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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2014 Aug 15;7(9):6231–6244.

Interaction of DNA repair gene polymorphisms and aflatoxin B1 in the risk of hepatocellular carcinoma

Jin-Guang Yao 1,*, Xiao-Ying Huang 1,*, Xi-Dai Long 1,2
PMCID: PMC4203246  PMID: 25337275

Abstract

Aflatoxin B1 (AFB1) is an important environmental carcinogen and can induce DNA damage and involve in the carcinogenesis of hepatocellular carcinoma (HCC). The deficiency of DNA repair capacity related to the polymorphisms of DNA repair genes might play a central role in the process of HCC tumorigenesis. However, the interaction of DNA repair gene polymorphisms and AFB1 in the risk of hepatocellular carcinoma has not been elucidated. In this study, we investigated whether six polymorphisms (including rs25487, rs861539, rs7003908, rs28383151, rs13181, and rs2228001) in DNA repair genes (XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and XRCC3) interacted with AFB1, and the gene-environmental interactive role in the risk of HCC using hospital-based case-control study (including 1486 HCC cases and 1996 controls). Genotypes of DNA repair genes were tested using TaqMan-PCR technique. Higher AFB1 exposure was observed among HCC patients versus the control group [odds ratio (OR) = 2.08 for medium AFB1 exposure level and OR = 6.52 for high AFB1 exposure level]. Increasing risk of HCC was also observed in these with the mutants of DNA repair genes (risk values were from 1.57 to 5.86). Furthermore, these risk roles would be more noticeable under the conditions of two variables, and positive interactive effects were proved in the followed multiplicative interaction analysis. These results suggested that DNA repair risk genotypes might interact with AFB1 in the risk of HCC.

Keywords: Aflatoxin B1, DNA repair gene, interaction effect, polymorphism, hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) is the most common malignant tumor in the Guangxi Zhuang Autonomous Region of China [1,2]. Epidemiological evidence has shown that exposure to aflatoxin B1 (AFB1), an important chemical carcinogen, is the most important cause of the high rate of HCC in this area [1,2]. This carcinogen was an important member of aflatoxin family highly substituted coumarins containing a fused dihydrofurofuran moiety [1-3]. It is mainly produced by some strains of the moulds Aspergillus parasiticus and Aspergillus flavus, and is structurally characterized by fusion of a cyclopentanone ring to the lactone ring of the coumarin moiety [1,2]. AFB1 was discovered as a contaminant of human and animal food, especially peanuts (ground nuts), core, soya sauce, and fermented soy beans 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. This type of toxin has three toxicological effects: a. genotoxicity, mainly inducing the formation of AFB1-DNA adducts and the hot-spot mutation of p53 gene; b. the attraction of specific organs, especially liver; and c. carcinogenicity, primarily causing hepatocellular carcinoma (HCC) [1-9]. Today, AFB1 has been classified as a known human carcinogen by the International Agency for Research on Cancer [1-3]. Increasing evidence has suggested that AFB1 can induce the formation of AFB1-DNA adducts and cause DNA strand breakage, DNA base damage, and oxidative damage that may ultimately lead to cancer. AFB1-induced DNA damage can be repaired by base excision repair, strand break repair, and nucleotide excision repair (NER) [1,2]. Recent studies have shown that DNA damage by AFB1 plays the central role of carcinogenesis of HCC-related to this toxin in the toxic studies [1,2,10-17]. Furthermore, AFB1-induced HCC risk value might be more noticeable under the conditions of more amounts of DNA damage [1,2,11,12,14-21]. This indicates DNA repair capacity (limited to DNA repair genes) might be able to interact with AFB1 exposure and this interaction might strengthen the risk effects of AFB1 exposure on HCC risk. Here, we investigated the interactive effects of AFB1 exposure and genetic polymorphisms in DNA repair genes XRCC1, XRCC3, XRCC4, XRCC7, XPD, and XPC (including rs25487, rs861539, rs7003908, rs28383151, rs13181, and rs2228001) on HCC risk.

Materials and methods

Study subject

This was a hospital-based case-control study, including 1486 HCC cases and 1996 controls [10]. All cases and controls were recruited from affiliated hospitals of the two main medical colleges in southwestern Guangxi (Guangxi Medical University and Youjiang Medical College for Nationalities) from January 2004 to December 2012. All cases and controls were residents of the Guangxi Zhuang Autonomous Region from AFB1 exposure areas and accepted enrollment in this study. The cases included in this study, representing a significant proportion (> 90%) of HCC patients in the Guangxi population, were identified by histopathological diagnosis in 100% of the HCC cases. During the same period of HCC investigation, controls without any evidence of liver disease were randomly selected from a pool of healthy volunteers who visited the general health check-up centers of the same hospitals for their routinely scheduled physical examinations supported by local governments.

To control the effects of confounders which were associated with the distribution of genotypes or the exposure of AFB1, controls were individually matched (1:1 or 2:1) to cases based on ethnicity (Han, Minority), sex, age (± years), and hepatic B virus (HBV) and hepatic C virus (HCV) infection. All patients and controls gave informed consent for participation and were interviewed uniformly before surgery by a well-trained interviewer. The questionnaire used in the interview sought detailed information on current and past living habits, occupational history, family disease history, dietary history and general demographic data. Demographic information (including age, sex, race, medical history, family disease history, dietary history, and living history), α-fetoprotein (AFP), hepatitis virus B (HBV) and hepatitis virus C (HCV) infection information, and therapeutic data were collected from medical records in the hospitals by a Youjiang Cancer Institution staff member. At the same time, 4 mL of peripheral blood was obtained for the extraction of genomic DNA. In this study, those hepatitis B surface antigen (HBsAg) positive and anti-HCV positive in their peripheral serum were defined as groups infected with HBV and HCV.

The study protocol was been carried out in accordance with Ethical Principles for Medical Research Involving Human Subjects (World Medical Association Declaration Of Helsinki, 2004) and approved by Institutional review boards from Guangxi Cancer Institute, and the Medical Research Council from the corresponding hospitals.

Nucleic acid isolation

Leukocytes from peripheral venous blood samples were isolated by standard procedures. DNA was then extracted from leukocyte samples by standard phenol-chloroform extraction and ethanol precipitation. DNA was stored at -20°C until additional analysis.

AFB1 exposure assay

In this study, AFB1 exposure levels were ascertained according to serum AFB1-albumin adducts levels of peripheral blood. AFB1-albumin adducts levels were tested using the comparative enzyme-linked immunosorbent assay (ELISA) as previously published [22].

Polymorphisms selection of DNA repair genes and genotyping

In this study, we only selected these single nucleotide polymorphisms (SNPs) in the DNA repair genes that might modify AFB1-related HCC risk. According to our previous results, a total of 6 SNPs, including rs25487 (in the XRCC1), rs861539 (in the XRCC3), rs7003908 (in the XRCC7), rs28383151 (in the XRCC4), rs13181 (in the XPD), and rs2228001 (in the XPC), were finally analyzed in the present study (Table 1) [10].

Table 1.

Characteristics of polymorphisms in the DNA repair genes

Polymorphism Gene Chr:bp Alleles Genotypes Codon No. Amino acid Genotyping
Wild Mutant Wild Heterozygotes mutant
rs25487 XRCC1 19:44055726 C T CC CT TT 399 Arg/Gln TaqMan-PCR
rs861539 XRCC3 14:104165753 G A GG GA AA 241 Thr/Met TaqMan-PCR
rs7003908 XRCC7 8:48770702 A C AA AC CC / / TaqMan-PCR
rs28383151 XRCC4 5:82406873 G A GG GA AA 56 Ala/Thr TaqMan-PCR
rs13181 XPD 19:45854919 T G TT TG GG 751 Lys/Gln TaqMan-PCR
rs2228001 XPC 3:14187449 T G TT TG GG 939 Lys/Gln TaqMan-PCR
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The genotypes of DNA repair genes were genotyped using the previous TaqMan-PCR methods on iCycler iQ™ real-time PCR detection system (iQ5, Bio-Rad Laboratories Inc.). Primer and probe sets and annealing temperatures used for TaqMan-PCR assay are shown in Table 2 [10]. For quality control, controls were included in each run, and repeated genotyping and sequencing of a random 5% subset yielded 100% identical genotypes.

Table 2.

Technical details of TaqMan-PCR analysis

Polymorphism Gene Primers Probes
rs25487 XRCC1 5’-GTGGGTGCTGGACTGTC-3’ 5’-FAM-CCTCCCGGAGGTAA-MGB-3’
5’-GCAGGGTTGGCGTGTGA-3’ 5’-VIC-CCCTCCCAGAGGTAA-MGB-3’
rs861539 XRCC3 5’-CCAGGGCCAGGCATCTG-3’ 5’-FAM-CAGCATGGCCCCCA-MGB-3’
5’-CAGCACAGGGCTCTGGA-3’ 5’-VIC-CAGCGTGGCCCCCA-MGB-3’
rs7003908 XRCC7 5’-CCTACCTCACGAACTCAGCAATT-3’ 5’-FAM-CTAAGAGTCCGCTGTTT-MGB-3’
5’-GCTGCCAACGTTCTTTCCTTATAGT-3’ 5’-Hex-CCTAAGAGTCAGCTGTTT-MGB-3’
rs28383151 XRCC4 C_58444701_10a C_58444701_10
rs13181 XPD 5’-AGTCACCAGGAACCGTTTATGG-3’ 5’-HEX-CTCTATCCTCTGCAGCG-MGB-3’
5’-TCTGTTCTCTGCAGGAGG ATC-3’ 5’-FAM-TATCCTCTTCAGCGTCT-MGB-3’
rs2228001 XPC 5’-AGCAGCTTCCCACCTGTTC-3’ 5’-FAM-CACAGCTGCTCAAAT-MGB-3’
5’-GTGGGTGCCCCTCTAGTG-5’ 5’-Hex-CTCACAGCTTCTCAAAT-MGB-3’
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a

From the Applied Biosystems.

Statistical analysis

All statistical analyses were done using SPSS version 18 (SPSS, Inc., Chicago, IL). In this study, genotype data were analyzed as trichotomous variables, including wild homozygotes (wild genotype), heterozygotes (heterozygotes genotype), and mutant homozygotes (mutant genotype). Frequency tables of independent variables were evaluated for statistical significance by Pearson’s χ2. To analyze the risk for gene mutation and HCC associated with each genotype while adjusting for confounders, multivariable logistic regression was done and odds ratios (OR) along with 95% confidence intervals (95% CI) generated. In this type of the additive model, we treated genotype as an ordinal variable (wild type coded as 0, heterozygote as 1, and homozygotes variant as 2). Based on individually matched design of case-control study, we did conditional logistic regression (with multivariate factors, including known causes of HCC among the Guangxi population) to estimate ORs for risk of HCC and their 95% CIs.

For environment-gene interaction analysis, joint effects between genotypes and AFB1 exposure status on HCC risk were assessed with the full regression model, which included all possible confounders. The interactive effects were evaluated according to the following formula [11,12,23]: OReg < OReg’ × ORe’g where OReg is the odds ratio for the presence of both high AFB1 exposure and risk genotypes of DNA repair genes (adjusted OR > 1), OReg’ is the odds ratio for high AFB1 exposure for patients with the low-risk genotypes of DNA repair genes, and Ore’g is the odds ratio for the high risk genotypes of DNA repair genes in patients with low AFB1 exposure. In the present study, a P-value of < 0.05 was considered statistically significant.

Results

Demographic characteristics of HCC cases

The 1486 HCC cases and 1996 controls were from high AFB1 exposure areas and included in the final analysis and the demographic data of these subjects is shown in Table 3. There were no significant differences between cases and controls in terms of distribution of age, sex, race, and HBV and HCV status as a result of individual matching (P > 0.05). These results suggest that HCC patient data were comparable to control data.

Table 3.

Demographic and Etiologic Characteristics of HCC Cases and Controls

Controls (n = 1996) Cases (n = 1486)
n % n % c2 P
Sex 0.157 0.692
    Male 1516 76.0 1120 75.4
    Female 480 24.0 366 24.6
Age (years)a 4.631 0.705
    ≤ 34 225 11.3 147 9.9
    35-40 309 15.5 261 17.6
    41-45 292 14.6 219 14.7
    46-50 296 14.8 221 14.9
    51-55 242 12.1 168 11.3
    56-60 218 10.9 160 10.8
    61-65 210 10.5 164 11.0
    ≥ 66 204 10.2 146 9.8
Ethnicity 2.089 0.148
    Han 891 44.6 700 47.1
    Zhuang 1105 55.4 786 52.9
HBV infection 2.424 0.119
    HBsAg (-) 588 29.5 402 27.1
    HBsAg (+) 1408 70.5 1084 72.9
HCV infection 0.312 0.576
    anti-HCV (-) 1640 82.2 1210 81.4
    anti-HCV (+) 356 17.8 276 18.6
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a

The mean ± S.D. ages were 49.32 ± 11.43 and 49.42 ± 11.30 for cases and controls, respectively.

AFB1 exposure and HCC risk

The AFB1 exposure information for the entire study population is shown in Table 4. We found that HCC cases (28.36 fmol/mg) had higher serum levels of AFB1-albumin adducts than controls (11.55 fmol/mg). For statistical analysis, values were logarithmically transformed and then were divided into three stratus: low (< 2.18 ln fmol/mg), medium (2.18-2.98 ln fmol/mg), and high (> 2.98 ln fmol/mg), according to the mean logit value of serum AFB1-albumin adducts among controls and cases (Table 4). Regression analysis showed that HCC risk gradually increased with an increasing number of exposure levels (adjusted OR = 2.08-6.52; P < 0.01).

Table 4.

AFB1 exposure and the risk of HCC

AFB1 exposureb Controls HCCs OR (95% CI)a P trend
n % n %
Lowc 1060 53.1 352 23.7 Reference
Medium 604 30.3 417 28.1 2.08 (1.75-2.47) 1.36 × 10-16
High 332 16.6 717 48.3 6.52 (5.46-7.79) 1.26 × 10-94
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a

OR conditional on matched set.

b

The mean ± S.D. level of serum AFB1-album adducts is 2.98 ± 0.80 and 2.18 ± 0.64 ln fmol/mg for cases and controls, respectively.

c

Adduct levels were: < 2.18 ln fmol/mg for low level; 2.18-2.98 ln fmol/mg for medium; > 2.98 ln fmol/mg for high level.

DNA repair genes polymorphisms and HCC risk

To explore the correlation between these six polymorphisms in DNA repair genes and HCC risk, we conducted a hospital-based case-control study according to our previously published methods [11,12]. A total of 1486 HCC cases and 1996 individually-matched (based on age, sex, race, and HBV and HCV infection status) controls were included in the present risk analysis (Table 5). Higher frequency of mutants of DNA repair genes was observed in the HCC patients than in the controls. Conditional logistic regression analysis exhibited that mutant alleles increased about 2 to 6 fold of HCC risk value. This risk was more noticeable under the conditions of mutant homozygotes (Table 5). For example, HCC risk for the genotype with XRCC4 rs28383151-GA was 2.248 (1.857-2.722); whereas risk value was 3.690 (2.708-5.029) for XRCC4 rs28383151-AA genotype. These results suggested the risk of HCC was associated with the number of mutant alleles of DNA repair genes XRCC1, XRCC3, XRCC7, XRCC4, XPD, and XPC.

Table 5.

Polymorphisms in DNA repair genes and HCC risk

Controls HCCs
Gene Polymorphism Genotype n % n % ORa 95% CI P
XRCC1 rs25487 CC 1437 71.99 777 52.29 Reference
CT 520 26.05 608 40.92 2.155 1.861-2.495 9.918 × 10-25
TT 39 1.95 101 6.80 4.774 3.264-6.981 7.614 × 10-16
XRCC3 rs861539 GG 1430 71.64 509 34.25 Reference
GA 539 27.00 634 42.66 3.321 2.848-3.872 5.671 × 10-53
AA 27 1.35 343 23.08 5.846 3.907-13.747 3.399 × 10-67
XRCC7 rs7003908 AA 1141 57.16 363 24.43 Reference
AC 608 30.46 663 44.62 3.434 2.921-4.037 1.664 × 10-50
CC 247 12.37 460 30.96 5.867 4.828-7.129 7.478 × 10-71
XRCC4 rs28383151 GG 1717 86.02 1047 70.46 Reference
GA 217 10.87 300 20.19 2.248 1.857-2.722 1.034 × 10-16
AA 62 3.11 139 9.35 3.690 2.708-5.029 1.341 × 10-16
XPD rs13181 TT 1214 60.82 549 36.94 Reference
TG 611 30.61 607 40.85 2.193 1.884-2.551 3.216 × 10-24
GG 171 8.57 330 22.21 4.270 3.458-5.273 1.841 × 10-41
XPC rs2228001 TT 988 49.50 546 36.74 Reference
TG 804 40.28 696 46.84 1.570 1.357-1.817 1.388 × 10-9
GG 204 10.22 244 16.42 2.185 1.764-2.706 8.231 × 10-13
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a

Adjusted by age, sex, race, HBV status, and HCV status.

To assess possible interactive effects of matching factors and polymorphisms in the DNA repair genes on HCC risk, we performed a series of bivariate stratified analyses by matching factors, such as HBV and HCV infection, age, race, and sex, on this polymorphism and did not find that these factors modulated the effect of these polymorphisms on HCC risk (P interaction > 0.05; Data not shown). This implied that these matching factors should be effectually manipulated and should not modify the association between these six polymorphisms and HCC in the present study.

Joint effects of DNA repair genes polymorphisms and AFB1 exposure on HCC risk

To study the correlation between the polymorphisms in the DNA repair genes and AFB1 exposure in the risk for HCC, we first analyzed the joint effects of AFB1 exposure levels and DNA repair genes genotypes on HCC risk (Table 6). In this analysis, we used as a reference the lowest risk group: those who had wild genotypes of DNA repair genes and low AFB1-exposure levels. We observed that increasing the number of exposure levels consistently increased HCC risk; moreover, this risk was more pronounced among subjects with the risk genotypes of DNA repair genes XRCC1, XRCC3, XRCC7, XRCC4, XPD, and XPC (OR, > 1). For example, risk value was 1.41 (1.02-1.95) for these individuals with risk genotypes with XRCC4-GA/AA (Stratum 2); whereas corresponding value was 8.81 (6.95-11.17) under the conditions of high AFB1 exposure levels (Stratum 4). We next analyzed the possible environment exposure-risk genotypic interaction using multiplicative models (Table 7) and found significantly evidence of multiplicatively interactive effects of genotypes and AFB1 exposure on HCC risk according to the previously published formula (OReg < OReg’ × ORe’g) [23].

Table 6.

Joint effects of Polymorphisms in DNA repair genes and AFB1 exposure on HCC risk

Controls HCCs
Variable n % n % ORa 95% CI P
AFB1/XRCC1
    Stratum 1b 769 38.5 200 13.5 Reference
    Stratum 2c 291 14.6 152 10.2 2.01 1.56-2.58 4.77 × 10-8
    Stratum 3d 668 33.5 577 38.8 3.32 2.74-4.02 1.10 × 10-34
    Stratum 4e 268 13.4 557 37.5 7.98 6.44-9.87 4.64 × 10-81
AFB1/XRCC3
    Stratum 1 782 39.2 185 12.5 Reference
    Stratum 2 278 13.9 167 11.2 2.54 1.98-3.26 2.72 × 10-13
    Stratum 3 648 32.5 324 21.8 2.12 1.72-2.61 1.95 × 10-12
    Stratum 4 288 14.4 810 54.5 11.9 9.65-14.88 1.14 × 10-118
AFB1/XRCC7
    Stratum 1 592 29.7 103 6.9 Reference
    Stratum 2 468 23.4 249 16.8 3.06 2.36-3.97 2.99 × 10-17
    Stratum 3 549 27.5 260 17.5 2.72 2.11-3.52 1.79 × 10-14
    Stratum 4 387 19.4 874 58.8 12.96 10.19-16.50 2.40 × 10-96
AFB1/XRCC4
    Stratum 1 916 45.9 288 19.4 Reference
    Stratum 2 144 7.2 64 4.3 1.41 1.02-1.95 3.66 × 10-2
    Stratum 3 801 40.1 759 51.1 3.09 2.55-3.55 8.24 × 10-39
    Stratum 4 135 6.8 375 25.2 8.81 6.95-11.17 3.36 × 10-72
AFB1/XPC
    Stratum 1 516 25.9 152 10.2 Reference
    Stratum 2 544 27.3 200 13.5 1.25 0.98-1.60 0.07
    Stratum 3 472 23.6 394 26.5 2.83 2.26-3.55 1.14 × 10-19
    Stratum 4 464 23.2 740 49.8 5.41 4.36-6.71 1.79 × 10-53
AFB1/XPD
    Stratum 1 654 32.8 143 9.6 Reference
    Stratum 2 406 20.3 209 14.1 2.36 1.84-3.01 9.15 × 10-12
    Stratum 3 560 28.1 406 27.3 3.32 2.66-4.14 2.74 × 10-26
    Stratum 4 376 18.8 728 49.0 8.87 7.11-11.05 4.27 × 10-84
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a

OR conditional on matched set.

b

Stratum 1 refers to the combination of low AFB1 exposure and wild genotypes of DNA repair genes.

c

Stratum 2 refers to the combination of low AFB1 exposure and genotypes of DNA repair genes with mutant alleles.

d

Stratum 3 refers to the combination of medium-high AFB1 exposure and wild genotypes of DNA repair genes.

e

Stratum 4 refers to the combination of medium-high AFB1 exposure and genotypes of DNA repair genes with mutant alleles.

Table 7.

Interactive effects of Polymorphisms in DNA repair genes and AFB1 exposure on HCC risk

Variable OReg ORe’g OReg’ ORe’g × OReg’ AFB1 exposure-Gene interaction
AFB1/XRCC1 7.98 2.01 3.32 6.67 Multiplication interaction
AFB1/XRCC3 11.9 2.54 2.12 5.38 Multiplication interaction
AFB1/XRCC7 12.96 3.06 2.72 8.32 Multiplication interaction
AFB1/XRCC4 8.81 1.41 3.09 4.36 Multiplication interaction
AFB1/XPC 5.41 1.25 2.83 3.54 Multiplication interaction
AFB1/XPD 8.87 2.36 3.32 7.84 Multiplication interaction
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To elucidate the environment-gene interactive effects on HCC risk, we calculated the interactive coefficient between AFB1 exposure and genotypes of DNA repair genes using logistic regression models with the environment-gene interactive variables (Table 8). Results showed that the corresponding interactive coefficients were about 1.6 for between AFB1 exposure and DNA repair genes XRCC1, XRCC3, XRCC4, and XRCC7 (P interaction < 0.01). Taken together, these results exhibited AFB1 exposure significantly and multiplicatively interacted with the polymorphisms in the DNA repair genes in the process of HCC carcinogenesis.

Table 8.

Interactive coefficient of Polymorphisms in DNA repair genes and AFB1 exposure on HCC risk

Interactive variable ORinteraction 95% CI P interaction
AFB1 × XRCC1 1.61 1.53-1.69 2.31 × 10-87
AFB1 × XRCC3 1.74 1.66-1.82 2.21 × 10-133
AFB1 × XRCC7 1.59 1.53-1.65 2.08 × 10-127
AFB1 × XRCC4 1.64 1.55-1.73 6.41 × 10-73
AFB1 × XPC 1.36 1.29-1.43 2.75 × 10-33
AFB1 × XPD 1.14 1.08-1.20 1.00 × 10-6
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Discussion

Evaluation of AFB1-DNA repair genes interaction

The main toxicological effect of AFB1 is to induce DNA damage, consisting of AFB1-DNA adducts and the hot-spot mutation of tumor suppressor gene p53 at codon 249 (TP53M) [5,20,24,25]. Our previous studies have shown the low DNA repair capacity from genetic polymorphisms in the DNA repair genes increases the toxicological effects of AFB1 exposure [10]. This suggests that there be environment-gene interaction in the process of HCC carcinogenesis induced by AFB1 exposure. In this study, we systematically investigated the possible interaction of this toxin and genotypes of DNA repair genes via three models: (1) joint effects model, (2) environment-gene interactive model, and (3) interactive coefficient model.

Because previous reports have exhibited that both environmental variable AFB1 exposure and genic variable DNA repair genes increase HCC risk [10-17], we analyzed the combination effects of AFB1 exposure and polymorphisms in the DNA repairgenes XRCC1, XRCC3, XRCC4, XRCC7, XPD, andXPC in the joint effects model. In this model, environmental variable and genotype variable were combined and divided into four subgroups: (1) Stratum 1, the combination of low AFB1 exposure and wild genotypes of DNA repair genes; (2) Stratum 2, the combination of low AFB1 exposure and genotypes of DNA repair genes with mutant alleles; (3) Stratum 3, the combination of medium-high AFB1 exposure and wild genotypes of DNA repair genes; and (4) Stratum 4, the combination of medium-high AFB1 exposure and genotypes of DNA repair genes with mutant alleles. For the estimate of cumulative risk value of the combination of AFB1 exposure and DNA repair genotypes, this combinative variable was treated as an ordinal variable (Stratum 1 coded as 0, Stratum 2 as 1, Stratum 3 as 2, and Stratum 4 as 3) and incorporated into multivariable logistic regression. To calculate risk value, we used as a reference the lowest risk group: those who had wild genotypes of DNA repair genes and low AFB1-exposure levels, namely “Stratum 1”. This model suggests possible additive or multiplicative effects of environmental and genic factors.

Based on the results in the first model, we obtained risk value of environmental variable AFB1 exposure and genic variables DNA repair genotypes. According to previous in interactive formula [23]: OReg < OReg’ × ORe’g.

We can analyze whether environmental variable AFB1 exposure and genic variables DNA repair genotypes are likely to act simultaneously at the same mutation sites. If the value of OReg is more than that of OReg’ × ORe’g, higher AFB1 exposure should interact with risk genotypes of DNA repair genes and they might simultaneously exert influence on HCC carcinogenesis [23]. Furthermore, interactive coefficient can reflect the strength of the interaction of environmental variable AFB1 exposure and genic variables DNA repair genotypes using. Because of the aforementioned reasons, the interactive effects of AFB1 exposure and DNA repair genes were evaluated through the following three methods: (1) joint effects, (2) interactive effects; and (3) interactive coefficient, in this study. Our results also exhibited that there is significantly interaction of environmental variable AFB1 exposure and genic variables DNA repair genotypes in the process of HCC carcinogenesis.

Interactive effects of XRCC1 polymorphism and AFB1 exposure on HCC

XRCC1 gene is one of three submits of DNA repair complex in the SSBR pathway (Gene dbase from PubMed). This gene spans about 32 kb on chromosome 19q13.2 and contains 17 exons and 16 introns. Its’ encoding protein (633 amino acids), consists of three functional domains: N-terminal 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) [26-28]. 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 single-strand break repair (SSBR) and base excision repair (BER) pathway [26-28]. More than 50 SNPs in the coding region of XRCC1 gene that lead to amino acid substitution have been described (SNP database). Among these polymorphisms, rs25487 polymorphism (also called codon Arg399Gln polymorphism) is of special concern, because this polymorphism resides in functionally significant regions (BRCT II) and may be related to decreasing DNA repair activity [26,29-31].

In the several decade years, increasing evidence has shown that XRCC1 rs25487 polymorphism interacts with environmental factors such as HBV infections, cigarettes and alcohol, and plays an important role in the carcinogenesis of cancers [29-31]. However, these studies about XRCC1 rs25487 polymorphism were reported with the results being contradictory in the several decades. To investigate the association between XRCC1 genotypes and HCC-related to AFB1 exposure and possible gene-environmental interaction, we designed and conducted a hospital-based case-control study in the high AFB1 exposure areas. We first evaluated the risk role of this polymorphism on the AFB1-related HCC. Results showed that the HCC patients with XRCC1 genotypes with rs25487 T alleles (namely: rs25487-CT or rs25487-TT) faced a significantly increasing risk of HCC than those with the wild-type homozygote of XRCC1 [namely, rs25487-CC, OR = 2.155, 95% CI = 1.861-2.495 for rs25487-CT; OR = 4.774, 95% CI = 3.264-6.981 for rs25487-TT, respectively]. Our results showed this polymorphism could modulate HCC risk. Interactive analysis of both environmental and genotypic variables showed a significant interactive effects on HCC risk (interactive risk value = 1.61, P interaction < 0.01). Previous several meta-analysis based on different AFB1 exposure levels supported our conclusion [13,30-32]. These results suggest that the decreasing capacity of DNA repair resulting from XRCC1 rs25487 polymorphism is able to interact with higher AFB1 exposure in the process of HCC carcinogenesis.

Interactive effects of XRCC3 polymorphism and AFB1 exposure on HCC

The protein encoded by XRCC3 gene is one of identified paralogs of the strand-exchange protein RAD51 in human beings. This protein associates directly with DNA breaks and facilitates of the formation of the RAD51 nucleoprotein filament, which is crucial both for homologous recombination and HRR [33,34]. Previous studies have shown that a common polymorphism (rs861539) at codon 241 of XRCC3 gene (Thr to Met) modifies the function of this gene [35-44]. Two reports from high AFB1-exposure areas all of world supported above-mentioned conclusions [16,45]. In the first frequent case-control study in Guangxiese, we observed that the higher-frequency of genotypes with XRCC3 codon 241 Met alleles (namely Thr/Met and Met/Met) was observed in controls (33.01%) than HCC cases (61.48%, P < 0.001). Regression analysis showed that 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) [45]. The followed relative size analysis and the present study not only found similar risk value of AFB1-related HCC [16], but also found this polymorphism significantly interacted with AFB1 exposure [19]. Our present study supported aforementioned results. These data exhibit that the polymorphism at codon 241 of XRCC3 gene is a genetic determinant in AFB1-induced HCC. Its deficient type strengthens the carcinogenic role of higher AFB1 exposure, and showed a gene-environment interactive effect in the process of HCC tumorigenesis induced by AFB1 exposure.

Interactive effects of XRCC7 polymorphism and AFB1 exposure on HCC

DNA repair gene XRCC7, also called DNAPK, DNPK1, HYRC, HYRC1, or p350) (Genbank ID. 5591), spans about 197 kb on chromosome 8q11 and contains 85 exons and 86 introns (Gene dBase in PubMed). The protein encoded by XRCC7 acts as DNA-dependent protein kinase catalytic subunit (DNA-PKcs) that constitutes the large catalytic subunit of the DNA-PK complex [46]. When DNA-PKcs is recruited to the site of DSBs by the Ku70/Ku80 heterodimer, DNA-PK complex changes into its active form and subsequently initiates the non-homologous end joining (NHEJ) repair, an important DSBR pathway [47]. Murine mutants defective in the XRCC7 have non-detectable DNA-PK activity, suggesting that XRCC7 is required for NHEJ pathway protein [48,49]. More than 100 polymorphisms have been reported in the XRCC7 gene, some of which are correlated with malignant tumors such as bladder cancer (dbSNP in NCBI Database). Of these genetic polymorphisms in XRCC7 gene, we only investigated the interactive relation between rs7003908 polymorphism and AFB1 toxicological effects, and found this polymorphism might be an important interactive variable for AFB1 toxic role. Supporting our findings, a previous study was also found that these individuals with XRCC7 rs7003908 G alleles increased HCC risk compared the homozygote of XRCC7 rs7003908 T alleles (XRCC7-TT), with OR value 3.45 (2.40-4.94) for XRCC7-TG and 5.04 (3.28-7.76) for XRCC7-GG, respectively. Furthermore, this genetic mutation was correlated with higher the levels of AFB1-DNA adducts (r = 0.142, P < 0.001) [18], suggesting an environment-gene interactive effect. Taken together, these results explored that genetic polymorphism of XRCC7 rs7003908 might decrease AFB1-related DSBR capacity and result in an increasing toxicological capacity of AFB1. This interaction of environmental and genic variables progressed risk value of themselves on HCC tumorigenesis, however, it inquires more studies to support this conclusion.

Interactive effects of XRCC4 polymorphism and AFB1 exposure on HCC

XRCC4, located on chromosome 5q14.2, is an important the nonhomologous end-joining (NHEJ) gene [50,51]. The encoded protein of this gene consists of 336 amino acid residues (DDBJ/EMBL/Genbank accession no. AAD47298) and interacts directly with Ku70/Ku80 in the NHEJ pathway [50,51]. It is hypothesized that XRCC4 serves as a flexible join between Ku70/Ku80 and its associated protein, Ligase IV [50,51]. XRCC4 is required for precise end-joining of blunt DNA DSBs in mammalian fibroblasts, and the mutant, XRCC4, results in more-deficient NHEJ capacity. A gene-targeted mutation study has also shown that differentiating neurons and lymphocytes strictly require XRCC4 end-joining proteins. The targeted inactivation of this gene leads to late embryonic lethality accompanied by defective neurogenesis and defective lymphogenesis. These results demonstrate that XRCC4 is essential for the DNA repair capacity of NHEJ [52-54]. More than 100 polymorphisms have been reported in the XRCC4 gene (SNP database), some of which are correlated with DNA adducts, gene mutation, and malignant tumors (such as oral, gastric, liver, and bladder cancers) [11,12,55-60]. In this study, we only analyzed rs28383151 polymorphism in the coding region of this gene because this polymorphism localizes at conserved sites of this gene. It changes the coded amino acids and may be associated with a decreased DNA repair capacity and an increased cancer risk [11,12]. Our previous and present studies exhibited that this polymorphisms increased AFB1-related HCC risk, especially under the conditions of higher AFB1 exposure. This is possibly associated with the fact that this polymorphism is associated with increasing AFB1-DNA adducts levels and the hot-spot mutation risk of TP53 gene. Given that the amount of AFB1-DNA adducts and TP53 gene mutation reflects the toxic capacity of AFB1 exposure, there is reason to believe that the interaction of AFB1 exposure and XRCC4 genotypes exerts an important effects on HCC carcinogenesis. Supporting aforementioned hypothesis, our present study showed a significantly interactive risk value OR of 1.64 (1.55-1.73), therefore, this role should not be neglected.

Interactive effects of XPD polymorphism and AFB1 exposure on HCC

XPD gene, also called excision repair cross-complementing rodent repair deficiency complementation group 2 (ERCC2), COFS2, EM9, or TTD.) (Genbank ID. 2068), spans about 20 kb on chromosome 19q13.3 and contains 23 exons and 22 introns [1,61]. Its encoded-protein is one of seven central proteins in the NER pathway and act as a DNA-dependent ATPase/helicase. This protein is associated with the TFIIH transcription-factor complex, and plays a role in NER pathway. During NER, XPD participates in the opening of the DNA helix to allow the excision of the DNA fragment containing the damaged base [61]. There are four described polymorphisms that induce amino acid changes in the protein: at codons 199 (Ile to Met), at codon 201 (His to Tyr), at codon 312 (Asp to Asn) and at codon 751 (Lys to Gln) [62]. Among these polymorphisms, we only analyzed codon 751 polymorphism (rs13181) in this study, mainly because our previous studies [10,15] found the variant XPD codon 751 genotypes (namely Lys/Gln and Gln/Gln) detected by TaqMan-MGB PCR was significantly different between HCC cases (35.9% and 20.1% for Lys/Gln and Gln/Gln, respectively) and controls (26.3% for Lys/Gln and 8.6% for Gln/Gln, P < 0.001). Individuals having 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). Our present study (based on relative large sample size) suggested that the genetic polymorphisms at conserved sequence of XPD gene such as at codon 751 may have potential effect on AFB1-related HCC susceptibility. Furthermore, risk role would be more noticeable under the conditions of higher AFB1 exposure (OR = 8.87, 95% CI = 7.11-11.05). Interactive analysis showed significant AFB1-XPD joint effects on HCC risk (corresponding P interaction < 0.01). This supports HCC risk might modified by the interactive effects of genetic polymorphisms at codon 751 in DNA repair gene XPD and AFB1 exposure.

Interactive effects of XPC polymorphism and AFB1 exposure on HCC

XPC gene (Genbank accession No. AC090645) spans 33kb on chromosome 3p25, and consists of 16 exons and 15 introns. This gene encodes a 940-amino acid protein, an important DNA damage recognition molecule which plays an important role in NER pathway [63-65]. XPC protein binds tightly with another important NER protein 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 [63-65]. Therefore, it may play a role in the decreasing toxic effects of AFB1 and its deficiency may interact with AFB1 exposure. Recent some studies have shown that low DNA repair capacity resulting from the genetic mutation of XPC rs2228001 can progress AFB1-induced HCC [63-65]. In the past several decade years, a total of three studies reported the interaction of XPC rs2228001 polymorphism and environmental variables including HBV and AFB1 exposure, involving in HCC tumorigenesis [14,66,67]. The first study is from Shunde area, Guangdong Province which is characterized by high AFB1 exposure and high incidence rate of HCC. In this study, researchers explored the correlation between this polymorphism and risk of HCC via an 1-1 case-control study (including 78 HCC patients and 78 age- and sex-matching controls) method, and found the mutation of XPC modified HCC risk (adjusted odds ratios [ORs] were 6.78 with 95% CI 2.03-22.69) [67]. They furthermore analyze the mutant genotype interacted with HBV infection status, suggesting this polymorphism could interact with such environmental variables as HBV infection status. Although they did not directly evaluated the interactive effects of XPC rs2228001 polymorphism and AFB1 exposure on HCC risk, study population in their study is from both high HBV infection areas and high AFB1 exposure areas and high risk of HCC for XPC mutant. The other two studies was conducted by our teams and showed XPC codon 939 Gln alleles increased about 2-times risk of HCC, this risk would be more noticeable under the conditions of higher AFB1 exposure [14,66]. In our present study, we not only observed this mutation increased AFB1-related HCC risk, but found more direct evidence of interactive effects of XPC polymorphism and AFB1 exposure on HCC risk. As a result, these data suggest that genetic polymorphism at codon 939 of XPC gene is a genetic determinant in the DNA repair process of DNA damage induced by AFB1 exposure, and its low activity might interact with different AFB1 exposure status in the carcinogenetic process of HCC.

Limitation and conclusions

This study had several limitations. First, because the present study is the hospital-based case-control study, potential selection bias might have occurred. Second, because the liver disease itself may affect the metabolism of AFB1 and modify the levels of AFB1-DNA adducts, the increased risk with AFB1 exposure status noted in this study was probably underestimated. Third, although six genetic mutations in DNA repair genes were analyzed, some other genes might interact with AFB1 exposure in the process of HCC carcinogenesis. Therefore, more genes deserve further elucidation based on a large sample and the combination of genes and AFB1 exposure.

In conclusion, to the best of our knowledge, this is the first report to systematically investigate the interactive effects of AFB1 exposure and polymorphisms in DNA repair genes XRCC1, XRCC3, XRCC4, XRCC7, XPC, and XPD on HCC risk among Guangxi population from a high AFB1-exposure area. We find that both AFB1 exposure and the genetic mutations in the DNA repair genes XRCC1, XRCC3, XRCC4, XRCC7, XPC, and XPD might increase the risk of HCC, and their environment-gene interaction would furthermore strengthens this risk value. Given that AFB1 is am important genic agent and a kind of I type carcinogen, our findings might have prevention implications through identifying population with low DNA repair capacity, once these findings are replicated by other studies based on a larger scale or prospective studies.

Acknowledgements

We thank Dr. Qiu-Xiang Liang, Dr. Yun Yi, and Dr. Yuan-Feng Zhou for sample collection and management; Dr. Hua Huang for molecular biochemical technique. We also thank all members of Department of Medical Test and Infective Control, Affiliated Hospital of Youjiang Medical College for Nationalities for their help. This study was supported in part by the National Natural Science Foundation of China (No. 81372639 and 81160255), the Innovation Program of Guangxi Municipal Education Department (No. 201204LX674), Innovation Program of Shanghai Municipal Education Commission (No. 13YZ035), the Natural Science Foundation of Guangxi (No. 2013GXNSFAA019251), Key Discipline and Specialty Foundation of Shanghai Municipal Commission of Health and Family Planning, and “Shu Guang” project (supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation, No. 13SG19).

Disclosure of conflict of interest

None.

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