**Abstract**

Our previous reports have shown that the genetic single-nucleotide polymorphisms (GSNPs) in the DNA repair gene X-ray repair cross complementing 4 (XRCC4) are involved in the carcinogenesis of hepatocellular carcinoma (HCC) induced by aflatoxin B1 (AFB1). However, the effects of GSNPs in the coding regions of XRCC4 on hepatic toxicity of AFB1 have been less investigated. We conducted a hospital-based clinic tissue samples with pathologically diagnosed HCC (n = 380) in a high AFB1 exposure area to explore the possible roles of GSNPs in the coding regions of XRCC4 in AFB1-induced HCC using liver toxicity assays. A total of 143 GSNPs were included in the present study and genotyped using the SNaPshot method, whereas the liver toxicity of AFB1 was evaluated using AFB1- DNA adducts in the tissues with HCC. In the clinicopathological samples with HCC, the average adduct amount is 2.27 1.09 μmol/mol DNA. Among 143 GSNPs of XRCC4, only rs1237462915, rs28383151, rs762419679, rs766287987, and rs3734091 significantly increased the levels of AFB1-DNA adducts. Furthermore, XRCC4 GSNPs (including rs28383151, rs766287987, and rs3734091) also increased cumulative hazard for patients with HCC. These results suggest that the liver toxicity of AFB1 may be modified by XRCC4 GSNPs.

**Keywords:** AFB1, liver toxicity, XRCC4, genetic single-nucleotide polymorphism, hepatocellular carcinoma

## **1. Introduction**

Aflatoxin B1 (AFB1) is an important type I chemical toxicant mainly produced by the toxigenic strains of *Aspergillus flavus* (*A. flavus*) and *Aspergillus parasiticus* (*A. parasiticus*) [1, 2]. This carcinogen is often taken into human body via contaminating human foods such as nuts and cereals and displays its toxic effects, especially hepatic toxicity [1–8]. AFB1-induced hepatic effects consist of acute toxic damages (such as severe DNA damage, severe liver degeneration and necrosis, and the failure of hepatic function) and chronic cumulative damages (such as a series of cumulative DNA damage, slight hepatocellular degeneration and necrosis, chronic inflammation, liver cirrhosis, and liver cancer) [3–5]. Increasing evidence has shown that under the same exposure of AFB1, some individuals feature severe hepatic damage; others have no noticeable damage [9–14]. This suggests that different individuals have different responses to the toxic effects of AFB1 and genetic factors may play a central role in the AFB1-induced hepatic toxicity.

**2.3 GSNP selection**

*DOI: http://dx.doi.org/10.5772/intechopen.88752*

for final analyses.

**2.4 Genotypic analyses**

included in each analysis.

**2.5 Statistical analysis**

**3. Results**

**155**

**3.1 The characteristics of subjects**

All GSNPs of XRCC4 gene were first screened from the SNPdatabase (http://

*X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms…*

Genomic DNA in all tumor tissue samples with HCC was standard phenolchloroform extraction binding with proteinase K. The GSNPs of XRCC4 were genotyped using SNaPshot method (Applied Biosystems [ABI], Foster City, CA) as previously described [28]. For quality control, all laboratory personnel were blind to the status of every sample with hepatocarcinoma, and controls were also

The test for genotypic distribution of XRCC4 GSNPs among HCC cases featuring different AFB1-DNA adducts was accomplished using student *t-*test or one-way analysis of variance (ANOVA) test. Multiple tests were adjusted using a *Bonferroni* correction, and the threshold for GSNP screening was defined as <sup>α</sup> = 3.53 <sup>10</sup><sup>4</sup>

Kaplan-Meier survival model with log-rank test and Cox regression model (the selection of significant varies based on forward-step method with likelihood ratio test) was used to analyze the association between XRCC4 GSNPs and HCC outcomes. Cumulative hazard value for the effects of XRCC4 GSNPs on the hepatic toxicity for AFB1 and corresponding 95% confidence interval (CI) were calculated using hazard ratio (HR) from significant multivariate Cox regression model

(including all significant variates). All statistical analyses were performed with SPSS

All subjects suffered from hepatic carcinoma, and **Table 1** summarized their characteristics. The mean age of all participants was 50.74 11.55 years, and more than 70% of them are male. For these cancer patients, 70.3% (267/380) and 26.3% (100/380) cases featured TNM II and III stages of tumor, and they also had an

A total of 143 GSNPs in the coding regions of XRCC4 gene were selected in our final analyses, and **Table 2** showed the genotypic distribution of all GSNPs. To evaluate the effects of these potential GSNPs on AFB1-DNA adducts, the role of each GSNP in the coding regions of XRCC4 gene was tested using Student *t-*test or ANOVA test with the adjustment of multiple test. Among these GSNPs, only rs1237462915 (cat#SNP016, at codon 38), rs28383151 (cat#SNP026, at codon 56), rs762419679 (cat#SNP069, at codon 127), rs766287987 (cat#SNP112, at codon 203),

statistical package (Version 18, SPSS Institute, Chicago, IL, USA).

average AFB1 exposure value of 2.27 1.09 μmol/mol DNA.

**3.2 XRCC4 GSNPs increased AFB1-DNA adducts**

.

asia.ensembl.org/Homo\_sapiens/Gene/Variation\_Gene/Table?db=core;g= ENSG00000152422;r=5:83077498-83353787). According to the data from SNPdatabase, a total of 143 GSNPs can result in missense mutations and the change of amino acids in XRCC4 protein, and thus they were ultimately selected

X-ray repair cross complementing 4 (XRCC4), an important DNA repair gene involved in nonhomologous end-joining (NHEJ) repair pathway, plays a scaffold function via stabilizing and localizing DNA repair enzymes LIG IV, Ku70/80 heterodimer, and the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) in the ends of DNA double-stranded breaks (DSBs) during NHEJ [15, 16]. In the past decades, growing reports have exhibited that the abnormal structures and functions of XRCC4 may alter the capacity of DNA repair and ultimately result in human diseases [17–22]. Several recent studies have also shown that the genetic alterations in the coding regions of XRCC4 can modify hepatocellular carcinoma (HCC) risk and prognosis [23–27]. However, the effects of this genetic alteration on the hepatic toxicity of AFB1 is unclear. Here, we conducted a clinical sample study exposure to explore whether the genetic single-nucleotide polymorphisms (GSNPs, a type of genetic alterations) in the coding regions of XRCC4 modified the effects of AFB1 on hepatic damage.

### **2. Materials and methods**

#### **2.1 Study population**

This was a hospital-based molecular epidemiological study conducted in high AFB1 exposure area, Guangxi Zhuang Region, China. All participants were newly diagnosed HCC cases and recruited from the Affiliated Hospitals of Youjiang Medical University for Nationalities (located at Bose region, a major AFB1 exposure area) between January 2010 and January 2013 inclusively. The inclusive criteria of cases consisted of (a) cases with ultimately histopathologically confirmed HCC; (b) cases without any evidence of hepatitis virus infection; (c) cases with the history of AFB1 exposure which was defined according to positive history of peripheral serum AFB1-albumin adducts [5, 24]; and (d) cases with available tumor tissue samples and clinicopathological data.

According to the criteria, a total of 380 cases with HCC were recruited in this study during the period. With informed consent, the tissue samples with HCC for all patients and clinicopathological data were collected. Additionally, survival follow-up information was also collected through cases themselves or their family contact. In this study, the last follow-up date was set on January 31, 2019. The protocol for clinical samples was approved by Youjiang Medical University for Nationalities Medical Ethics Committee.

#### **2.2 The evaluation of AFB1-related hepatic toxicity**

Hepatic toxicity of AFB1 was evaluated using AFB1-DNA adducts in the tissue samples with HCC, and the amounts of AFB1-DNA adducts were tested by the previously described enzyme-linked immunosorbent assay (ELISA).

*X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms… DOI: http://dx.doi.org/10.5772/intechopen.88752*

## **2.3 GSNP selection**

the failure of hepatic function) and chronic cumulative damages (such as a series of

X-ray repair cross complementing 4 (XRCC4), an important DNA repair gene involved in nonhomologous end-joining (NHEJ) repair pathway, plays a scaffold function via stabilizing and localizing DNA repair enzymes LIG IV, Ku70/80 heterodimer, and the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) in the ends of DNA double-stranded breaks (DSBs) during NHEJ [15, 16]. In the past decades, growing reports have exhibited that the abnormal structures and functions of XRCC4 may alter the capacity of DNA repair and ultimately result in human diseases [17–22]. Several recent studies have also shown that the genetic alterations in the coding regions of XRCC4 can modify hepatocellular carcinoma (HCC) risk and prognosis [23–27]. However, the effects of this genetic alteration on the hepatic toxicity of AFB1 is unclear. Here, we conducted a clinical sample study exposure to explore whether the genetic single-nucleotide polymorphisms (GSNPs, a type of genetic alterations) in the coding regions of XRCC4 modified the effects of AFB1 on

This was a hospital-based molecular epidemiological study conducted in high AFB1 exposure area, Guangxi Zhuang Region, China. All participants were newly diagnosed HCC cases and recruited from the Affiliated Hospitals of Youjiang Medical University for Nationalities (located at Bose region, a major AFB1 exposure area) between January 2010 and January 2013 inclusively. The inclusive criteria of cases consisted of (a) cases with ultimately histopathologically confirmed HCC; (b) cases without any evidence of hepatitis virus infection; (c) cases with the history of AFB1 exposure which was defined according to positive history of peripheral serum AFB1-albumin adducts [5, 24]; and (d) cases with available tumor

According to the criteria, a total of 380 cases with HCC were recruited in this study during the period. With informed consent, the tissue samples with HCC for all patients and clinicopathological data were collected. Additionally, survival follow-up information was also collected through cases themselves or their family contact. In this study, the last follow-up date was set on January 31, 2019. The protocol for clinical samples was approved by Youjiang Medical University for

Hepatic toxicity of AFB1 was evaluated using AFB1-DNA adducts in the tissue samples with HCC, and the amounts of AFB1-DNA adducts were tested by the

cumulative DNA damage, slight hepatocellular degeneration and necrosis, chronic inflammation, liver cirrhosis, and liver cancer) [3–5]. Increasing evidence has shown that under the same exposure of AFB1, some individuals feature severe hepatic damage; others have no noticeable damage [9–14]. This suggests that different individuals have different responses to the toxic effects of AFB1 and genetic factors may play a central role in the AFB1-induced

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

hepatic toxicity.

hepatic damage.

**2. Materials and methods**

tissue samples and clinicopathological data.

Nationalities Medical Ethics Committee.

**154**

**2.2 The evaluation of AFB1-related hepatic toxicity**

previously described enzyme-linked immunosorbent assay (ELISA).

**2.1 Study population**

All GSNPs of XRCC4 gene were first screened from the SNPdatabase (http:// asia.ensembl.org/Homo\_sapiens/Gene/Variation\_Gene/Table?db=core;g= ENSG00000152422;r=5:83077498-83353787). According to the data from SNPdatabase, a total of 143 GSNPs can result in missense mutations and the change of amino acids in XRCC4 protein, and thus they were ultimately selected for final analyses.

### **2.4 Genotypic analyses**

Genomic DNA in all tumor tissue samples with HCC was standard phenolchloroform extraction binding with proteinase K. The GSNPs of XRCC4 were genotyped using SNaPshot method (Applied Biosystems [ABI], Foster City, CA) as previously described [28]. For quality control, all laboratory personnel were blind to the status of every sample with hepatocarcinoma, and controls were also included in each analysis.

#### **2.5 Statistical analysis**

The test for genotypic distribution of XRCC4 GSNPs among HCC cases featuring different AFB1-DNA adducts was accomplished using student *t-*test or one-way analysis of variance (ANOVA) test. Multiple tests were adjusted using a *Bonferroni* correction, and the threshold for GSNP screening was defined as <sup>α</sup> = 3.53 <sup>10</sup><sup>4</sup> . Kaplan-Meier survival model with log-rank test and Cox regression model (the selection of significant varies based on forward-step method with likelihood ratio test) was used to analyze the association between XRCC4 GSNPs and HCC outcomes. Cumulative hazard value for the effects of XRCC4 GSNPs on the hepatic toxicity for AFB1 and corresponding 95% confidence interval (CI) were calculated using hazard ratio (HR) from significant multivariate Cox regression model (including all significant variates). All statistical analyses were performed with SPSS statistical package (Version 18, SPSS Institute, Chicago, IL, USA).
