Role of epigenetic alterations in aflatoxin‐induced hepatocellular carcinoma

Aflatoxins are produced by Aspergillus flavus and Aspergillus parasiticus and are toxic carcinogens. These ‘fungal molds’ grow on corn, groundnuts, cereals and other grains. Of all the aflatoxins, Aflatoxin‐B1 (AFB1) is considered the most toxic. Long‐term exposure of AFB1 forms DNA adducts causing many genetic mutations and epigenetic alterations, ultimately leading to hepatocellular carcinoma (HCC). The liver is the major site of Aflatoxin detoxification; wherein cytochrome P‐450 (CYP450) enzymes process the AFB1 into its epoxide AFB1‐Exo‐8,9‐Epoxy (ABFO) and other less toxic metabolites. ABFO, in turn, reacts with DNA, RNA and protein molecules forming AFB adducts. The AFB1‐DNA adducts in turn will induce various mutations, mainly mediated by G→T transversions. Aflatoxins are also known to cause HCC cell proliferation, growth, and invasion as well as angiogenesis by various epigenetic mechanisms including DNA methylation, histone post‐translational modifications and non‐coding RNA deregulation, etc. In this review, we will be emphasizing on epigenetic mechanisms by which aflatoxins induce hepatocarcinogenesis. In the last section, we will also discuss various methodologies to control aflatoxin contamination and detoxification of aflatoxin adducts using natural substances that are potentially anti‐aflatoxins.

because of HCC. 1 In the United States, there has been ~fourfold increase in HCC incidence over the last four decades (1.6 per 100 000 population in 1975-1977 to 4.8 per 100 000 in 2005-2007, and 6.7 per 100 000 population by 2012). A similar rate in increase has also been reported in 2016. 4,5 Incidence of HCC is also high in Asia and Sub-Saharan Africa because of endemic hepatitis B, compared to the United States and other developing countries. Mongolia has the highest reported incidence at 93.7 per 100 000, but China has the greatest number of cases, because of both an elevated rate (18.3 per 100 000) and the world's largest population (1.4 billion persons). 3 Males get affected higher with HCC than females. 6 HCC is most likely linked with hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, Aflatoxin exposure, or alcohol consumption. 6  Mycotoxins are fungal metabolites that contaminate human food and animal feed. There are more than 20 different types of Aflatoxins that occur in nature, of which aflatoxin B1 (AFB1) is the most toxic carcinogen. AFB1 causes HCC by forming DNA adducts.
Liver cells are largely affected as hepatocytes, since they produce cytochrome P-450 enzymes that metabolize aflatoxins into toxic substances along with other complex chemicals. Cytochrome 450 enzymes like CYP3A4 convert aflatoxin into epoxy-aflatoxin that reacts with amines such as guanine, which damages DNA, causing cancer. Globally 83% of HCC cases occurring in Asia and sub-Saharan Africa are because of aflatoxin exposure. 8

| RIS K FAC TOR S
Hepatocellular carcinoma develops because of mutations and epigenetic alterations that avoid normal apoptosis as well as induce uncontrolled cell division. The most prominent factors associated with HCC include hepatitis B and hepatitis C infection, chronic alcohol consumption, aflatoxin-B1-contaminated food intake, gender differences as well as metabolic disorders like diabetes, etc. [9][10][11] Chronic infection with HBV and HCV accounts for nearly 80% of HCCs globally. 12,13 In most cases, HBV or HCV infection leads to cirrhosis, which develops into chronic hepatitis. Nearly, in 2%-5% of the patients, cirrhosis subsequently transforms into HCC. 14 It is important to note that in HBV patients, HCC can develop without cirrhosis. In Asia and Africa, the HBV infection is prevalent, and 30%-50% of patients develop HCC without cirrhosis, whereas in the USA, wherein the HBV is not endemic, >90% of patients with HBVassociated HCC have the cirrhotic liver disease. 15,16 Metabolic disorders contribute to a major segment (32%) of the population attributable fractions over time that result in HCC development. 17 Metabolic disorders and genetic diseases associated with HCC include Porphyrias, α-1 antitrypsin disease, tyrosinemia, hemochromatosis, glycogen-storage disease types I and II, as well as Wilson's disease. Individuals with hemochromatosis, non-alcoholic fatty liver disease (NAFLD), type 2 diabetes (T2DM) pose an increased risk of developing HCC. NAFLD is a major risk factor for HCC in developed countries like the USA and attributes to 10%-20% of HCCs. [18][19][20] It was proposed that NAFLD is associated with a 2.6fold increase in HCC risk, 18 however, rigorous population studies are missing in this regard. On the other hand, alcoholic cirrhosis is the second most common risk factor for HCC development in the USA and Europe. 21 Furthermore, in alcoholic cirrhosis patients, older age (>55 years) and thrombocytopenia (platelet count <125 000 per mm 3 of blood) become independent HCC risk factors. 22 Obesity and/or diabetes are the major risk factors with the highest population-attributable fraction of over 37% in the United States. 17 Similar to HBV, NAFLD-associated HCC can also occur in the absence of cirrhosis. 17,23,24 Diabetes increases the chance of HCC by two to threefold. Diabetes primes insulin resistance and reactive oxygen production, which are in turn leading to hepatic inflammation, triggering carcinogenesis. 14,25 Besides, hereditary Hemochromatosis increases HCC risk by 100-to 200-fold. 26 Males are 2.4 times more affected by HCC than females, suggesting a crucial role of sex in HCC progression. 6 Chronic exposure to the fungal toxin, AFB 1 is strongly associated with HCC development. Aflatoxins are poisonous, carcinogenic mycotoxins produced from molds that grow on decaying vegetation, grains, hay and soil. When improperly stored, they grow on edible commodities such as wheat, groundnuts, sorghum, chili peppers, cottonseed, millet, sesame seeds, peanuts, rice, cassava, corn, etc and release aflatoxins. 6 Because of inapt post-harvest processing, aflatoxin exposure to humans is prominent in several west African countries, whereas in the western countries, Aflatoxin exposure is minimal.

| MECHANIS MS OF AFL ATOXIN -INDUCED H CC
Aflatoxins once ingested, interact with and affect a wide range of biomolecules, organs and tissues. 27,28 Aflatoxins mainly interact with nucleic acids and other metabolic enzyme systems. They target DNA, RNA, as well as proteins and interfere with transcription, translation and other cellular pathways [29][30][31] (Figure 1). In the liver, enzyme CYP3A4 metabolizes AFB1 into highly reactive epoxide; AFB0, which is later transformed to AFB1-8,9-diol that binds to the lysine present in albumin forming AFB-Lysine adduct. This AFB-Lysine adduct is validated as a biomarker of aflatoxin exposure in humans. 30 Epoxy-Aflatoxin also reacts with amines such as guanine, forming AFB1-DNA adduct, which damages DNA. The evidence supports the interpretation that the formation of AFB 1 -DNA adducts in hepatocytes leads to a series of mutations, mainly G:C → T:A.
More importantly, it was observed that 25% of all mutations were G → T in the CGC trinucleotide context, which leads to the substitution of Arginine to Serine (R249S) in p53 gene. 32 In the regions of high aflatoxin-exposed areas. HCCs show tp53 R249S mutation as high as 50%-90%, whereas in regions such as the USA, where aflatoxin exposure is low, R249S mutation drops down to <6% of HCCs. 32,33 In addition to p53 hot-spot mutation, mutational activation of proto-oncogenes such as H-RAS is also observed in HCC. 34 Rieswijk et al performed the whole-genome DNA methylation changes and the whole genome transcriptomic analysis in response to AFB1 exposure and found that TXNRD1, PCNA, CCNK, DIAPH3, RAB27A and HIST1H2BF are up-regulated because of promoter hypomethylation. 35 Hypermethylation of RASSF1A promoter was also shownto be associated with AFB 1 exposure. 36,37 Zhu et al identified that AFB1 causes impairment in miRNA biogenesis. 38 These authors also showed that AFB1 down-regulates Wnt/β-catenin signalling pathway by up-regulating miR-34a and is responsible for liver tumourigenesis. 38 AFB 1 also enhances HCC cell proliferation through an IGF-2-dependent signal axis. 39 Finally, activation of oxidative stress and inflammatory factors accounts for histopathological progression of AFB1-induced hepatocarcinogenesis. 40 Unlike HCV-and alcohol-induced hepatocarcinogenesis, there is no clear connection between AFB1 exposure and the development of cirrhosis, 41 indicating that the mutational actions of this toxin might be the primary driver of HCC development. AFB1 exposure often coexists with HBV infection and such individuals possess a 5 to 10-fold increased risk of developing HCC compared with exposure to only one of these factors. 42 Recently, AFB1 exposure was also shown to increase the risk of HCC associated with HCV infection or alcohol consumption. 43 The mechanistic basis for this synergy is not yet established, it seems plausible that cooperation would derive from AFB 1 -induced mutagenesis and continuous hepatocyte turnover and regeneration during chronic infection. Aflatoxin-mediated DNA damage even affects the pro-apoptotic and cellular pathways such as c-Myc, p53, NF-kB, CDK, protein kinase A (PKA), pRb, Ras, protein kinase C (PKC), BCl 2 , Cyclins and CKI's, deregulating the cell cycle and causing HCC. 35,44 AFBO is also known to affect other metabolites, telomere length, oxidative phosphorylation and electron transport chain of carbohydrate metabolism. [45][46][47][48][49]

| EPI G ENE TI C MECHANIS MS IN AFL ATOXIN -INDUCED H CC
Epigenetics is the study of heritable changes in gene expression without undergoing any alteration in its DNA sequence. Epigenetics is a Greek word, which means 'over and above the genome'. [50][51][52] ie, a change in the phenotype is observed without any change in its genotype. Epigenetic modifications naturally occur in the developmental F I G U R E 1 Effects of aflatoxins and induction of hepatocellular carcinoma. Aflatoxins react with DNA, RNA, proteins and other compounds to form adducts. These Aflatoxin adducts cause many genetic mutations and epigenetic alterations leading to the deregulation of many cellular metabolic pathways affecting growth and normal functioning of cells process, during cellular differentiation, but they are largely influenced by several factors such as age, environmental and lifestyle changes leading to damaging effects such as cancer. 53 Epigenetic modifications induced by the aflatoxins include aberrant DNA methylation, histone post-translational modifications (methylation, acetylation, phosphorylation, ubiquitylation and sumoylation, etc) and irregular expression of ncRNA's ( Figure 2). These epigenetic modifications will, in turn, alter the gene expression profiles leading to HCC. 54-56

| DNA methylation
Methylation of DNA is a natural event that occurs in both prokaryotes and eukaryotes. In the eukaryotes, DNA methylation helps to regulate the gene expression. 57,58 In cancer, aberrant DNA methylation is a widespread phenomenon and may be among the earliest changes to occur during the onset of oncogenesis. [59][60][61] It also plays crucial roles in cell cycle regulation, genomic imprinting, X-chromosome inactivation and embryonic development.
DNA methylation is generally considered as a heritable event and regulates gene expression by methylating cytosine at position-5.
Methylation of critical regulatory regions such as promoters leads to the silencing of the gene expression, while the loss of methylation is associated with gene activation. 62 DNA methylation occurs by a post-replication enzymatic modification and is carried by two enzymatic classes, namely maintenance methylation and de novo methylation. 62 DNMT1 is said to be the maintenance methyltransferase, which during the DNA replication, copies methylation patterns to the newly synthesized daughter strand. DNMT3a and DNMT3b are the de novo methyltransferases that lead to DNA methylation, generally during the development. The methylation of cytosine occurs primarily at 5′-CpG-3′ sites in the genome.
The CpG sites or also called CG sites are the regions of DNA where a cytosine is followed by a guanine in the linear base sequence along its 5′→3′ direction. CpG islands are regions that consist of a high number of CpG sites. CpG island is the region in DNA with at least 200 bp, a GC percentage of more than 50%, and an observed to expected CpG ratio >60%. These CpG islands are frequently found in the upstream region of the gene. Many mammalian genes have CpG islands in their promoter regions (70%), which helps F I G U R E 2 Epigenetic alterations in aflatoxin-induced HCC. Epigenetic alterations include changes in DNA methylation, histone modifications such as acetylation and methylation, and Non-coding RNAs that play a crucial role in gene regulation and expression. Aflatoxins alter these key regulatory mechanisms leading to HCC in gene annotation and prediction. In the human genome, it is estimated that there are about 28 million CpG islands. 63,64 Methylation of multiple CpG sites in these islands of promoters leads to the stable gene silencing. 65 DNA methylation at CpG is associated with gene regulation and is known to importantly relate to many cancers, including HCC. 66,67 This gene's functional regulation by methylation is done in two ways.
Firstly, the DNA methylation may physically impede the transcriptional protein binding to the DNA and secondly, methyl-CpG-binding domain proteins (MBDs) may bind to these DNA methylated regions.
These MBDs recruit histone deacetylases and other chromatin remodeling proteins, which modify the histones leading to the formation of heterochromatin. 65 Aflatoxins are shown to promote the methylation of the CpG islands near the promoters and bring changes in nucleosome occupancy. 68 Methylation status of 92 cancer-associated genes was analysed by Zhu et al, in liver cancer patients, using the MSP method.
In their study, they found that promoters of seven genes (MAGEA1, ing. Histone methylation is done by histone methyltransferases, which can activate or repress the transcription. 77 Histones are methylated on lysine (K) and arginine (R) residues only, but predominantly methylation occurs at lysine on H3 and H4 tails. 78 Common histone modification sites for methylation for gene ac- Several in vitro studies proved the HDAC6 role in HCC invasion and metastasis. 28,87 Therefore, it can be a good biomarker for HCC progression. HDAC8 role in HCC was studied by a comparative study of HDAC8 expression levels in HCC cells and normal hepatocytes. HDAC8 repression causes inhibition of HCC cell proliferation, elevation in the expression and the acetylation of the p53 gene in lysine 382. HDAC8, when inhibited is also proved to increase the apoptotic rate in HCC cells, which can act as a good therapeutic target. 88 Modifications such as histone H3 ly-sine9 methylation (H3K9me) or lysine27 methylation (H3K27me) are generally repressive marks, while the H3 lysine9 or lysine14 acetylation (H3K9Ac and H3K14Ac) and H3 lysine4 methylation (H3K4me) are generally considered as activation marks. 89 Scientists have discovered that there is an increase in the levels of H3K27ac and H3K27me3 in HCC patients, when compared to normal individuals 88 and hence can be used as markers in HCC diagnosis. AFB1 also activates AHR (Hydrocarbon receiver) in the liver that plays a major role in the high expression of HDAC8 leading to epigenetic deregulation and HCC. 35 RNAs not only to function as sequence-specific RNA substrates but also to carry out catalytic processes. [96][97][98][99] Most of the mammalian genome with estimated 5880 human transcription clusters from both sense and anti-sense strands are known to pair with anti-sense transcripts being ncRNA. 100,101 Evidence suggests that RNA signalling supports chromatin remodeling and epigenetic memory, although the mechanisms were not established. 102 Transcription itself is regulated by ncRNAs. 103 RNA polymerase II is also known to be regulated by ncRNA signalling. 104 ncRNAs also plays an important role in stress responses. Small non-coding transcript, B2, is produced by RNA polymerase III from murine, short interspersed elements (SINEs) under heat shock and this B2 RNA binds to RNA polymerase II and represses the transcription after heat shock. 105 Hence, changes caused by the Aflatoxins in these ncRNA can lead to the up or down-regulation of genes. The silencing of genes by miRNA takes place when the duplex ribbon miRNA binds with the mRNA molecule in the 3′-UTR regions of the mRNA. 106 miR-34 directly represses the p53 gene, thus regulating its function. miR-1, miR-191, miR-124, miR-125b and miR-203 are found to be silenced in HCC tissues. 107 The miRNAs can be both oncogenes or can also act as tumour suppressors. 35,105,107 Other important miRNA that play a crucial role in Aflatoxin-induced HCC is miR-21, which modulates PTEN expression and PTEN-dependent pathways, essentially enhancing the AKT pathway. miR-21 is known to promote cell proliferation, avoid apoptosis and promote invasion as well as angiogenesis. 28,108,109 miR-221 overexpression causes inhibition of HDAC6, acts as a pro-apoptotic enzyme, and also a tumour suppressor gene. 109
Systemic administration of miR-26a inhibits cancer cell proliferation and induces apoptosis, 110 while the miR-195, miR-17-5p expression leads to proliferation and migration. 112 miRNAs are epigenetically regulated and about 60% of human proteins are regulated by these miR-NAs and about 50% of these miRNAs are known to be associated with CpG islands. When these CpG islands are repressed by methylation or any other epigenetic alterations such as histone modifications, miRNA expression is also affected causing HCC. 113 Aflatoxin exposure also modulates LncRNA expression. Lv et al showed that AFB1 upregulates the expression of LncRNA-H19, which in turn promotes the growth and invasion of Hepatocarcinoma cells in vitro. 114 Data from these authors convey that AFB1 induces E2F1-mediated transcriptional upregulation of H19 and either the overexpression of E2F1 or downregulation of H19 decreases the growth and invasion of HCC cells. 114 Recent transcriptomic analysis of chickens fed with AFB1 showed differential expression of ~164 LncRNA genes in their livers, through which AFB1 was shown to regulate hepatic fat deposition and hepatocyte apoptosis. 115 Shi et al observed an extensive alteration in LncRNA expression in rat liver cells exposed to AFB1. 116 These authors compared the LncRNA expression between the control and AFB1 exposed as well as AFB1 resistant samples and confirmed that AFB1 regulates the LncRNA expression differently between the carcinogenesis and resistance  121 By identifying mutations in the circulating DNA, it is possible to segregate the high-risk population that is exposed to Aflatoxin and can develop HCC.

| PRE VENTI ON AND CONTROL
Aflatoxins are the most toxic and carcinogenic substances, which possess a significant risk for human health. The ability to control and eliminate Aspergillus fungi contamination in food is essential to reduce human exposure. To minimize Aflatoxin contamination in

ACK N OWLED G EM ENTS
We acknowledge the contributions of all the authors for their excellent research that we have cited in this review. We apologize, because of space constraints, we have omitted some of the studies. We

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was disclosed by the author.