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

*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

DNA Repair Capacity-Related to Genetic Polymorphisms of DNA Repair

(88, 94, 98-100).

induced by AFB1.

stimulates the activity of PNK (103).

**risk of HCC** 

Genes and Aflatoxin B1-Related Hepatocellular Carcinoma Among Chinese Population 511

Several groups have done genotype-phenotype analyses with these two polymorphisms and have shown that the variant allele genotypes are associated with low DNA repair ability (96, 97). Recent studies have showed the polymorphisms at codon 312 and 751 of XPD are correlated with DNA-adducts levels, p53 gene mutation, and cancers risk

In a hospital-based case-control study in Guangxi (39), we found that the variant XPD codon 751 genotypes (namely Lys/Gln and Gln/Gln) detected by TaqMan-MGB PCR was significantly different between controls (26.3% and 8.6% for Lys/Gln and Gln/Gln, respectively) and HCC cases (35.9% and 20.1% for Lys/Gln and Gln/Gln, respectively, *P* < 0.001). Individuals with variant alleles had about 1.5- to 2.5-fold risk of developing the cancer (adjusted OR 1.75 and 95% CI 1.30-2.37 for Lys/Gln; adjusted OR 2.47 and 95% CI 1.62-3.76 for Gln/Gln). Based on relative sample size (including 618 HCC cases and 712 controls), we stratified genotypes of XPD codon 751 according to matching factors and observed some evidence of interaction between XPD codon 751 Gln alleles and sex. These female having Gln alleles, compared to those without these alleles, featured increased HCC risk. Furthermore, the interactive effects of between variant genotypes of XPD gene codon 751 environment variant AFB1 or another NER gene XPC on HCC risk were also found, with interactive value 0.85, 1.04, and 1.71 for AFB1-exposure years, AFB1-exposure levels, and XPC gene codon 939 risk genotypes (*P*interaction < 0.05). Therefore, the XPD gene codon 751 polymorphism may have potential effect on AFB1-related HCC susceptibility among Chinese population. However, the study from AFB1-exposure areas don't exhibit polymorphism at codon 312 of XPD gene significantly associates with the risk of HCC

**5. Genetic polymorphisms in genes involved in SSBR pathway and** 

SSB is a common type of DNA damage produced by AFB1 exposure (36). If not repaired, it can disrupt transcription and replication and can be converted into potentially clastogenic and/or lethal DSBs. This DNA damage is repaired via SSBR pathway (101, 102). SSBR pathway includes four basic steps: *a.* SSB detection and signaling, through poly (ADPribose) polymerase (PARP); *b.* DNA break end processing, through the role of polynucleotide kinase (PNK), AP endonuclease-1 (APE1), DNA polymerase β (Pol β), tyrosyl phosphodiesterase 1 (TDP1), and flap endonuclease-1 (FEN-1); *c.* gap filling, involving in multiple DNA polymerases; *d.* DNA ligation, involving in multiple DNA ligases. Of the later three steps of SSBR pathway, x-ray repair cross complementary 1 (XRCC1) is indispensible, because it not only acts as the scaffolding protein of SSBR, but also

XRCC1 gene encoding protein (633 amino acids), consists of three functional domains — Nterminal domain (NTD), central breast cancer susceptibility protein-1 homology C-terminal (BRCT I), and C-terminal breast cancer susceptibility protein-1 homology C-terminal (BRCT II) (103-106). This protein is directly associated with Pol β, DNA ligase III, and PARP, via their three functional domains and is implicated in the core processes in SSBR and BER pathway (103). Mutant hamster ovary cell lines that lack XRCC1 genes are hypersensitive to DNA damage agents such as ionizing radiation, hydrogen peroxide, and alkylating agents (103). Furthermore, this kind of cells usually face increasing frequency of spontaneous

of HCC-related AFB1. Some recent studies have showed that defects in XPC have been related to many types of malignant tumors (73-82). Transgenic mice studies also revealed predisposition to many types of tumors in XPC gene knockout mice (83). Furthermore, pathological and cellular researches have exhibited that the abnormal expression of this gene is related to hepatocarcinogenesis (84). These studies suggests the polymorphisms localizing at conserved sites of XPC gene might modify the risk of HCC induced by AFB1 exposure. Recently, four studies from high AFB1-exposure areas of China have approved aforementioned hypothesis (4, 70, 71, 85).

The first study conducted by Cai *et al.*(85) is from Shunde area, Guangdong Province. In this 1-1 case-control study (including 78 HCC cases and 78 age- and sex-matching controls), researchers analyzed between two common polymorphisms—Ala499Val and Lys939Gln of XPC gene and risk of HCC and found these two polymorphisms modified HCC risk [adjusted odds ratios (ORs) were 3.77 with 95% confidence interval (CI)1.34-12.89 for Ala499Lys and 6.78 with 95% CI 2.03-22.69], especially under HBV and HCV infection condition. Although they evaluated the effects of XPC-hepatitis viruses interaction on HCC risk, they did not elucidate the possible interaction of AFB1 exposure.

The other three studies are from Guangxi Zhuang Autonomous Region (4, 70). Li *et al.*(71), Wu *et al.* (70), and Long *et al*. (4) investigated the modifying effects of genetic polymorphisms XPC on HCC based hospitals. The results showed XPC codon 939 Gln alleles increased about 2-times risk of HCC. Furthermore, Wu, *et al.*(70), and Long, *et al*. (4) quantitatively elucidated AFB1 exposure years and levels and their interactive effects with XPC Lys939Gln polymorphism. They found some evidence of AFB1 exposure-risk genotypes of XPC codon 939 on HCC risk (22.33 > 1.88 × 8.69 for the interaction of AFB1 exposure years and XPC risk genotypes and 18.38 > 1.11 × 4.62 for the interaction of AFB1 exposure levels and XPC risk genotypes). Additionally, Gln alleles at codon 939 of XPC gene are observed to be correlated with the decrease of XPC expression levels in cancerous tissues (*r* = - 0.369, *P* < 0.001) and with the overall survival of HCC patients (the median survival times are 30, 25, and 19 months for patients with XPC gene codon 939 Lys/Lys, Lys/Gln, and Gln/Gln respectively). This decreasing 5-years survival rates would be noticeable under high AFB1 exposure conditions (the median survival times are 15 months for the joint of XPC gene codon 939 Gln/Gln and long-term AFB1-exposure years and 17 month for the joint of XPC gene codon 939 Gln/Gln and high AFB1-exposure level) (4).

These results demonstrate that polymorphism at codon 939 of XPC gene is not only a genetic determinant in the development of HCC induced by AFB1 exposure in Chinese population, but also is an independent prognostic factor influencing the survival of HCC, like AFB1 exposure. However, Li *et al.* (71) reported that the proportional distribution of the Val/Val genotype at codon 499 of XPC gene did not differ between cases with HCC and controls in Guangxi Zhuang Autonomous Region, China (*P* > 0.05), dissimilar to the data from another area of China, Guangdong Province (85). Possible explanations for these inconsistent finding may be either due to unknown confounders or due to small sample size.

*XPD.* XPD gene-encoding protein, a DNA-dependent ATPase/helicase, is associated with the TFIIH transcription-factor complex and plays a role in NER pathway (86, 87). During NER, XPD participates in the opening of the DNA helix to allow the excision of the DNA fragment containing the damaged base. There are two described polymorphisms that induce amino acid changes in the protein: at codons 312 (Asp to Asn) and 751 (Lys to Gln) (87-89). To date, these two polymorphisms have been extensively studied (87, 88, 90-95).

of HCC-related AFB1. Some recent studies have showed that defects in XPC have been related to many types of malignant tumors (73-82). Transgenic mice studies also revealed predisposition to many types of tumors in XPC gene knockout mice (83). Furthermore, pathological and cellular researches have exhibited that the abnormal expression of this gene is related to hepatocarcinogenesis (84). These studies suggests the polymorphisms localizing at conserved sites of XPC gene might modify the risk of HCC induced by AFB1 exposure. Recently, four studies from high AFB1-exposure areas of China have approved

The first study conducted by Cai *et al.*(85) is from Shunde area, Guangdong Province. In this 1-1 case-control study (including 78 HCC cases and 78 age- and sex-matching controls), researchers analyzed between two common polymorphisms—Ala499Val and Lys939Gln of XPC gene and risk of HCC and found these two polymorphisms modified HCC risk [adjusted odds ratios (ORs) were 3.77 with 95% confidence interval (CI)1.34-12.89 for Ala499Lys and 6.78 with 95% CI 2.03-22.69], especially under HBV and HCV infection condition. Although they evaluated the effects of XPC-hepatitis viruses interaction on HCC

The other three studies are from Guangxi Zhuang Autonomous Region (4, 70). Li *et al.*(71), Wu *et al.* (70), and Long *et al*. (4) investigated the modifying effects of genetic polymorphisms XPC on HCC based hospitals. The results showed XPC codon 939 Gln alleles increased about 2-times risk of HCC. Furthermore, Wu, *et al.*(70), and Long, *et al*. (4) quantitatively elucidated AFB1 exposure years and levels and their interactive effects with XPC Lys939Gln polymorphism. They found some evidence of AFB1 exposure-risk genotypes of XPC codon 939 on HCC risk (22.33 > 1.88 × 8.69 for the interaction of AFB1 exposure years and XPC risk genotypes and 18.38 > 1.11 × 4.62 for the interaction of AFB1 exposure levels and XPC risk genotypes). Additionally, Gln alleles at codon 939 of XPC gene are observed to be correlated with the decrease of XPC expression levels in cancerous tissues (*r* = - 0.369, *P* < 0.001) and with the overall survival of HCC patients (the median survival times are 30, 25, and 19 months for patients with XPC gene codon 939 Lys/Lys, Lys/Gln, and Gln/Gln respectively). This decreasing 5-years survival rates would be noticeable under high AFB1 exposure conditions (the median survival times are 15 months for the joint of XPC gene codon 939 Gln/Gln and long-term AFB1-exposure years and 17 month for the

These results demonstrate that polymorphism at codon 939 of XPC gene is not only a genetic determinant in the development of HCC induced by AFB1 exposure in Chinese population, but also is an independent prognostic factor influencing the survival of HCC, like AFB1 exposure. However, Li *et al.* (71) reported that the proportional distribution of the Val/Val genotype at codon 499 of XPC gene did not differ between cases with HCC and controls in Guangxi Zhuang Autonomous Region, China (*P* > 0.05), dissimilar to the data from another area of China, Guangdong Province (85). Possible explanations for these inconsistent finding

*XPD.* XPD gene-encoding protein, a DNA-dependent ATPase/helicase, is associated with the TFIIH transcription-factor complex and plays a role in NER pathway (86, 87). During NER, XPD participates in the opening of the DNA helix to allow the excision of the DNA fragment containing the damaged base. There are two described polymorphisms that induce amino acid changes in the protein: at codons 312 (Asp to Asn) and 751 (Lys to Gln) (87-89). To date, these two polymorphisms have been extensively studied (87, 88, 90-95).

risk, they did not elucidate the possible interaction of AFB1 exposure.

joint of XPC gene codon 939 Gln/Gln and high AFB1-exposure level) (4).

may be either due to unknown confounders or due to small sample size.

aforementioned hypothesis (4, 70, 71, 85).

Several groups have done genotype-phenotype analyses with these two polymorphisms and have shown that the variant allele genotypes are associated with low DNA repair ability (96, 97). Recent studies have showed the polymorphisms at codon 312 and 751 of XPD are correlated with DNA-adducts levels, p53 gene mutation, and cancers risk (88, 94, 98-100).

In a hospital-based case-control study in Guangxi (39), we found that the variant XPD codon 751 genotypes (namely Lys/Gln and Gln/Gln) detected by TaqMan-MGB PCR was significantly different between controls (26.3% and 8.6% for Lys/Gln and Gln/Gln, respectively) and HCC cases (35.9% and 20.1% for Lys/Gln and Gln/Gln, respectively, *P* < 0.001). Individuals with variant alleles had about 1.5- to 2.5-fold risk of developing the cancer (adjusted OR 1.75 and 95% CI 1.30-2.37 for Lys/Gln; adjusted OR 2.47 and 95% CI 1.62-3.76 for Gln/Gln). Based on relative sample size (including 618 HCC cases and 712 controls), we stratified genotypes of XPD codon 751 according to matching factors and observed some evidence of interaction between XPD codon 751 Gln alleles and sex. These female having Gln alleles, compared to those without these alleles, featured increased HCC risk. Furthermore, the interactive effects of between variant genotypes of XPD gene codon 751 environment variant AFB1 or another NER gene XPC on HCC risk were also found, with interactive value 0.85, 1.04, and 1.71 for AFB1-exposure years, AFB1-exposure levels, and XPC gene codon 939 risk genotypes (*P*interaction < 0.05). Therefore, the XPD gene codon 751 polymorphism may have potential effect on AFB1-related HCC susceptibility among Chinese population. However, the study from AFB1-exposure areas don't exhibit polymorphism at codon 312 of XPD gene significantly associates with the risk of HCC induced by AFB1.
