**2. Progression of HCC**

Liver cancer progression is slow and the median age of onset is 60–65 years [11]; populations in areas that are at high risk for environmental and life-style factors are exceptions. For example, the incidence of liver cancer in the Qidong province of China peaks at 45 years [9, 11]. HCC generally develops as a consequence of liver injury, whether caused by chronic hepatitis or cirrhosis, which leads to chronic inflammation and deposition of connective tissue. Chronic hepatitis leads to upregulation of mitogenic pathways, partially through epigenetic mechanisms [23]. Monoclonal populations of dysplastic hepatocytes may exhibit telomere erosion and re-expression of telomerase to maintain viability. Eventual malignant cells accumulate irreversible genetic alterations [23]. As the transformed phenotype advances, the rate in the accumulation of genetic alterations increases [24]. The exact threshold for the number of mutations or alterations present in liver cancer has not been established. Thus, the progression of liver cancer is associated and is accelerated with the accumulation of genetic mutations and altered gene expression patterns.

### **2.1 Mutations that contribute to liver cancer**

Understanding which HCC-associated genetic changes are associated with AFB1 exposure requires a comparison of the genomic alterations that occur in sporadic HCC or HCC associated with other causes. For sporadic HCC, similar to solid tumors, there is both a multiplicity and heterogeneity in genetic alterations in HCC [23–25]. In general, these genetic alterations can be grouped into those that result in loss of function and those that result in gain of function. Genetic alterations that result in loss of function include dominant negative mutations and recessive mutations, which are expressed after loss of heterozygosity (LOH).

 Among sporadic tumors, both loss of heterozygosity (LOH) and mutations have been found in HCC tumors. Among 363 patients, The Cancer Genome Atlas Research Network [25], report that the most heavily mutated gene was TP53 (31%), encoding p53, followed by WNT pathway member CTNNB1 (27%), encoding β-catenin, and AXIN (8%), encoding a WNT signaling scaffolding protein, and chromatin remodeling genes (12%) [25]. In greater than 10% of HCC, mutations are found in CDH1, TP53, IGF2H, RB1, CDKN2A, PTEN, KLC, TP73, EXT, MLH1, THRB, THRA, E2F5, and CTNNB1 [23]. Whether these mutations occur early or late in the etiology of liver cancer is still not understood. While the p53 gene functions in controlling the DNA damage response and apoptosis, the WNT pathway is important in controlling cell proliferation [23]. Many of the mutagenic events result from G to T transversions, unlike events found other tumors. The strong bias for G to T transversions suggests that these genetic alterations likely result from chemical DNA damaging agents, rather than spontaneous events, such as cytosine deamination [23]. While the heterogeneity in genetic mutations may reflect multiple mechanisms for liver cell transformation, identifying alterations in HCC are informative in understanding the etiology and possible treatment of individual cancer cases. For example, β-catenin defective liver cancer may be easier to treat than liver cancer resulting from multiple mutations [23–25].

In addition to mutation and LOH events, gain-of-function genetic alterations may confer higher levels of oncogene expression and thereby accelerate carcinogenesis [26–29]. Such alterations could include gene amplification events, such as c-N-methyl-N′-nitro-N-nitroso-guanidine HOS transforming gene (c-MET) and cyclin D (CCND1) [27]. Other gains of function mutations include mutations in the promoter for telomerases reverse transcriptase (TERT) promoter. TERT mutations frequently were shown to be among the earliest and most prevalent neoplastic events in HCC [28, 30].

Both epidemiological and molecular pathology studies have facilitated the identification of which genetic alterations are likely to be associated AFB1 exposure. Mutated genes found in HCC from areas with high AFB1 exposure include p53 and β-catenin [30]. The p53 249ser mutation shows a strong correlation with HCC associated with AFB1 exposure, while is less frequent or absent in HCC from localities where there is little AFB1 exposure [19, 20]. For example, among have HCCs from southern Guangxi province of China, an area of high AFB1 exposure, the p53 249ser mutation was found in 36% of tumors [30]. *CTNNB1* mutations and β-catenin protein accumulation in human hepatocellular carcinomas is also associated with high exposure to AFB1, although it is less clear whether these mutations directly result from AFB1-associated DNA adducts [30]. Whether these mutations must occur early or late in cancer progression is still unclear. One hypothesis is that initial mutations confer a higher level genetic instability that is aggravated by further exposure to genotoxic agents.

To determine whether mutations found in HCC confer higher levels of genetic instability and a higher probability of liver cancer when present in a non-cancerous liver, scientists have constructed transgenic mice that exhibit similar genotypes found in human cancer. Ghebranious and Sell [31] constructed transgenic mice that were both homozygous and heterozygous for the p53ser246 gene, equivalent to the human p53 249ser mutations. Male mice expressing p53ser246 increased the incidence of AFB1-associated high-grade tumors to 14%, compared to 0% exhibited by p53+ (wild type) mice [31]. These studies indicate that the mutant p53 249ser may also be a driver of AFB1-associated liver cancer.

 The role of inflammation in liver cancer has led to insights into the gender bias of its incidence. Men are afflicted more than women in nearly all age groups; however the prognosis of liver cancer in either sex is about the same [9]. Naugler et al. [18], have shown that inflammatory cytokines, such as IL-6, are more prevalent in men than women, estrogen having a negative effect on IL-6 production. This gender difference is not only true for humans [32, 33] but also for rodents, including mice and rats [31]. The gender bias underscores the notion that inflammatory responses play a role in liver cancer etiology.

#### **2.2 Associated risk factors that accelerate AFB1-associated liver cancer: role of HBV and HCC virus**

 The incidence of HCC synergistically increases when individuals are both exposed to AFB1 and infected with either HBV or HCV virus. Interestingly, the incidence of high grade tumors in p53ser246 transgenic mice that are HBsAg-positive is 100% [31]. The common molecular mechanisms by which HBV and HCV infection stimulates AFB1-associated genetic instability phenotypes are still not completely understood; HBV is a DNA virus that replicates by reverse transcription while HCC is a RNA virus that replicates by RNA replication and encodes a single polycistronic message [34, 35]. While 257 million individuals are estimated to be infected with HBV, 140 million individuals are estimated to be infected with HCV; and chronic HBV and HCV infection is the leading cause for 60–70% of HCC [35, 36]. Although HCC contains no oncogenes *per se*, HCV-associated carcinogenesis is associated with increase in reactive oxygen species (ROS), ROS-associated genetic instability, inflammation, and hepatocyte proliferations [36]. Similarly, HBV-associated HCC is associated with inflammation and necro-inflammatory liver damage [16, 36]. Both viruses are not cytopathic *per se*; liver damage caused by HCV and HBV is likely induced by viral-specific CD8+ T and natural killer cells (NK) [35, 36]. Thus, both HCV and HBV create an inflammatory cellular environment that stimulates repopulation of hepatocytes, enhancing AFB1-associated genetic instability.

However, different pathologies of HBV and HCV infection may accelerate HCC progression at different rates. While the median onset age for HBV-associated HCC is 55 years that of HCV is 65 years [11]. HBV can chronically infect children after transmission from the mother [11, 36]. Once HBV is stably integrated into the host genome, HBV can promote chromosomal rearrangements and mutations in cancerassociated genes and interfere with checkpoint controls [37, 38]. For example, HBV integration can occur in TERT promoters, stimulating expression of telomerase, and near LINE sequences [39]. The HBV-encoded oncogene HBx can activate both Src and Ras signaling and is essential for viral DNA (cccDNA) replication. To facilitate replication, HBx mediates chromatin changes by recruiting histone acetyltransferases to acetylate histone H3. HBx is also thought to interact with p53 249ser, and attenuate DNA repair and apoptosis [20].

Besides stimulating host cell replication, HBx may also interfere with the host cell's DNA repair pathways and promote genetic instability and replication [38, 40–44]. HBx binds to DNA damage binding protein 1 (DDB1) and cullin-4 (Cul4), which form a ubiquitinase complex, and can perturb the stability of structural

#### *Cellular Responses to Aflatoxin-Associated DNA Adducts DOI: http://dx.doi.org/10.5772/intechopen.81763*

maintenance of chromosome proteins 5 and 6 (Smc5/6) and thus affect DNA replication and DNA damage tolerance [35]. HBx may also interfere with nucleotide excision repair (NER) of AFB1-associated DNA adducts [40–42, 44]. Although less substantiated, HBx is also thought to interfere with PARP1 and decrease excision repair of DNA adducts. Thus HBx drives carcinogenesis by multiple mechanisms that accelerate carcinogenesis.

## **2.3 Gene polymorphisms associated with AFB1-associated liver cancer**

While HBV and HCV infections are the primary factors that aggravate the risk for AFB1-associated HCC, genetic risk factors have also been postulated [45]. With the advent of technologies that accelerate genome sequencing, such as next generation sequencing (NGS), epidemiologists have identified candidate polymorphic genes that increase the risk for aflatoxin-associated liver cancer. Single nucleotide polymorphisms (SNPs) may be located in the amino acid coding region, the introns, or the promoter regions of the candidate genes. Risk factors generally can be grouped into those that (1) are associated with AFB1 metabolic activation and detoxification and (2) that function in DNA repair or DNA damage tolerance genes.

 To understand genetic risk factors that affect metabolic activation and detoxification of AFB1 it is necessary to identify genes involved in these pathways. AFB1 is activated by cytochrome P450 enzymes that hydroxylate AFB1 so that the metabolized carcinogen can be rendered hydrophilic and effectively excreted; for review, see [46–48]. Referred to as phase I enzymes and monooxygenases, the cytochrome P450 enzymes contain a heme group at their active sites and catalyze the transfer of single oxygen to specific sites on the target molecule [46]. Cytochrome P450 enzymes require NADPH oxidoreductase (POR) to maintain activity [46]. The P450 enzymes are located in the endoplasmic reticulum in the vicinity of the POR [46]. Of the characterized enzymes expressed by 57 CYP450 genes, CYP1A2 is liver specific and has a high affinity for AFB1, while CYP3A4 constitutes approximately 50% of the hepatic P450 activity. While there have been disagreements over which cytochrome P450 enzymes is chiefly responsible for AFB1 activation in the liver [46, 49, 50], several reports favored CYP3A4 [50, 51], while another report suggested that CYP3A5 has the highest catalytic activity [52]. Among extrahepatic CYPs, CYP2A13 activates AFB1 in the lung, while CYP1A1 catalyzes the formation of AFM1, a hydroxylated AFB1 derivative that can be excreted in milk, which is still carcinogenic [2]. A transient intermediate in the hydroxylation pathway is a highly reactive epoxide, referred to as AFB1-8,9-exo-epoxide (AFBO) (**Figure 1**). This epoxide can be effectively detoxified by either epoxide hydrolases (EHs) or glutathione S-transferases (GSTs), referred to as phase II enzymes [47, 48]. While multiple cytochrome P450s can activate AFB1, the highly reactive epoxide is thought to be the predominant reactive intermediate in all P450 reactions. Thus, gene polymorphisms that increase the risk of HCC could: (1) increase P450 enzyme levels or activation, (2) downregulate phase II enzymes, (3) decrease the repair of DNA existing lesions, and (4) channel the repair of the DNA lesions into mutagenic pathways.

 One source of polymorphic enzymes that can influence the fate of AFB1 is glutathione S-transferases that are present in the liver [53]. In the mouse, knock-out of GSTa3 confers extreme AFB1-associated toxicity [54] and GSTa3 expression levels correlate with AFB1-associated liver cancer in young mice [55]. In humans, HCC risk is dramatically increased by SNPs in glutathione S transferase mu1 (GSTMI) and (glutathione-S-transferase theta1) (GSTT1) [56]. Expression of epoxide hydrolase in yeast also leads to detoxification of AFBO [57]; however, polymorphisms associated with epoxide hydrolase only have a weak association with liver

#### **Figure 1.**

*CYP-mediated metabolic activation of AFB1 to the activated AFB1-8,9 epoxide and adduct formation and conversion to AFB1-Fapy. Adapted from Ref. [130].* 

cancer [45]. These studies support the idea that detoxification of the highly reactive epoxide is critical in reducing AFB1 toxicity.

While diminished ability to detoxify AFBO is a risk factor for AFB1-associated liver cancer, higher or altered P450 activity could also increase HCC risk. HBx activates the pregnane receptor (PXR) and stimulates expression of CYP3A4 [58]. Particular CYP3A5 alleles, such as CYP3A5\*3, are correlated with higher levels of expression and aflatoxin-protein adducts in individuals from Gambia, Africa [59]. CYP3A5\*3 is present in a high percentage of individuals in Gambia but not in the Caucasian population [59]; the allele found in the Caucasian population confers an altered spliced mRNA, which is poorly expressed [60]. However, establishing correlations between HCC and increased expression of other P450 genes is complicated by the multiple interactions between P450 enzymes.

 Genetic risk factors have also been identified among polymorphic alleles of DNA repair and cell cycle checkpoint genes, which may increase chromosomal instability in cells chronically exposed to AFB1. These risk factors have been found in p53, XRCC1, XRCC3, and ERCC1. The combination of p53 codon 72 Arg72Pro and MDM2 (mouse double minute 2 homolog) SNP309 (T>G) increases the risk of HCC in individuals infected with HBV [61]; p53 codon 72 Arg72Pro affects the frequency of double strand breaks and is associated with hyper-methylation of promoters in tumor suppressor genes [61]. XRCC3 (X-ray complementing defective repair in Chinese hamster cells) encodes a Rad51 paralog which is involved in double-strand break repair and could be involved in error-free by pass of AFB1 associated DNA lesions. The XRCC3 rs861539 allele (codon Thr241Met polymorphism) is a risk factor for HCC, and the risk is aggravated if individuals are exposed to AFB1 [62–64]. Other alleles that have been associated with higher risk for HCC include those participating in the base excision repair (BER) and NER pathways, such as XRCC1 rs25487 polymorphism (codon Arg399Gln polymorphism) [65, 66] and XPD rs25487 polymorphism, respectively [67]. These studies reinforce the idea that AFB1-associated genotoxicity can accelerate HCC progression. To understand the genotoxicity in more detail it is important to understand the nature of the AFB1 associated DNA adducts.
