Molecular Mechanisms of Hepatocellular Carcinoma Related to Aflatoxins: An Update

Hepatocellular carcinoma (hepatocarcinoma) is a major type of primary liver cancer and one of the most frequent human malignant neoplasms. Aflatoxins are I-type chemical carcinogen for hepatocarcinoma. Increasing evidence has shown that hepatocarcinoma induced by aflatoxins is the result of interaction between aflatoxins and hereditary factor. Aflatoxins can induce DNA damage including DNA strand break, adducts formation, oxidative DNA damage, and gene mutation and determine which susceptible individuals feature cancer. Inheritance such as alterations may result in the activation of protooncogenes and the inactivation of tumor suppressor genes and determine individual susceptibility to cancer. Interaction between aflatoxins and genetic susceptible factors commonly involve in almost all pathologic sequence of hepatocarcinoma: chronic liver injury, cirrhosis, atypical hyperplastic nodules, and hepatocarcinoma of early stages. In this review, we discuss the biogenesis, toxification, and epidemiology of aflatoxins and signal pathways of aflatoxin-induced hepatocarcinoma. We also discuss the roles of some important genes related to cell apoptosis, DNA repair, drug metabolism, and tumor metastasis in hepatocarcinogenesis related to aflatoxins.


Introduction
Hepatocellular carcinoma (also called hepatocarcinoma or liver carcinoma) is a major type of primary liver cancer and one of the most frequent human malignant neoplasms. This malignancy has been proved to correlate with aflatoxins, especially aflatoxin B1 (AFB1) [1][2][3].
Increasing evidence has exhibited that several mechanisms, including the toxic production from metabolism, the accumulation of DNA damage and genic mutation-induced aflatoxins, the decreasing DNA repair capacity, and dysregulation of signal pathways may play a central role in the tumorigenesis of aflatoxin-induced hepatocarcinoma [4][5][6]. In this review, we discuss the biogenesis, metabolism, and genic toxification of aflatoxins. We also discuss the molecular mechanisms of aflatoxin-induced hepatocarcinoma, involving in aflatoxin toxification, abnormal change of tumor relative genes, the interaction of aflatoxins and genetic factors, and signal pathway for tumorigenesis. The roles of some important genes related to cell apoptosis, DNA repair, drug metabolism, and tumor metastasis in hepatocarcinogenesis related to aflatoxins are further emphasized.

Aflatoxin biosynthesis
The biosynthesis of aflatoxins has been fully summarized in several previous reviews [7,8].
In brief, aflatoxins are an important type of mycotoxins, which were the most early identified in the Aspergillus flavus (A. flavus) and regarded as causative agents of "turkey X" disease in the late 1950s and early 1960s. Thus, these toxins were named as "aflatoxins (namely A. flavus toxins)" according to their origin fungus [9]. Until now, 17 related aflatoxin isoforms and aflatoxin metabolites have been identified, and 4 of them often contaminated a number of agricultural commodities [10]. According to the amounts and fluorescent reactions, four aflatoxins primarily identified in foodstuffs are named as AFB1, aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). Among these four known aflatoxins, AFB1 and AFB2 are named as B-type aflatoxins because they are attached to a pentanone and can produce blue-color fluorescent under UV light, whereas AFG1 and AFG2 are termed as G-type aflatoxins because of their attachment to a 6-membered lactone and producing green fluorescent color feature. These aflatoxins are mainly produced by A. flavus, Aspergillus parasiticus (A. parasiticus), Aspergillus nidulans (A. nidulans), Aspergillus pseudotamarii (A. pseudotamarii), and Aspergillus bombycis (A. bombycis) [7,8].
Toxigenic strains of A. flavus produce only B-type aflatoxins, but do not synthesize G-type aflatoxins due to the deletion of an unstable microsome enzyme and a-220 kDa cytosolic protein. The other aflatoxigenic species including A. parasiticus, A. nidulans, A. pseudotamarii, and A. bombycis can produce all four aflatoxins [8].
Numeral synthetical genes, such as aflatoxin regulatory protein gene (aflR), are required for aflatoxin biosynthesis and act as a huge neighbor gene cluster consisting of about 60-70 kb in original fungi (Figure 1) [8][9][10]. All corresponding gene-encoding enzymes and transcription factors produce aflatoxin production and regulate biosynthesis. Increasing evidence has proved that aflatoxin biosynthesis involves in at least 3 stages and 18 enzyme steps (Figures 2-4). The first stage, including the first (R01) to eighth reaction (R08) of biosynthesis, refers from acetyl CoA to hydroxyversicolorone. The primary product hydroxyversicolorone will be formed and regulated by transcription factors aflR and aflJ (Figure 2) [8,10]. The second (biosynthesis reaction: R09-R12) (Figure 3) and third stages (biosynthesis reaction: R13-R18) (Figure 4) refer from hydroxyversicolorone to versicolorin B and from versicolorin B (VB) to the formation of ultimate products, respectively. These two stages involve in the formation of hydroxy-and non-hydroxy-versicolorone, and toxins. During the aflatoxin synthesis, more than 10 nicotinamide-adenine dinucleotide phosphate reduced form (NAPDH), one nicotinamide-adenine dinucleotide (NAD), and 2S-adenosylmethionine (SAM) are required. These cofactors may play a critical role in the control of aflatoxin biosynthesis [7][8][9][10].

The metabolism of aflatoxins in liver
Aflatoxins synthesized in the mycelia are finally excreted into such mediums as cereals (maize, wheat, sorghum, rice, and millet), nuts (peanuts, pistachios, walnuts, Brazil nut, and coconut), spices (chili, turmeric, paprika, black pepper, and ginger), and seeds. Epidemiological studies have exhibited that AFB1 is the most common in contaminated human foods [8,10]. Once this aflatoxin in the mediums is taken into body, it is metabolized via two-stage reactions in the liver. The first-stage metabolisms include reduction reaction (ketoreduction to aflatoxicol), oxidative reaction (O-dealkylation to aflatoxin P1), and hydrolytic reactions (hydroxylation to aflatoxin M1, aflatoxin Q1, and aflatoxin B2). This stage reaction involves numerous enzymes such as cytochromes P450 (CYP450), monooxygenases, amino-oxidases,  alcohol dehydrogenases, epoxide-hydrolases, aldehyde-reductases, and ketone-reductases. The second-stage reaction mainly comprises covalent binding reaction (toxic produces) and conjugation reaction (excretion and detoxification). Through these metabolites, aflatoxins ultimately transform into nontoxic secretions and toxic products [10,11].

The toxification of aflatoxins in liver
Toxification of aflatoxins in liver is mainly divided into acute and chronic toxic effects. Data from epidemiological, experimental, and clinical studies have shown that above 6000 mg exposure of aflatoxin through digestion will cause acute severe liver damage and subsequent illness or death. This kind of acute effect is mainly associated with malfunction of the liver induced by toxic metabolic products. For chronic toxic effects, chronic exposure of aflatoxins can induce DNA damage and produce genotoxicity and carcinogenicity. In the past decades, increasing evidence has proved that AFB1 as aflatoxins often induce genic mutations such as TP53 and are among the most carcinogenic substances known and the major cancerous hepatocarcinoma risk factor.  phosphate (reduced form). Noted: adapted from Yabe and Nakajima [7].

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.
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][19][20][21][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.

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][26][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

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]  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][30][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].

The abnormality of tumor suppressor genes 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.

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 AFB1and 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][67][68][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.

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

Liver Research and Clinical Management
tematically reviewed by several studies [73][74][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][78][79][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][79][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][83][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 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] Abbreviations. HNP, hepatic neoplasms; HCC, hepatocarcinoma. 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][91][92].

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][94][95][96][97][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][99][100][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.

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.

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

Summary
It has been shown that increasing exposure of aflatoxins may promote the carcinogenesis of hepatocarcinoma. Molecular mechanisms of aflatoxin-induced hepatocarcinoma involve in DNA damage, gene mutations, the inactivation of such tumor suppressor gene as TP53, the activation of proto-oncogenes, abnormal immunoreaction, and the interaction between aflatoxins and other carcinogens such as HBV. However, an understanding of aflatoxin-induced hepatocarcinoma is far from complete, and further research in this field is looked forward to elucidating more detailed mechanisms responsible for hepatocarcinoma related to aflatoxins in the future.