**2. Epidemiology of AFB1-related HCC in China**

In China, HCC is the third or fourth most common malignant tumors and accounts for about 55% of the world's HCC cases, more than 340,000 each year (1, 13). This tumor occurs more often in eastern and southeastern China, including Jiangsu, Shanghai, Zhejiang, Fujiang, Guangdong, and Guangxi, mainly because of high AFB1 exposure and/or chronic infection of HBV and HCV (13). In the high AFB1-exposure areas such as Guangxi Zhuang Autonomous Region, this tumor is the most common occuring cancer (13, 14). Moreover, the incidence rate gradually increases with age increasing in abovementioned AFB1-exposure areas (15). Males are always more frequently affected than females but high male to female ratios of > 3 in the high AFB1-exposure areas (15). Although the incidence rates of this tumor in low AFB1-exposure areas in China have markedly decreased (because of the control of hepatitis virus infection), they have changed little in high AFB1-exposure areas (13, 15). For example, during May 2007 to April 2008, incidence rates were 117.8/100,000 and 103.1/100,000 for Xiangzhou and Fusui (two main high AFB1-exposure areas of China), respectively (13, 16). This was similar to the results before ten years (17).

Because of the very poor prognosis, HCC is the second most common cause of death from cancer in China (18). In the past thirty years, total mortality rate of HCC gradually increased from 12.5/100,000 to 26.26/100,000 (Fig 1A), regardless of countryside areas or urban areas (Fig 1B). This trend was more noticeable in male population than female population (Fig 1C), possibly because male individuals featured more high AFB1 exposure. Supporting aforementioned hypothesis, a recent study from high AFB1-exposure areas has demonstrated these having longer exposure years or higher exposure levels of AFB1 would face lower 5-years survival rate (4).

### **3. AFB1 exposure and DNA damage and repair**

AFB1 is an important mycotoxin produced by the moulds Aspergillus parasiticus and Aspergillus flavus (19). This toxic agent has been found as contaminants of human and animal food, particularly ground nuts (peanuts) and core, in tropical areas such as the Southeastern China as a result of fungal contamination during growth and after harvest which under hot and humid conditions (8, 14, 19, 20). Epidemiological evidence has shown dietary ingestion of high levels of AFB1 presents a significant environmental hazard of HCC (16, 17, 21). Experimental animal models have also shown that AFB1 can induce HCC; whereas DNA damage should play an important role during hepatocellular carcinogenesis (19, 22, 23). Therefore, AFB1 has been classified as a category I known human carcinogen by the International Agency for Research on Cancer (24).

AFB1 is metabolized by cytochrome P450 enzymes to its reactive form, AFB1-8,9-epoxide (AFB1-epoxide), which covalently binds to DNA and induces DNA damage (19, 25-28). DNA damage induced by AFB1 includes AFB1-DNA adducts, oxidative DNA damage,

carcinogenesis may be of importance in determining individual susceptibility to HCC (3, 9, 12). Therefore, the existence of low penetrate genetic polymorphisms may explain the reason why only a small portion of individuals, even in high-risk areas, develop HCC in their life span. This study reviews recent efforts in identifying genetic variants which may

In China, HCC is the third or fourth most common malignant tumors and accounts for about 55% of the world's HCC cases, more than 340,000 each year (1, 13). This tumor occurs more often in eastern and southeastern China, including Jiangsu, Shanghai, Zhejiang, Fujiang, Guangdong, and Guangxi, mainly because of high AFB1 exposure and/or chronic infection of HBV and HCV (13). In the high AFB1-exposure areas such as Guangxi Zhuang Autonomous Region, this tumor is the most common occuring cancer (13, 14). Moreover, the incidence rate gradually increases with age increasing in abovementioned AFB1-exposure areas (15). Males are always more frequently affected than females but high male to female ratios of > 3 in the high AFB1-exposure areas (15). Although the incidence rates of this tumor in low AFB1-exposure areas in China have markedly decreased (because of the control of hepatitis virus infection), they have changed little in high AFB1-exposure areas (13, 15). For example, during May 2007 to April 2008, incidence rates were 117.8/100,000 and 103.1/100,000 for Xiangzhou and Fusui (two main high AFB1-exposure areas of China), respectively (13, 16). This was

Because of the very poor prognosis, HCC is the second most common cause of death from cancer in China (18). In the past thirty years, total mortality rate of HCC gradually increased from 12.5/100,000 to 26.26/100,000 (Fig 1A), regardless of countryside areas or urban areas (Fig 1B). This trend was more noticeable in male population than female population (Fig 1C), possibly because male individuals featured more high AFB1 exposure. Supporting aforementioned hypothesis, a recent study from high AFB1-exposure areas has demonstrated these having longer exposure years or higher exposure levels of AFB1 would

AFB1 is an important mycotoxin produced by the moulds Aspergillus parasiticus and Aspergillus flavus (19). This toxic agent has been found as contaminants of human and animal food, particularly ground nuts (peanuts) and core, in tropical areas such as the Southeastern China as a result of fungal contamination during growth and after harvest which under hot and humid conditions (8, 14, 19, 20). Epidemiological evidence has shown dietary ingestion of high levels of AFB1 presents a significant environmental hazard of HCC (16, 17, 21). Experimental animal models have also shown that AFB1 can induce HCC; whereas DNA damage should play an important role during hepatocellular carcinogenesis (19, 22, 23). Therefore, AFB1 has been classified as a category I known human carcinogen by

AFB1 is metabolized by cytochrome P450 enzymes to its reactive form, AFB1-8,9-epoxide (AFB1-epoxide), which covalently binds to DNA and induces DNA damage (19, 25-28). DNA damage induced by AFB1 includes AFB1-DNA adducts, oxidative DNA damage,

have impact on risk of HCC.

**2. Epidemiology of AFB1-related HCC in China** 

similar to the results before ten years (17).

face lower 5-years survival rate (4).

**3. AFB1 exposure and DNA damage and repair** 

the International Agency for Research on Cancer (24).

Fig. 1. The mortality rates of HCC in China during 1973 and 2005. Total mortality rates (A), regardless of in urban areas or countryside areas (B), were significantly increasing from during 1973 and 1975 to during 1990 and 1992 or to during 2004 and 2005. This increasing mortality rates were more remarkable among male population (C).

DNA Repair Capacity-Related to Genetic Polymorphisms of DNA Repair

HCC induced by AFB1 exposure (54-56).

including AFB1-related HCC.

xeroderma pigmentosum D (XPD)(4, 39, 70, 71).

Genes and Aflatoxin B1-Related Hepatocellular Carcinoma Among Chinese Population 509

rare or absent in low or no AFB1-exposure areas (49, 51, 52). Using the human p53 gene in an in vitro assay, codon 249 has been exhibited to be a preferential site for formation of AFB1-N7-Gua adducts, evidence consistent with a role for AFB1 in the mutations observed in HCC (50, 53). Therefore, the codon 249 mutation of p53 gene has been defined as the hotspot mutation of p53 gene resulting from AFB1 and has become the molecular symbol of

A wide diversity of DNA damage produced by AFB1 exposure, if not repaired, may cause chromosomal aberrations, micronuclei, sister chromatid exchange, unscheduled DNA synthesis, and chromosomal strand breaks, and can be converted into gene mutations and genomic instability, which in turn results in cellular malignant transformation (19). Nevertheless, human cells have evolved surveillance mechanisms that monitor the integrity of genome to minimize the consequences of detrimental mutations (54). AFB1 induced DNA damage can be repaired through the following pathways: nucleotide excision repair (NER), base excision repair (BER), single-strand break repair (SSBR), and double-strand break repair (DSBR) (12, 28, 57). During the process of damage removed by aforementioned repair pathways, DNA repair genes play a central role, because their function determines DNA repair capacity (12). It has been shown that reduction in DNA repair capacity related to DNA repair genes is associated with increased risk of cancers (4, 39-41, 58-62). Thus, genetic polymorphisms in DNA repair genes which contribute to the variation in DNA repair capacity may be correlated with risk of developing cancers,

**4. Genetic polymorphisms in genes involved in NER pathway and risk of HCC**  NER pathway, a major DNA repair pathways in human cells featuring genomic DNA damage, can remove structurally such diverse lesions as pyrimidine dimers, irradiative damage, and bulky chemical adducts, and DNA damage from carcinogens and some chemotherapeutic drugs (63, 64). To date, the mechanism of this pathway is well understood and has been reconstituted in vitro. It consists of several sequential steps: lesion sensing, opening of a denaturation bubble, incision of the damaged strand, displacement of the lesion-containing oligonucleotide, gap filling, and ligation (63, 64). In the fibroblast cells with the deficiency of xeroderma pigmentosum A (XPA) gene, conversion of the initial AFB1-N7-Gua adduct to the AFB1-FAPy adduct has been found to be more extensive (53). This suggests that NER should be a major mechanism for enzymatic repair of AFB1 adducts (12). It's defects lead to severe diseases related AFB1 exposure, including liver injury and HCC. Accumulating evidence has implied that genetic polymorphisms in NER genes are associated with DNA repair capacity and modulate the risk of cancers (65-69). Molecular epidemiology studies of AFB1-related HCC in China have investigated the associations with several genes involved in NER pathway such as xeroderma pigmentosum C (XPC) and

*XPC.* XPC gene spans 33kb on chromosome 3p25 and contains 16 exons and 15 introns (Genbank accession no. AC090645). This gene encodes a 940-amino acid protein, an important DNA damage recognition molecule which plays an important role in NER pathway (72). It binds tightly with HR23B to form a stable XPC-HR23B complex, the first protein component that recognizes and binds to the DNA damage sites. XPC-HR23B complex can recognize a variety of DNA adducts formed by exogenous carcinogens such as AFB1 and binds to the DNA damage sites (72). Thus, it may play a role in the pathogenesis

and gene mutation (Fig. 2). Among these AFB1-DNA adducts, 8,9-di-hydro-8-(N7-guanyl)- 9-hydroxy–AFB1 (AFB1-N7-Gua) adduct is the most common type identified and confirmed in vivo researches (19, 25-27, 29, 30). The formation of this adduct proceeds by a precovalent intercalation complex between double-stranded DNA and the highly electrophilic, unstable AFB1-epoxide isomer (31, 32). After that, the induction of a positive charge on the imidazole portion of the formed AFB1-N7-Gua adduct gives rise to another important a DNA adduct, a ring-opened formamidopyridine AFB1 (AFB1-FAPy) adduct (33, 34). Accumulation of AFB1-FAPy adduct is characterized by time-dependence, nonenzyme, and may be of biological basis of genes mutation because of its apparent persistence in DNA (19, 33, 34). Furthermore, above adducts are capable of forming subsequent repair-resistant adducts, depurination, or lead to error-prone DNA repair resulting in single-strand breaks (SSBs), double-strand breaks (DSBs), base pair substitution, or frame shift mutations (35, 36). Additionally, AFB1 exposure also induces the formation of such oxidation DNA damage as 8-oxodeoxyguanosine (8-oxodG), a common endogenous DNA adduct (36-38). Although these DNA adducts are mainly produced in liver cells, they are also found in the peripheral blood white cells (39, 40). Recent studies have shown that the levels of AFB1-DNA adduct of the peripheral blood white cells are positively and lineally correlated with that of liver cells, implying analysis of AFB1-DNA adducts in the peripheral blood white cells may substitute for the elucidation of tissular levels of adducts (39, 41).

Fig. 2. The DNA damage induced by AFB1.

For genes mutations induced by AFB1 exposure, the experimental and theoretical researches are briefly on the p53 gene (42-49). Reaction with DNA at the N7 position of guanine preferentially causes a G:C > T:A mutation in codon 249 of this gene, leading to an amino acid substitution of arginine to serine (44-50). In high AFB1-exposure areas, this mutation is present in more than 40% of HCC and can be detected in serum DNA of patients with preneoplastic lesions and HCC (41). While codon 249 transversion mutations are either very

and gene mutation (Fig. 2). Among these AFB1-DNA adducts, 8,9-di-hydro-8-(N7-guanyl)- 9-hydroxy–AFB1 (AFB1-N7-Gua) adduct is the most common type identified and confirmed in vivo researches (19, 25-27, 29, 30). The formation of this adduct proceeds by a precovalent intercalation complex between double-stranded DNA and the highly electrophilic, unstable AFB1-epoxide isomer (31, 32). After that, the induction of a positive charge on the imidazole portion of the formed AFB1-N7-Gua adduct gives rise to another important a DNA adduct, a ring-opened formamidopyridine AFB1 (AFB1-FAPy) adduct (33, 34). Accumulation of AFB1-FAPy adduct is characterized by time-dependence, nonenzyme, and may be of biological basis of genes mutation because of its apparent persistence in DNA (19, 33, 34). Furthermore, above adducts are capable of forming subsequent repair-resistant adducts, depurination, or lead to error-prone DNA repair resulting in single-strand breaks (SSBs), double-strand breaks (DSBs), base pair substitution, or frame shift mutations (35, 36). Additionally, AFB1 exposure also induces the formation of such oxidation DNA damage as 8-oxodeoxyguanosine (8-oxodG), a common endogenous DNA adduct (36-38). Although these DNA adducts are mainly produced in liver cells, they are also found in the peripheral blood white cells (39, 40). Recent studies have shown that the levels of AFB1-DNA adduct of the peripheral blood white cells are positively and lineally correlated with that of liver cells, implying analysis of AFB1-DNA adducts in the peripheral blood white cells may substitute for the

For genes mutations induced by AFB1 exposure, the experimental and theoretical researches are briefly on the p53 gene (42-49). Reaction with DNA at the N7 position of guanine preferentially causes a G:C > T:A mutation in codon 249 of this gene, leading to an amino acid substitution of arginine to serine (44-50). In high AFB1-exposure areas, this mutation is present in more than 40% of HCC and can be detected in serum DNA of patients with preneoplastic lesions and HCC (41). While codon 249 transversion mutations are either very

elucidation of tissular levels of adducts (39, 41).

Fig. 2. The DNA damage induced by AFB1.

rare or absent in low or no AFB1-exposure areas (49, 51, 52). Using the human p53 gene in an in vitro assay, codon 249 has been exhibited to be a preferential site for formation of AFB1-N7-Gua adducts, evidence consistent with a role for AFB1 in the mutations observed in HCC (50, 53). Therefore, the codon 249 mutation of p53 gene has been defined as the hotspot mutation of p53 gene resulting from AFB1 and has become the molecular symbol of HCC induced by AFB1 exposure (54-56).

A wide diversity of DNA damage produced by AFB1 exposure, if not repaired, may cause chromosomal aberrations, micronuclei, sister chromatid exchange, unscheduled DNA synthesis, and chromosomal strand breaks, and can be converted into gene mutations and genomic instability, which in turn results in cellular malignant transformation (19). Nevertheless, human cells have evolved surveillance mechanisms that monitor the integrity of genome to minimize the consequences of detrimental mutations (54). AFB1 induced DNA damage can be repaired through the following pathways: nucleotide excision repair (NER), base excision repair (BER), single-strand break repair (SSBR), and double-strand break repair (DSBR) (12, 28, 57). During the process of damage removed by aforementioned repair pathways, DNA repair genes play a central role, because their function determines DNA repair capacity (12). It has been shown that reduction in DNA repair capacity related to DNA repair genes is associated with increased risk of cancers (4, 39-41, 58-62). Thus, genetic polymorphisms in DNA repair genes which contribute to the variation in DNA repair capacity may be correlated with risk of developing cancers, including AFB1-related HCC.
