumption featured 10-times the mortality rate compared with those with low exposure consumption. Results from multivariable interactive analyses have further convinced that AFB1 multiplicatively interacted with HBV status for promoting hepatocarcinoma risk [77–80]. For example, Williams et al. reported that the risk of developing hepatocarcinoma was 6.37 for aflatoxin exposure, 11.3 for HBV infection, and 73.0 for the combination of aflatoxin and HBV [77]. The following several molecular epidemiological studies with large-size samples from areas with high aflatoxin exposure and high HBV infection in China showed remarkably multiplicative effect for hepatocarcinoma risk (multiplicative interaction: 63.2 (both positive) > 1.9 (AFB1 positive) × 9.5 (HBV positive) [78–80].

This interaction of two hepatocarcinogenic causes has been proved in the transgenic mice models with overexpressing HBV large envelope polypeptide [81]. Results from this study exhibited that animals will produce more rapid and extensive hepatic dysplasia and hepatocarcinoma under the conditions with aflatoxin consumption [81]. Similar findings have also shown in the studies based on woodchuck and duck models [82–84].

The aflatoxins interacting with HBV infection promoting hepatocarcinoma development mechanically involve in the following aspects. First, HBV infection directly or indirectly increases the sensitivity of hepatocytes on the toxification of aflatoxins. Evidence from observation studies have displayed that HBV-positive carriers have more amount of aflatoxin adducts than those with negative HBV status, although they are from the same high aflatoxin exposure area [85, 86]. The active product of aflatoxin AFBE is found to significantly increase the risk of viral DNA integrating into damaged DNA strand [87]. This promotes malignant transformation of damaged hepatocytes by aflatoxins. Second, HBV infection increases the mutation frequency at codon 249 of TP53 gene and coordinates with aflatoxins for abrogating the normal functions of TP53 (such as the control of cell cycle, DNA damage repair, and cell apoptosis), which contributes to multisteps of hepatic carcinogenesis [64, 88]. Third, the HBV X gene–expressing protein inhibits base excision repair potential and results in an increasing accumulation of aflatoxin-DNA adducts [89]. Finally, HBV infections can cause hepatocytic necrosis, inflammatory proliferation, and oxygen/ nitrogen active products, which may increase the likelihood of aflatoxin-induced mutations and the cellular clonal expansion containing mutations [90–92].

#### **3.6. The interaction of aflatoxins and inheritance alterations promoting hepatocarcinogenesis**

Increasing evidence has exhibited that the genetic alterations in DNA repair genes increase the amount of AFB1-DNA adducts and the frequency of hot-spot mutation at codon 249 of TP53 gene and may promote hepatic toxification of aflatoxins [1, 19, 20, 22, 37, 93–98]. Joint analyses based on meta-analyses further showed this kind of toxic effects (**Table 4**) [1, 22]. The genetic variants in other genes, such as CYP450, glutathione *S*-transferase T1 (GSTT1), glutathione *S*-transferase M1 (GSTM1), and microsomal epoxide hydrolase (HEHY), also display similar modificative effects on aflatoxin-induced hepatocarcinoma [98–101]. Interestingly, the multiplicatively interactive effects between aflatoxins and genetic alterations in these genes have been identified in the risk elucidation of hepatocarcinoma related to aflatoxins [22]. Taken together, genetic deficiency in the DNA repair and detoxification capacity may play a vital role in the carcinogenetic process of aflatoxin-induced hepatocarcinoma.

#### **3.7. The aflatoxin-caused immunosuppression promoting hepatocarcinogenesis**

Increasing evidence from *in vitro* and *in vivo* studies has proved that the immunosuppression induced by aflatoxins plays an important role in the carcinogenesis of hepatocarcinoma. Several known mechanisms may involve in this progression step. First, aflatoxins can significantly suppress the functions of macrophages via affecting the expression and secretions of cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-2, IL-3, IL-6, and reactive intermediates (including nitric oxide, hydrogen peroxide, and superoxide anion) [102, 103]. The suppression of macrophages by aflatoxins may be also correlated with the arrest in the G1/G0 phase [104] and altered expression of CD14 (a cell surface protein functionally regulating immunoreaction) [105]. This suppression may result in the dysregulation of the immune response and homeostasis, which contributes to the accumulation of abnormal cells with DNA damage and altered genome induced by aflatoxins, and ultimately progresses tumorigenesis. Second, aflatoxin exposure can decrease the secretion of antibody such as IgA [106]. For example, Turner et al. investigated effects of aflatoxin exposure on antibody production based on a large molecular epidemiological study [106]. In their study, they tested the levels of saliva secretory IgA (sIgA) in Gambian children (n = 472) with different degree exposure of aflatoxins and found that these individuals with high aflatoxin exposure featured lower level of sIgA in their saliva compared to those without high exposure (50.4 vs. 70.2 μg/mg protein). Finally, aflatoxins may alter T-cell functions (including decreased T-cell populations and suppressed CD4+ T-cell function) and increase individuals' susceptibility to other carcinogens [77, 107].

Altogether, the data available to date make it clear that aflatoxins can exert an immunosuppressive effect via different pathways. However, more detailed mechanisms by which this

*Noted:* Adapted from Refs. [13] and [84]. *Abbreviations*. TP53M, hot-spot mutation at codon 249 of TP53 gene; RS#, the

**Gene RS# Genotype TP53M DNA adducts**

XRCC1 rs25487 CC 46.51 Reference 3.276

XRCC3 rs861539 GG 32.17 Reference 2.990

XRCC7 rs7003908 AA 21.24 Reference 2.879

XRCC4 rs28383151 GG 67.03 Reference 3.308

XRCC4 rs3734091 GG 72.31 Reference 3.229

XPD rs13181 TT 34.41 Reference 2.926

XPC rs2228001 TT 34.05 Reference 3.083

**% Risk** *P* **Mean** *P*

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CT 45.25 2.419 3.371 × 10−11 3.264 0.899 TT 8.24 5.028 6.651 × 10−6 3.640 0.026

GA 43.55 1.380 0.018 3.216 0.025 AA 24.28 1.524 0.011 3.897 4.962 × 10−14

AC 46.06 1.883 1.372 × 10−5 3.347 1.663 × 10−5 CC 32.71 2.089 4.368 × 10−6 3.550 1.751 × 10−8

GA 21.68 1.688 0.001 3.405 0.069 AA 11.29 3.829 7.387 × 10−6 3.721 2.867×10−4

GT 17.56 2.799 9.191 × 10−7 3.439 0.095 TT 10.13 5.104 3.826 × 10−6 3.654 0.005

TG 41.85 1.458 0.005 3.253 0.011 GG 23.75 1.744 0.001 4.062 4.265 × 10−6

TG 48.30 1.500 0.002 3.332 0.001 GG 17.65 1.818 0.001 3.666 3.404 × 10−22

In the past decades, the advance in pathological mechanisms of aflatoxin-related hepatocarcinoma held great promise. However, we are still far from a comprehensive view of this kind of potentials. First, the detailed metabolic step and corresponding enzymes, especially the first-stage

effect is mediated remain unknown.

number of polymorphism.

**4. Limitation and further direction**

**Table 4.** Polymorphisms in DNA repair genes and HCC risk.


*Noted:* Adapted from Refs. [13] and [84]. *Abbreviations*. TP53M, hot-spot mutation at codon 249 of TP53 gene; RS#, the number of polymorphism.

**Table 4.** Polymorphisms in DNA repair genes and HCC risk.

infection increases the mutation frequency at codon 249 of TP53 gene and coordinates with aflatoxins for abrogating the normal functions of TP53 (such as the control of cell cycle, DNA damage repair, and cell apoptosis), which contributes to multisteps of hepatic carcinogenesis [64, 88]. Third, the HBV X gene–expressing protein inhibits base excision repair potential and results in an increasing accumulation of aflatoxin-DNA adducts [89]. Finally, HBV infections can cause hepatocytic necrosis, inflammatory proliferation, and oxygen/ nitrogen active products, which may increase the likelihood of aflatoxin-induced mutations

Increasing evidence has exhibited that the genetic alterations in DNA repair genes increase the amount of AFB1-DNA adducts and the frequency of hot-spot mutation at codon 249 of TP53 gene and may promote hepatic toxification of aflatoxins [1, 19, 20, 22, 37, 93–98]. Joint analyses based on meta-analyses further showed this kind of toxic effects (**Table 4**) [1, 22]. The genetic variants in other genes, such as CYP450, glutathione *S*-transferase T1 (GSTT1), glutathione *S*-transferase M1 (GSTM1), and microsomal epoxide hydrolase (HEHY), also display similar modificative effects on aflatoxin-induced hepatocarcinoma [98–101]. Interestingly, the multiplicatively interactive effects between aflatoxins and genetic alterations in these genes have been identified in the risk elucidation of hepatocarcinoma related to aflatoxins [22]. Taken together, genetic deficiency in the DNA repair and detoxification capacity may play a

and the cellular clonal expansion containing mutations [90–92].

**hepatocarcinogenesis**

124 Liver Research and Clinical Management

**3.6. The interaction of aflatoxins and inheritance alterations promoting** 

vital role in the carcinogenetic process of aflatoxin-induced hepatocarcinoma.

**3.7. The aflatoxin-caused immunosuppression promoting hepatocarcinogenesis**

Increasing evidence from *in vitro* and *in vivo* studies has proved that the immunosuppression induced by aflatoxins plays an important role in the carcinogenesis of hepatocarcinoma. Several known mechanisms may involve in this progression step. First, aflatoxins can significantly suppress the functions of macrophages via affecting the expression and secretions of cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-2, IL-3, IL-6, and reactive intermediates (including nitric oxide, hydrogen peroxide, and superoxide anion) [102, 103]. The suppression of macrophages by aflatoxins may be also correlated with the arrest in the G1/G0 phase [104] and altered expression of CD14 (a cell surface protein functionally regulating immunoreaction) [105]. This suppression may result in the dysregulation of the immune response and homeostasis, which contributes to the accumulation of abnormal cells with DNA damage and altered genome induced by aflatoxins, and ultimately progresses tumorigenesis. Second, aflatoxin exposure can decrease the secretion of antibody such as IgA [106]. For example, Turner et al. investigated effects of aflatoxin exposure on antibody production based on a large molecular epidemiological study [106]. In their study, they tested the levels of saliva secretory IgA (sIgA) in Gambian children (n = 472) with different degree exposure of aflatoxins and found that these individuals with high aflatoxin exposure featured lower level of sIgA in their saliva compared to those without high exposure (50.4 vs. 70.2 μg/mg protein). Finally, aflatoxins may alter T-cell functions (including decreased T-cell populations and suppressed CD4+ T-cell function) and increase individuals' susceptibility to other carcinogens [77, 107].

Altogether, the data available to date make it clear that aflatoxins can exert an immunosuppressive effect via different pathways. However, more detailed mechanisms by which this effect is mediated remain unknown.

### **4. Limitation and further direction**

In the past decades, the advance in pathological mechanisms of aflatoxin-related hepatocarcinoma held great promise. However, we are still far from a comprehensive view of this kind of potentials. First, the detailed metabolic step and corresponding enzymes, especially the first-stage reaction and toxicity mechanisms, have not been elucidated. Second, although the activation of aflatoxins is found to act as a crucial step, it is unclear how the tumorigenesis of hepatocarcinoma is triggered by aflatoxins. Third, the vast literature for aflatoxin-induced hepatocarcinoma mainly focuses on the studies on AFB1, and some important information may have been lost. Fourth, in spite of some evidence of AFB1 inducing abnormal immunoreaction and interacting with hepatitis virus and genetic factors, they are at the primary stage and still far from elucidation. Therefore, the detailed toxicity mechanisms of aflatoxins and corresponding carcinogenesis mechanism will greatly benefit our understanding of aflatoxin-related hepatocarcinoma.

AFG2 aflatoxin G2

AFP α-fetoprotein

*A. flavus Aspergillus flavus*

*A. parasiticus Aspergillus parasiticus*

*A. nidulans Aspergillus nidulans*

*A. bombycis Aspergillus bombycis*

HBV hepatitis virus B

HCV hepatitis virus C

*A. pseudotamarii Aspergillus pseudotamarii*

Hepatocarcinoma hepatocellular carcinoma

SAM *S*-adenosylmethionine

CYP450 cytochromes P450

, Yan Deng4†

 and Qiang Xia2 \*Address all correspondence to: sjtulongxd@263.net

† These authors contributed equally to this work.

, Juan Wang1†

**Author details**

Xi-Dai Long1,2,3\*†

Bing-Chen Huang1

Nationalities, Baise, China

University, Shanghai, China

Nationalities, Baise, China

Xue-Min Wu1†

NAD one nicotinamide-adenine dinucleotide

, Xiao-Ying Huang1†

1 Department of Pathology, the Affiliated Hospital of Youjiang Medical University for

2 Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong

4 Department of Epidemiology, Youjiang Medical University for Nationalities, Baise, China

5 Department of Medicine, the Affiliated Hospital of Youjiang Medical University for

, Qun-Qing Xu<sup>3</sup>

3 Guangxi Clinic Research Center of Hepatobiliary Diseases, Baise, China

, Jin-Guang Yao1†

Molecular Mechanisms of Hepatocellular Carcinoma Related to Aflatoxins: An Update

http://dx.doi.org/10.5772/intechopen.72883

127

, Xiao-Ying Zhu<sup>3</sup>

, Qun-Ying Su1†

,

, Chao Wang5

,

NAPDH nicotinamide-adenine dinucleotide phosphate reduced form
