**3.2 Blood AFB1-DNA adducts correlating with PHCC risk**

The amount of AFB1-DNA adducts in the peripheral white blood cells were calculated using ELISA technique. Compared to controls, patients with PHCC featured a higher level of blood AFB1-DNA adducts (0.98 ± 0.63 vs. 2.01 ± 0.71 μmol/DNA), suggesting blood AFB1-DNA adducts may play an important role in the PHCC carcinogenesis. To investigate possible correlation between AFB1-DNA adducts and PHCC risk, the levels of blood AFB1-DNA adducts were divided into three groups. Results from multivariable logistic regression analyses showed that these individuals with medium AFB1-DNA adduct level (MAL) had an increasing risk of PHCC compared to those with low AFB1-DNA adduct level (LAL) (OR = 1.82 and 95% CI = 1.34–2.48), whereas risk value for high AFB1-DNA adduct level (HAL) was 3.82 (2.71–5.40) (**Table 2**). Altogether, these data were indicative of important potential risk role of blood AFB1-DNA adducts in the carcinogenesis of PHCC.

### **3.3 Blood AFB1-DNA adducts correlating with PHCC outcome**

To explore the effects of blood AFB1-DNA adducts on the prognosis of patients with PHCC, we accomplished two survival model analyses. Kaplan-Meier's

#### **Figure 1.**

*The aflatoxin B1 (AFB1)-DNA adducts in peripheral blood white cells significantly correlating with the overall survival (OS) and tumor recurrence-free survival (RFS) of primary hepatocellular carcinoma (PHCC). Cumulative hazard function was plotted by Kaplan-Meier's methodology, and P value was calculated with two-sided log-rank tests. The relative hazard ratio (HR) values for genotypes were calculated using multivariable cox regression models (with all significant variables) based on forward-step method with likelihood ratio test. LAL, low AFB1-DNA adduct level; MAL, medium low AFB1-DNA adduct level; HAL, high low AFB1-DNA adduct level.*

**111**

*The Blood AFB1-DNA Adduct Acting as a Biomarker for Predicting the Risk and Prognosis…*

survival analyses first tested the association between blood AFB1-DNA adducts and patients' OS and results displayed that increasing level of adducts signifi-

Results from multivariate Cox's regression models further exhibited that these cases with an increasing level of blood AFB1-DNA adducts faced an increasing risk of death [HRs (95% CIs) = 1.44 (1.11–1.86) for MAL and 1.93 (1.47–2.54) for HAL, respectively] (**Figure 1**, left). For RFS, the corresponding tumor-recurrence risk was 1.49 (1.18–1.89) for MAL and 2.98 (1.93–4.60) for HAL, respectively

In this study, we explored the relationship between the blood AFB1-DNA adducts and the risk and prognosis of PPHCC. We found that individuals with an increasing level of AFB1-DNA adducts in peripheral blood white cells would feature higher PHCC risk (OR = 1.82 for MAL and 3.82 for HAL, respectively). Furthermore, the blood AFB1-DNA adduct levels were significantly associated with

AFB1 acts as a major cause of PHCC in the southeast areas of China and is taken into human bodies through its contaminating staple foods [2]. AFB1 is transferred into AFB1-DNA adducts and displays its genic toxicity and hepato carcinogenicity [3, 19]. Mechanically, PHCC induced by AFB1 is mainly concerned with DNA damage (including DNA single-/double-strand breaks, base damage, adduct formation, genic mutation), the dysregulation of DNA repair, the activation of cancer genes (such as ras and myc), the inactivation of cancer suppressor genes (such as TP53, BP1, H2AX, bcl2, p21, and p27), inheritance alterations, and/or abnormal immunoreaction [1, 20–25]. Among these knowledge mechanisms and pathways, AFB1-DNA adducts and mutations at codon 249 of TP53 gene (also termed as hot-spot mutation induced by AFB1) have been especially concerned in the past decades [26–29]. This is mainly because AFB1-DNA adducts are the key central forms in the metabolism of AFB1 in human bodies [19, 26, 30], whereas spot mutations at codon 249 of TP53 gene are highly frequent in HCC patients with AFB1 exposure [31–36]. Evidence from clinical epidemiology and experimental animal models has exhibited that they are constantly tested in biopsy samples, such as liver tissues, tumor tissues, placenta tissues, and blood cells, of individuals from AFB1 exposure areas

For example, Hsieh and Hsieh [8] examined the amounts of AFB1-DNA adducts in the 120 placenta tissue samples from women in Taipei, a high AFB1 exposure area, and observed that 57.5% (69/120) of samples were positive AFB1- DNA adducts with the range of 0.6 and 6.3 μmol/mol DNA. Furthermore, they found higher amount of AFB1-DNA adducts in samples collected in the summer than in the winter. Shirabe et al. [37] investigated the association between AFB1- DNA adducts in hepatocyte nuclei and TP53 mutation in PHCC among Japanese population. They found that 6% (118/279) patients with PHCC and 16% (13/83) patients with HBV- and HCV-negative PHCC were positive for AFB1-DNA adducts. Higher hot-spot mutations in the TP53 gene were also found in these with positive AFB1-DNA adduct status [37]. A relatively large-size sample clinical study, including 501 PHCC cases with different AFB1 exposure, also shows that positive status of AFB1-DNA adducts in the tumor tissues significantly increases the risk of TP53 mutations (OR = 3.38 and 95% CI = 2.23–5.11) [7].

) (**Figure 1**, left). Similar

) (**Figure 1**, right).

*DOI: http://dx.doi.org/10.5772/intechopen.88666*

poor OS and RFS of patients with PHCC.

(**Figure 1**, right).

**4. Discussion**

[6, 8, 11, 12, 15–17].

cantly shorten the OS time of patients (*P* = 1.33 × 10<sup>−</sup><sup>5</sup>

effects were also found in the RFS analyses (*P* = 2.88 × 10<sup>−</sup><sup>7</sup>

*The Blood AFB1-DNA Adduct Acting as a Biomarker for Predicting the Risk and Prognosis… DOI: http://dx.doi.org/10.5772/intechopen.88666*

survival analyses first tested the association between blood AFB1-DNA adducts and patients' OS and results displayed that increasing level of adducts significantly shorten the OS time of patients (*P* = 1.33 × 10<sup>−</sup><sup>5</sup> ) (**Figure 1**, left). Similar effects were also found in the RFS analyses (*P* = 2.88 × 10<sup>−</sup><sup>7</sup> ) (**Figure 1**, right). Results from multivariate Cox's regression models further exhibited that these cases with an increasing level of blood AFB1-DNA adducts faced an increasing risk of death [HRs (95% CIs) = 1.44 (1.11–1.86) for MAL and 1.93 (1.47–2.54) for HAL, respectively] (**Figure 1**, left). For RFS, the corresponding tumor-recurrence risk was 1.49 (1.18–1.89) for MAL and 2.98 (1.93–4.60) for HAL, respectively (**Figure 1**, right).

#### **4. Discussion**

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

*AFB1, aflatoxin B1; PHCC, primary hepatocellular carcinoma.*

*Associations between AFB1-DNA adduct levels and PHCC risk.*

**3.2 Blood AFB1-DNA adducts correlating with PHCC risk**

risk role of blood AFB1-DNA adducts in the carcinogenesis of PHCC.

**3.3 Blood AFB1-DNA adducts correlating with PHCC outcome**

The amount of AFB1-DNA adducts in the peripheral white blood cells were calculated using ELISA technique. Compared to controls, patients with PHCC featured a higher level of blood AFB1-DNA adducts (0.98 ± 0.63 vs. 2.01 ± 0.71 μmol/DNA), suggesting blood AFB1-DNA adducts may play an important role in the PHCC carcinogenesis. To investigate possible correlation between AFB1-DNA adducts and PHCC risk, the levels of blood AFB1-DNA adducts were divided into three groups. Results from multivariable logistic regression analyses showed that these individuals with medium AFB1-DNA adduct level (MAL) had an increasing risk of PHCC compared to those with low AFB1-DNA adduct level (LAL) (OR = 1.82 and 95% CI = 1.34–2.48), whereas risk value for high AFB1-DNA adduct level (HAL) was 3.82 (2.71–5.40) (**Table 2**). Altogether, these data were indicative of important potential

**AFB1-DNA adduct levels Controls PHCCs OR (95% CI)a** *P* **n % n %** Low 316 53.7 122 32.1 1 — Medium 186 31.6 131 34.5 1.82 (1.34–2.48) 1.20 × 10<sup>−</sup><sup>4</sup> High 86 14.6 127 33.4 3.82 (2.71–5.40) 2.35 × 10<sup>−</sup><sup>14</sup>

To explore the effects of blood AFB1-DNA adducts on the prognosis of patients

with PHCC, we accomplished two survival model analyses. Kaplan-Meier's

*The aflatoxin B1 (AFB1)-DNA adducts in peripheral blood white cells significantly correlating with the overall survival (OS) and tumor recurrence-free survival (RFS) of primary hepatocellular carcinoma (PHCC). Cumulative hazard function was plotted by Kaplan-Meier's methodology, and P value was calculated with two-sided log-rank tests. The relative hazard ratio (HR) values for genotypes were calculated using multivariable cox regression models (with all significant variables) based on forward-step method with likelihood ratio test. LAL, low AFB1-DNA adduct level; MAL, medium low AFB1-DNA adduct level; HAL,* 

**110**

**Figure 1.**

*high low AFB1-DNA adduct level.*

*a*

**Table 2.**

*OR conditional on matched set.*

In this study, we explored the relationship between the blood AFB1-DNA adducts and the risk and prognosis of PPHCC. We found that individuals with an increasing level of AFB1-DNA adducts in peripheral blood white cells would feature higher PHCC risk (OR = 1.82 for MAL and 3.82 for HAL, respectively). Furthermore, the blood AFB1-DNA adduct levels were significantly associated with poor OS and RFS of patients with PHCC.

AFB1 acts as a major cause of PHCC in the southeast areas of China and is taken into human bodies through its contaminating staple foods [2]. AFB1 is transferred into AFB1-DNA adducts and displays its genic toxicity and hepato carcinogenicity [3, 19]. Mechanically, PHCC induced by AFB1 is mainly concerned with DNA damage (including DNA single-/double-strand breaks, base damage, adduct formation, genic mutation), the dysregulation of DNA repair, the activation of cancer genes (such as ras and myc), the inactivation of cancer suppressor genes (such as TP53, BP1, H2AX, bcl2, p21, and p27), inheritance alterations, and/or abnormal immunoreaction [1, 20–25]. Among these knowledge mechanisms and pathways, AFB1-DNA adducts and mutations at codon 249 of TP53 gene (also termed as hot-spot mutation induced by AFB1) have been especially concerned in the past decades [26–29]. This is mainly because AFB1-DNA adducts are the key central forms in the metabolism of AFB1 in human bodies [19, 26, 30], whereas spot mutations at codon 249 of TP53 gene are highly frequent in HCC patients with AFB1 exposure [31–36]. Evidence from clinical epidemiology and experimental animal models has exhibited that they are constantly tested in biopsy samples, such as liver tissues, tumor tissues, placenta tissues, and blood cells, of individuals from AFB1 exposure areas [6, 8, 11, 12, 15–17].

For example, Hsieh and Hsieh [8] examined the amounts of AFB1-DNA adducts in the 120 placenta tissue samples from women in Taipei, a high AFB1 exposure area, and observed that 57.5% (69/120) of samples were positive AFB1- DNA adducts with the range of 0.6 and 6.3 μmol/mol DNA. Furthermore, they found higher amount of AFB1-DNA adducts in samples collected in the summer than in the winter. Shirabe et al. [37] investigated the association between AFB1- DNA adducts in hepatocyte nuclei and TP53 mutation in PHCC among Japanese population. They found that 6% (118/279) patients with PHCC and 16% (13/83) patients with HBV- and HCV-negative PHCC were positive for AFB1-DNA adducts. Higher hot-spot mutations in the TP53 gene were also found in these with positive AFB1-DNA adduct status [37]. A relatively large-size sample clinical study, including 501 PHCC cases with different AFB1 exposure, also shows that positive status of AFB1-DNA adducts in the tumor tissues significantly increases the risk of TP53 mutations (OR = 3.38 and 95% CI = 2.23–5.11) [7].

Following epidemiological studies on based clinical samples further prove that the amount of AFB1-DNA adducts is higher in the tumor tissues than in the peritumor tissues [6]. This increasing tissular AFB1-DNA adducts are significantly associated with poor OS and RFS of patients with PHCC [6].

In this study, we designed and finished a hospital-based case-control study in the southwestern of Guangxi, a knowledge-high AFB1 exposure area. Our data exhibited that increasing the amount of AFB1-DNA adducts in peripheral white blood cells not only increased PHCC risk, but also modified the OS and RFS of patients with PHCC. Supporting our findings through several studies from high AFB1 exposure areas, the amount of blood AFB1-DNA adducts can reflect the levels of AFB1 exposure information and may be related to PHCC risk and prognosis [11, 12, 14, 15, 17, 38]. Taken together, these results suggest that AFB1-DNA adducts in the blood as well as in the tumor tissues may be potential biomarkers for PHCC risk and outcome.

This study has several strengthens. We accomplished the predictive value analyses using these individuals only with AFB1 exposure but without HBV or HCV. This is done mainly because both HBV and HCV infection will alter effects of AFB1-DNA adducts predicting the risk and outcome of PHCC. Additionally, to control potential confounders such as age, gender, and race, the individually matched design was finished in this study. Therefore, our study may represent a relatively more actual predictive role of blood AFB1-DNA adducts.

To conclude, this study explored the association between blood AFB1-DNA adducts and the risk and prognosis of PHCC using a retrospective clinic-sample research approach and displayed that blood AFB1-DNA adduct may be a potential biomarker for HCC risk and outcome. Several limitations should be focused for our study. First, relatively small-size samples may underestimate the effects of blood AFB1-DNA adducts on PHCC risk and outcome. Second, selective bias may happen because of this hospital-based retrospective investigative design. Finally, the mechanical analyses for AFB1-DNA adducts predicting PHCC risk and prognosis were not finished. Thus, the blood AFB1-DNA adducts may be valuable biomarkers for predicting the risk and prognosis of PHCC once the present findings were proved by larger clinic samples and functional analyses.
