**5. Genetic polymorphisms in genes involved in SSBR pathway and risk of HCC**

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 stimulates the activity of PNK (103).

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

DNA Repair Capacity-Related to Genetic Polymorphisms of DNA Repair

Genes and Aflatoxin B1-Related Hepatocellular Carcinoma Among Chinese Population 513

Fig. 3. The meta-analysis of the relationship between XRCC1 codon Lys399Gln

observed in the binding of Arg/Gln and Gln/Gln genotypes (C).

polymorphism and HCC risk among China population. Compared with Arg/Arg genotype, Arg/Gln (A) and Gln/Gln (B) genotypes increased HCC risk. This risk effect was also

chromosome aberrations and deletions. Three single nucleotide polymorphisms in the coding region of XRCC1 gene that lead to amino acid substitution have been described and investigated (12). Of these polymorphisms, the codon 399 polymorphism is of special concern, because this polymorphism resides in functionally significant regions (BECT II) and may be related to decreasing DNA repair capacity, increasing genes mutation, and running-up risk of cancers (12, 107-114).

In AFB1-exposure areas from China, a total of six molecular epidemiological studies were found in PubMed database, Wangfang Database, and Weipu database (61, 62, 115-118). However, associations between XRCC1 gene codon 399 polymorphism and individual susceptibility to HCC have been reported in these case-control studies with the results being contradictory. We analyzed the possible causes of contradictory using meta-analysis method (Comprehensive Meta Analysis Version 2, http://www.meta-analysis.com/). Fig. 3 showed the meta-analysis results of the modifying effects of XRCC1 gene codon 399 polymorphism on HCC risk. We found these subjects with Gln alleles had increasing risk of HCC (total crude adjusted OR = 1.34, *P* < 0.01), moreover, there were larger relative weight to assign to those studies with OR-value more than 1. Actually, although Yang *et al.* (116) and Ren *et al.* (118) did not observed significantly risk of XRCC1 gene codon 399 polymorphism in crude logistic regression, they found Gln alleles would increase HCC risk in stratified analysis with susceptive environment variants. A individually matching case-controls demonstrated that subjects having Gln alleles might feature remarkably increasing risk of HCC under longer-term AFB1-exposure years or higher AFB1-exposure levels conditions (adjusted OR > 10) (61). This suggests that the genotypes with codon 399 Gln alleles of XRCC1 should be a risk biomarker of Chinese HCC related to AFB1 exposure.

#### **6. Genetic polymorphisms in genes involved in BER pathway and risk of HCC**

Of the oxidative DNA damage resulting from AFB1 exposure, the formation of 8-oxodG is thought to be important due to being abundant and highly mutagenic and hepatocarcinogenesis (21, 36-38). The 8-oxodG lesions are repaired primarily through the BER pathway (119). The BER pathway facilitates DNA repair through two general pathways: *a.* the short-patch BER pathway, leading to a repair tract of a single nucleotide; *b.* the longpatch BER pathway, producing a repair tract of at least two nucleotides (120). In these two repair sub-pathways, DNA glycosylases play a central role because they can recognize and catalyze the removal of damaged bases (120). This suggests that the defect of DNA glycosylases should be related to the decreasing capacity of the BER pathway and might increase the risk of such cancers as HCC.

Human oxoguanine glycosylase 1(hOGG1) is a specific DNA glycosylase that catalyzes the release of 8-oxodG and the cleavage of DNA at the AP site (121, 122). Genetic structure study has revealed the presence of several polymorphisms within hOGG1 locus (123). Among them, the polymorphism at position 1245 in exon 7 causes an amino acid substitution (Ser to Cys) at codon 326, suggesting this polymorphism may glycosylase function (123). A functional complementation activity assay showed that hOGG1 protein encoded by the 326 Cys allele had substantially lower DNA repair activity than that encoded by the 326 Ser allele (124). Similar results were observed in human cells in vivo (122, 125). Therefore, low capacity of 8-oxodG repair resulting from hOGG1 326Cys polymorphism might contribute to

chromosome aberrations and deletions. Three single nucleotide polymorphisms in the coding region of XRCC1 gene that lead to amino acid substitution have been described and investigated (12). Of these polymorphisms, the codon 399 polymorphism is of special concern, because this polymorphism resides in functionally significant regions (BECT II) and may be related to decreasing DNA repair capacity, increasing genes mutation, and

In AFB1-exposure areas from China, a total of six molecular epidemiological studies were found in PubMed database, Wangfang Database, and Weipu database (61, 62, 115-118). However, associations between XRCC1 gene codon 399 polymorphism and individual susceptibility to HCC have been reported in these case-control studies with the results being contradictory. We analyzed the possible causes of contradictory using meta-analysis method (Comprehensive Meta Analysis Version 2, http://www.meta-analysis.com/). Fig. 3 showed the meta-analysis results of the modifying effects of XRCC1 gene codon 399 polymorphism on HCC risk. We found these subjects with Gln alleles had increasing risk of HCC (total crude adjusted OR = 1.34, *P* < 0.01), moreover, there were larger relative weight to assign to those studies with OR-value more than 1. Actually, although Yang *et al.* (116) and Ren *et al.* (118) did not observed significantly risk of XRCC1 gene codon 399 polymorphism in crude logistic regression, they found Gln alleles would increase HCC risk in stratified analysis with susceptive environment variants. A individually matching case-controls demonstrated that subjects having Gln alleles might feature remarkably increasing risk of HCC under longer-term AFB1-exposure years or higher AFB1-exposure levels conditions (adjusted OR > 10) (61). This suggests that the genotypes with codon 399 Gln alleles of XRCC1 should be a risk biomarker of Chinese HCC related to AFB1

**6. Genetic polymorphisms in genes involved in BER pathway and risk of HCC**  Of the oxidative DNA damage resulting from AFB1 exposure, the formation of 8-oxodG is thought to be important due to being abundant and highly mutagenic and hepatocarcinogenesis (21, 36-38). The 8-oxodG lesions are repaired primarily through the BER pathway (119). The BER pathway facilitates DNA repair through two general pathways: *a.* the short-patch BER pathway, leading to a repair tract of a single nucleotide; *b.* the longpatch BER pathway, producing a repair tract of at least two nucleotides (120). In these two repair sub-pathways, DNA glycosylases play a central role because they can recognize and catalyze the removal of damaged bases (120). This suggests that the defect of DNA glycosylases should be related to the decreasing capacity of the BER pathway and might

Human oxoguanine glycosylase 1(hOGG1) is a specific DNA glycosylase that catalyzes the release of 8-oxodG and the cleavage of DNA at the AP site (121, 122). Genetic structure study has revealed the presence of several polymorphisms within hOGG1 locus (123). Among them, the polymorphism at position 1245 in exon 7 causes an amino acid substitution (Ser to Cys) at codon 326, suggesting this polymorphism may glycosylase function (123). A functional complementation activity assay showed that hOGG1 protein encoded by the 326 Cys allele had substantially lower DNA repair activity than that encoded by the 326 Ser allele (124). Similar results were observed in human cells in vivo (122, 125). Therefore, low capacity of 8-oxodG repair resulting from hOGG1 326Cys polymorphism might contribute to

running-up risk of cancers (12, 107-114).

increase the risk of such cancers as HCC.

exposure.

Fig. 3. The meta-analysis of the relationship between XRCC1 codon Lys399Gln polymorphism and HCC risk among China population. Compared with Arg/Arg genotype, Arg/Gln (A) and Gln/Gln (B) genotypes increased HCC risk. This risk effect was also observed in the binding of Arg/Gln and Gln/Gln genotypes (C).

DNA Repair Capacity-Related to Genetic Polymorphisms of DNA Repair

individuals and, therefore, targeting prevention of this malignant tumor.

Key Construction Project of Laboratory Room (NO. 2009).

exposure among Chinese population.

**8. Summary** 

risk for this cancer.

**9. Acknowledgments** 

**10. Abbreviations** 

Genes and Aflatoxin B1-Related Hepatocellular Carcinoma Among Chinese Population 515

of hepato-tumorigenesis. These results exhibits that the polymorphism at codon 241 of XRCC3 gene is a genetic determinant in the development of HCC induced by AFB1

Like most other human malignant tumors, HCC is a complex disease attributed to environment variation and genetic susceptive factors. In high incidence areas of HCC in China, AFB1 is an important environment variation as well as chronic HBV and HCV infection. This toxic variation is characterized by: *a.* the attraction of specific organs, especially liver; *b.* genotoxicity, mainly inducing the formation of AFB1-DNA adducts and the hot-spot mutation of p53 gene; and *c.* carcinogenicity, primarily causing HCC. In the process of AFB1 hepatocarcinogenesis, AFB1-DNA adducts play a central role because of their genotoxicity and interactions with genetic susceptive factors. Numerous studies reviewed in this paper have demonstrated that the hereditary variations in DNA repair genes are associated with susceptibility to AFB1-related HCC among Chinese population. These molecular epidemiological studies have significantly contributed to our knowledge of the importance of genetic polymorphisms in DNA repair genes in the etiology of HCC related to AFB1 exposure. It would be expected that genetic susceptibility factors involved in DNA repair genes for HCC could serve as useful biomarkers for identifying at-risk

However, there are several issues to be noted. Firstly, the conclusions should be drawn carefully, because of conflicting data existing in the same ethnic population in view of between some genotypes of DNA repair genes and the risk of HCC. Secondly, caution should be taken particularly in extrapolating these data to other ethnic populations, because of the difference of population frequencies corresponding to genetic polymorphisms that depends on ethnicity. Thirdly, when risk of a specific polymorphism is considered, AFB1 exposure should be stressed because AFB1 exposure may differ from areas to areas and from individuals to individuals. Lastly, because of the fact that AFB1-related hepatocarcinogenesis is polygenic, no single genetic marker may sufficiently predict HCC risk. Therefore, a panel of susceptive biomarkers is warranted to define individuals at high-

We are grateful to Yuan-Feng Zhou for the collection and management of data. This study is supported by the Youth Science Foundation of Guangxi (NO. 0832097), the Science Foundation of Youjiang Medical College for Nationalities (NO. 2005 and 2008), and Guangxi

AFB1, Aflatoxin B1; AFB1-epoxide, AFB1-8,9-epoxide; AFB1-N7-Gua, 8,9-di-hydro-8-(N7 guanyl)-9-hydroxy–AFB1; AFB1-FAPy, ring-opened formamidopyridine AFB1; APE1, AP endonuclease-1; BER, base excision repair; CI, confidence interval; DSB, double-strand break; DSBR, double-strand break repair; HBV, hepatitis virus B; HCV, hepatitis virus C; HCC, hepatocellular carcinoma; hOGG1, Human oxoguanine glycosylase 1; NER,

the persistence of 8-oxodG in genomic DNA in vivo, which, in turn, could be associated with increased cancer risk (125, 126).

In 2003, Peng *et al.* (126) investigated the correlation among 8-oxodG levels, hOGG1 expression, and hOGG1 Cys326Ser polymorphism in Guangxi Autonomous Region. They found that individuals with genotypes with hOGG1 codon 326 Cys alleles faced lower level of hOGG1 expression and higher 8-oxodG levels. Supporting their results, Cheng *et al.* (21) reported that hOGG1 expression was significantly linear correlated with HCC. Recently, using the molecular epidemiological methods, Zhang *et al.*(127) found that the distribution of Cys alleles at codon 326 of hOGG1 in HCC cases (43.0%) significantly differed from in controls (33.1%). Logistic regression analysis showed that the genotypes with Cys alleles, compared to without this alleles, increased HCC risk of Chinese population, with adjusted OR-value (95% CI) 1.5 (0.79-2.93) for Cys/Ser and 1.9 (0.83-4.55) for Cys/Cys. These findings suggested pathogenic role of hOGG1 Cys326Ser polymorphism in the hepatocarcinogenesis.

#### **7. Genetic polymorphisms in genes involved in DSBR pathway and risk of HCC**

DSBs, although only make up a very small proportion of AFB1-induced DNA damage, are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss (19, 128, 129). This type damage is repaired DSBR consisting of non-homologous end-joining (NHEJ) and homologous recombination (HR) (130-133). There are several decades DNA repair genes involves in DSBR pathway and the defects in these genes cause genome instability and promote tumorigenesis (128, 134, 135). In published molecular epidemiological studies, only XRCC3 gene codon Thr241Met polymorphism effects the risk of AFB1-related HCC risk among Chinese population (58, 60).

The product of the XRCC3 gene is one of identified paralogs of the strand-exchange protein RAD51 in human beings (136). This protein correlates directly with DNA breaks and facilitates of the formation of the RAD51 nucleoprotein filament, which is crucial both for homologous recombination and HRR (136-138). Previous studies have shown that a common polymorphism at codon 241 of XRCC3 gene (Thr to Met) modifies the function of this gene ad increases cancers risk (139-143). Two reports from high AFB1-exposure areas of China supported above-mentioned conclusions (58, 60).

In the first frequent case-control study in Guangxiese (58), we observed that the genotypes with XRCC3 codon 241 Met alleles (namely Thr/Met and Met/Met) was significantly different between controls (33.01%) and HCC cases (61.48%, *P* < 0.001). Met alleles increases about 2- to 10-fold risk of HCC and this running-up risk is modulated by the number of Met alleles (adjusted OR 2.48 and 10.06 for one and two this alleles). Considering small sample size in this study, we recruited, in another independent frequent case-control study (60), a relatively larger sample size to compare the results. Subjects included in this study, 491 HCC cases and 862 age-, sex, race, hepatitis virus infection information-matching controls, were permanent residents of Guangxi areas. Similar to the results of the first report, the distribution of XRCC3 codon 241 Met allele frequency was found to be significantly different between cases (59.7%) and controls (32.1%). Individuals having the Thr/Met or Met/Met were at a 2.22-fold or 7.19 fold increased risk of developing HCC cancer. Above two studies showed this allele multiplicatively interacted with AFB1 exposure in the process of hepato-tumorigenesis. These results exhibits that the polymorphism at codon 241 of XRCC3 gene is a genetic determinant in the development of HCC induced by AFB1 exposure among Chinese population.
