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Carcinogenesis Advance Access originally published online on August 27, 2007
Carcinogenesis 2007 28(11):2347-2354; doi:10.1093/carcin/bgm193
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Evaluation of oxidative stress in a group of adolescents exposed to a high level of aflatoxin B1—a multi-center and multi-biomarker study

Tao Peng1,2,{dagger}, Le-Qun Li1, Min-Hao Peng1, Zhi-Ming Liu1, Tang-Wei Liu1, Ya Guo1, Kai-Yin Xiao1, Zhong Qin1, Xin-Ping Ye1, Xin-Shao Mo1, Lu-Nan Yan3, Bee-Lam Lee4, Han-Ming Shen4, Kazuyoshi Tamae5, Lian Wen Wang2, Qiao Wang2, Khalid M. Khan2, Kai-Bo Wang6, Ren-Xiang Liang6, Zong-Liang Wei6, Hiroshi Kasai5,{dagger}, Choon Nam Ong4,{dagger} and Regina M. Santella2,{dagger},*

1 Department of Hepatobiliary Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Province, China
2 Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY 10032, USA
3 Department of General Surgery, West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, China
4 Department of Community, Occupational and Family Medicine, National University of Singapore, Singapore 119260, Singapore
5 Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan
6 Fusui Cancer Institute, Fusui County 530021, Guangxi Province, China

* To whom correspondence should be addressed. Tel: 212 305 1996; Fax: 212 305 5328; Email: rps1{at}columbia.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
The association between aflatoxin B1 (AFB1) exposure and oxidative stress was extensively examined in 84 adolescents from an area at high risk for hepatocellular carcinoma in China. Plasma level of aflatoxin B1–albumin adducts (AAAs) was associated with AFB1 excretion in urine (r = 0.394, P < 0.001). Urinary AFB1 was also associated with both the urinary excretion of 8-hydroxydeoxyguanosine (8-OHdG) (r ≥ 0.479, P < 0.001) and 8-OHdG and hOGG1 levels in peripheral leukocytes (r ≥ 0.308, P ≤ 0.005). Similarly, AAA was significantly associated with both the urinary excretion of 8-OHdG (r ≥ 0.259, P ≤ 0.018) and the 8-OHdG and hOGG1 levels in peripheral leukocytes (r ≥ 0.313, P ≤ 0.004). In addition, urinary 8-OHdG was correlated with both the level of DNA 8-OHdG (r ≥ 0.24, P ≤ 0.05) and the expression of hOGG1 in peripheral leukocytes (r ≥ 0.429, P < 0.001). Protein carbonyl content (PCC) level was significantly associated with not only the level of DNA 8-OHdG (r ≥ 0.366, P < 0.001) and the urinary 8-OHdG (r ≥ 0.258, P ≤ 0.018) but also the expression of hOGG1 in peripheral leukocytes (r = 0.485, P < 0.001). A significant but weak association was found between high-performance liquid chromatograph–electrochemical detection (HPLC–ECD) and enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG (r = 0.334, P = 0.002) and between HPLC–ECD and flow cytometry assays for 8-OHdG in leucocytes (r = 0.395, P < 0.001). Significant associations were observed between AAA and PCC and liver function indices (alanine aminotransferase and aspartate aminotransferase). These findings suggest significant contribution from AFB1 exposure to oxidative stress and subsequent repair among adolescents that may impose substantial risk for hepatocarcinogenesis in adulthood in this region.

Abbreviations: AAA, aflatoxin B1–albumin adduct; AFB1, aflatoxin B1; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BSA, body surface area; BW, body weight; ELISA, enzyme-linked immunosorbent assay; FCM, flow cytometry; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HPLC–ECD, high-performance liquid chromatograph–electrochemical detection; HV, hepatitis virus; 8-OHdG, 8-hydroxydeoxyguanosine; PCC, protein carbonyl content; ROS, reactive oxygen species; TP, total protein; WBC, white blood cell


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a leading cause of death in many countries, especially in East Asia and sub-Saharan Africa (1). It is also increasing in morbidity in some developed countries among younger generations (2). The broad characteristics of HCC can be partially explained by the patterns of exposure to the key risk factors in different regions (3). In Asia and Africa where the majority of cases live, aflatoxin and hepatitis viruses (HVs) [hepatitis B virus (HBV) and HCV] are important factors giving rise to the extraordinarily high incidence rates (24.2–35.5/100 000) of HCC in these areas (3,4). Aflatoxins are a group of structurally diverse metabolites of the common fungi Aspergillus flavus and Aspergillus parasiticus. Among them, aflatoxin B1 (AFB1) attracts most concern as a health risk factor because of its wide distribution and high concentration in human and animal foods in HCC endemic areas, its high potency as a human and animal hepatotoxin and hepatocarcinogen and its strong implication in the etiology of human HCC in endemic areas (5). AFB1 produces chromosomal aberrations, micronuclei, sister chromatid exchanges, unscheduled DNA synthesis and chromosomal strand breaks, as well as DNA-adduct formation in rodent and human cells (6).

The role of reactive oxygen species (ROS) has been postulated in the development of aging, chronic degenerative diseases, inflammatory diseases and cancers (7). There is evidence that oxidative stress is involved in AFB1-relevant hepatocarcinogenesis. For example, it has been noted that there is free radical generation during AFB1 metabolism (8), and oxidative damage is one type of damage caused by AFB1 in human lymphocytes (9). An elevated level of ROS was induced by AFB1 in rat hepatocytes (10). Lipid peroxidation (11) and 8-hydroxydeoxyguanosine (8-OHdG) (12) formation were also observed in rat liver after AFB1 administration, with time- and dose-dependent responses of 8-OHdG (11,12). However, evidence in humans is rare and complicated by the coexistence of HV, chemical and physical carcinogens and many other physiologic/pathologic conditions contributing to the generation of ROS (13).

We found in our pilot study (14) an association between AFB1 exposure and oxidative protein damage in a Chinese population at high risk for HCC (15). This, together with the finding of Wild et al. that high exposure to aflatoxins in utero and continuing throughout childhood in West Africa children critically impairs their growth and development (16), raises the possibility that aflatoxin-related oxidative damage to macromolecules in adolescents might ultimately increase risk for hepatocarcinogenesis in adulthood. To test this hypothesis, substantial effort in recent years has been made in this multi-center study to examine oxidative stress status using multiple biomarkers in a group of HBV-free adolescents from an area with known exposure to high levels of AFB1 and also at high risk for HCC.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Sample collection
Study subjects came from Guangxi Zhuang Autonomous Region of People's Republic of China , a region of elevated HCC risk and AFB1 contamination (15,17). Details of subjects' recruitment have been described elsewhere (18,19). In brief, after screening for HV infection, 21 HV (–) adolescents from Fusui County and 63 age- (±3 years) and gender-matched and HV (–) subjects from Nanning city were recruited into this study (mean ± SD, 11.45 ± 3.0 years, male:female = 56:28). Prior epidemiologic studies determined that the HCC mortality for males was 92–97/100 000 in Fusui and 32–47/100 000 in Nanning. The aims of the study were explained in detail and written consent was obtained from the parents. Overnight urine and 10 ml venous fasting blood (with heparin as anticoagulant) was collected in the morning. Buffy coat and plasma were separated in the field after blood collection, kept together with urine in liquid nitrogen during transportation and stored at –80°C till analysis. Body weight (BW), height, age, gender, occupation and family history of hepatitis infections were also recorded using a structured questionnaire approved by the Guangxi Medical University. Subjects were screened for HV (HBV, HCV, HDV, HEV and HGV) infections and liver biochemistry [alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alkaline phosphatase, r-glutamyl transpeptidase, 5'-nucleotidase, albumin (ALB), globulin, total bilirubin, direct bilirubin, indirect bilirubin and bile acid levels as well as plasma total protein (TP)] as described previously (14,18). Cases positive for HV markers were excluded in order to rule out the possible contribution of ROS from infection (20). Furthermore, subjects with known exposure to known environmental or occupational hazards (such as cigarette smoking, alcohol or pesticides), as well as those under medication or known to have a chronic illness, were also excluded. There were 84 adolescents who were born and grew up in this region, met the above-mentioned criteria and were selected for this investigation. All adolescent samples were collected within 1 month during May and June.

Biomarker levels in plasma (AAA, PCC, WBC-8-OHdG-HPLC, WBC-8-OHdG-FCM and WBC-hOGG1-FCM)
Aflatoxin B1–albumin adducts (AAAs) and protein carbonyl content (PCC) levels in plasma were quantified by ELISA as described previously (14,21). Values were normalized to the amount of ALB for AAA (fmol/mg ALB) and TP for PCC (nmol/mg TP), and then by ALB and TP concentration in plasma (fmol/ml plasma for AAA and nmol/ml plasma for PCC). 8-OHdG, the most studied DNA oxidation product (22), in peripheral white blood cells (WBCs) was quantified by high-performance liquid chromatograph–electrochemical detection (HPLC–ECD) as described previously (12). WBC-8-OHdG-HPLC values were expressed as per 105 dG. Using a flow cytometry (FCM) assay as described previously (19), we simultaneously determined the leukocyte DNA 8-OHdG and expression levels of hOGG1, a DNA glycosylase/ß-lyase that recognizes 8-OHdG opposite cytosine (23) and a key component of the base excision repair pathway (24). The values were reported as percentage of positive-staining cells (WBC-8-OHdGP-FCM and WBC-hOGG1P-FCM) and mean of relative fluorescence intensity (relative fluorescence intensity for WBC-8-OHdGI-FCM and WBC-hOGG1I-FCM) of 10 000 cells counted by the flow cytometer. Since both dosimetries gave similar results in the statistic analysis, only the latter were presented in this report.

Biomarker levels in urine (urinary AFB1 and urinary 8-OHdG)
Urinary AFB1 levels were quantified by ELISA as described previously (25), and expressed as fmol/ml urine and further converted to fmol/mg urinary creatinine. Urinary 8-OHdG was determined by HPLC–ECD and ELISA methods in two laboratories by two investigators (K.T. and K.M.K.) independently. For the ELISA assay, crude urine was applied directly to ELISA without immunoaffinity purification (26). Protocols had been described elsewhere (22,26). Both results were expressed as ng/ml urine and further converted to fmol/mg urinary creatinine. Urinary creatinine was determined in a commercial company as described previously (22).

Statistical analysis
Levels of biomarkers in subjects from Fusui and Nanning were compared by Mann–Whitney U non-parametric test. To obtain a normal distribution and to stabilize the variance, levels of AAA, PCC and liver chemistry indices were log transformed in the linear regression model. Spearman non-parametric correlation was used to test the association between AAA and PCC and the association between PCC and liver chemistry indices in each group. In addition, a partial correlation analysis was used to calculate the strength of the association between AAA and PCC (adjusted for ALB and TP level separately), while controlling for confounding factors [geography location, age, gender, body surface area (BSA), HV, smoking status, alcohol consumption and ethnicity]. Biomarker levels were further adjusted for anthropologic factors by BSA (27–30). BSA was calculated by Dubois–Dubois formula: S = 71.74BW(0.425)Body-Height(0.725), where surface is in square centimeter, weight (BW) in kilogram and length (Body-Height) in centimeter (31). All analyses were performed using the SPSS 10.0 program (Chicago, IL). A two-tailed P value < 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Comparison of biomarker levels between adolescents from Fusui and Nanning
There was no significant difference in age, BW , body mass index and BSA between Fusui and Nanning adolescents. But adolescents from Fusui had significantly higher levels of AAA and PCC in plasma and hOGG1 expression in WBCs than those from Nanning (Mann–Whitney U non-parametric test, P < 0.01). However, neither the level of 8-OHdG staining (FCM) nor 8-OHdG content in WBCs DNA (HPLC) was statistically different between the two populations (P > 0.05) (Figure 1).


Figure 1
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  		Fig. 1. Comparison of AFB1 and oxidative biomarkers' levels between adolescents from Fusui and Nanning. Mann–Whitney U non-parametric test; values are expressed as mean ± SD; only significant P values are marked. AAA (fmol/ml plasma/m2 BSA); urine AFB1 (fmol/mg creatinine/m2 BSA); PCC (nmol/ml plasma/m2 BSA); WBC-8-OHdG-HPLC (per 105 dG/m2 BSA); WBC-8-OHdGI-FCM (+RFI/m2 BSA); WBC-hOGG1I-FCM (+RFI/m2 BSA); urine 8-OHdG-HPLC (ng/mg creatinine/m2 BSA); urinary 8-OHdG-ELISA (ng/mg creatinine/m2 BSA). RFI: relative fluorescence intensity.

 
Association among biomarkers in blood
Consistent with our finding in the parent study that included 404 adults from the same areas, a significant association was found between AAA and PCC (Table I) (14). Furthermore, when all markers were adjusted for BSA, there were positive associations among the levels of AAA, PCC, WBC 8-OHdG and hOGG1 expression, as well as agreement on 8-OHdG by the HPLC and FCM assays.


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  		Table I. Associations between AFB1 and oxidative biomarkers in blood in 84 adolescents from Guangxi, southern China

 
Association among biomarkers in urine
There were significant correlations between urinary AFB1 (fmol/ml urine) and urinary 8-OHdG by HPLC (ng/ml urine) (r = 0.495, P < 0.001) and by ELISA (ng/ml urine) (r = 0.479, P < 0.001). However, a 10- to 15-fold difference in urinary 8-OHdG levels was observed when measured by HPLC versus ELISA.

Association between blood and urinary biomarkers
Plasma AAA level was associated with urinary excretion of AFB1 (r = 0.394, P < 0.001). Protein oxidative damage in blood (PCC) is also proportional to relatively recent AFB1 exposure (urinary AFB1) (r = 0.395, P < 0.001). Levels of the more chronic AFB1 exposure biomarker (AAA) are associated with the urinary excretion of 8-OHdG (r = 0.376, P < 0.001, by HPLC–ECD and r = 0.259, P = 0.018, by ELISA). Further, protein damage levels in blood (PCC) and 8-OHdG excretion in urine are significantly associated (r = 0.390, P < 0.001, by HPLC–ECD and r = 0.258, P = 0.018, by ELISA). With regard to the associations between levels of DNA damage biomarkers (8-OHdG) in leukocytes (WBC-8-OHdG-HPLC/FCM) and in urine (urinary 8-OHdG-HPLC/ELISA), an association was also observed (Table II, r > 0.241, P < 0.05). Finally, in view of DNA damage repair in WBCs and 8-OHdG excretion in urine, there were significant associations between WBC-hOGG1I-FCM and urinary 8-OHdG by HPLC and ELISA (r = 0.440 for HPLC and r = 0.429 for ELISA, P < 0.001) (Table II).


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  		Table II. Associations between urinary and blood biomarkers in 84 adolescents from Guangxi, southern China

 
Association between biomarkers and liver function indices
Consistent with our finding in the study of adults, significant associations were observed between AAA and PCC and liver function indices (Table III) (18). However, the associations between urinary biomarkers and liver function indices were not significant. One and 10 children had abnormal ALT and AST levels, respectively. There was a significant difference in PCC levels between the children with normal versus abnormal AST (t = 2.346, P = 0.021) but the differences for AAA and urinary AFB1 were not significant.


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  		Table III. Spearman's association between biomarkers and liver function indices in 84 adolescents from Guangxi, southern China

 
Consistency between biomarkers' assays
There were significant yet weak correlations between WBC 8-OHdG-HPLC and 8-OHdG-FCM (r = 0.395, P < 0.001) and between urinary 8-OHdG-HPLC and 8-OHdG-ELISA (r = 0.334, P = 0.002). For the urinary assays, however, the ELISA value (mean ± SD) of urinary 8-OHdG (83.4 ± 62.8 ng/mg creatinine) was ~16-fold higher than that by HPLC (5.26 ± 3.73 ng/mg creatinine), and overall, the HPLC assay values achieved higher r values for associations with the other blood biomarker values than the ELISA method (Table II).

Multivariate analysis
In a linear regression model, age (ß = –0.199), gender (ß = –0.303) and plasma AAA levels (ß = 0.178) were significant predictors of AFB1 excretion in urine, suggesting that elder and male children excrete less AFB1 in urine than younger, female children in this population. Using oxidative stress biomarkers as biologic end points (dependent variable), several models can be constructed to simulate the causes of oxidative stress as summarized in Table IV. Despite various biomarkers tested, consistent with prior monovariate analysis, age, gender, AAA and urinary AFB1 and hOGG1 expression in leukocytes are significant factors that predict oxidative stress status in this population. The diagram in Figure 2 summarizes the associations among biomarkers of oxidative stress and AFB1 exposure in this population.


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  		Table IV. Linear regression analysis of predictors of oxidative stress in 84 adolescents from Guangxi, China

 


Figure 2
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  		Fig. 2. Diagram of the association profile among biomarkers of oxidative stress and AFB1 exposure. Only significant associations are indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The impact of fetal and infant exposures might be modified by childhood environment and is considered critical for growth and risk of disease in later life (32). In this multi-center study, we found association between exposure to aflatoxin, a potent environmental carcinogen, and various oxidative damage biomarkers among adolescents from an HCC endemic area, providing new evidence for the importance of early life exposures.

AFB1 metabolism/excretion and liver parenchyma injury
AAA has already been proven to be a good indicator of HCC risk (21), and the significant association between AAA and urinary AFB1 we found in this study suggests a link between recent and chronic AFB1 exposure in adolescents. In contrast to AAA that reflects exposure over a period of weeks/months because of the relatively long half-life of ALB (~20 days) in humans (33), urinary excretion of aflatoxin metabolites is very rapid and hence is dependent on the consumption of aflatoxin in the proceeding 24 h (34). The significant association between several measures of liver function and AAA (but not urinary AFB1) suggests the importance of chronic, repeated exposures versus transient, single exposures to this end point. A good correlation between dietary intake of AFB1 and urinary excretion of aflatoxin metabolites and the major aflatoxin–DNA adduct has been observed in people from this region (35,36). Two large-scale cohort studies conducted independently in Taiwan and Shanghai region (25 618 and 18 244 subjects, respectively) have shown that the levels of urinary AFB1 metabolites were associated with HCC risk [OR = 1.3–23.4 (25), RR = 1.0–5.9 (37), respectively]. Another finding in our study is that older and male children tend to excrete less AFB1 in urine than younger, female children. HCC is more common among males (male:female = 4–6:1) with peak age 40–50 years in this area, even though there is similar positive rate of HBV surface antigen in both genders (15,17). It is therefore reasonable to speculate that growth- or gender-related factors might alter AFB1 metabolism and alter risk in a subset of the population. Consistent with our finding in the parent study (14,18), the significant associations between liver function indices and plasma AAA and PCC suggest parenchyma cell damage by AFB1 and/or AFB1-relevant oxidative stress in these children. It is also in agreement with the observation of Wild et al. (38) on AFB1 toxicity among African children aged 3–4 years in which a significant correlation between AAA and ALT (r = 0.4, P < 0.001) was found. Although those with HV infection, known environmental or occupational hazards as well as using various medications had been carefully excluded from the present study, one and 10 children had abnormal ALT and AST levels, respectively. The significant difference of PCC but not AAA or urinary AFB1 levels between the children with normal AST versus abnormal AST suggests that, despite the significant contribution from AFB1 exposure, there may be many other substantial sources of inducers of oxidative stress.

AFB1 and oxidative DNA damage
Given the small number of subjects and the recruitment criteria [HBV (–), exclusion of exposure to environmental and occupational hazards], we did not see significantly higher levels of 8-OHdG in subjects from Fusui than from Nanning, as expected based on HCC mortality. However, we found significant associations between both urinary (indicating relatively recent exposure) and plasma (representing chronic exposure) AFB1 biomarkers and DNA damage biomarkers in both urine and peripheral leukocytes. The excretion rate of 8-OHdG is assumed to represent the rate of repair of oxidative DNA damage throughout the body, and therefore also the rate of damage input (since these are generally in equilibrium) (29). Although debatable, it has been proposed that in addition to the base excision repair pathway, 8-OHdG might be derived from 8-OHdGTP in the cellular pool of DNA precursors, or alternatively, originate in the DNA of dead/apoptotic cells undergoing further oxidation during breakdown and excretion (39,40). In this situation, it would reflect overall oxidative stress, but not directly indicate the input of damage to cellular DNA (29). Our study revealed that oxidative DNA damage levels in this adolescent population are linear with respect to both recent and chronic exposure to environmental carcinogens. However, it is still an open question whether the DNA damage that accumulates throughout childhood might ultimately contribute to adult onset of HCC in this region.

It has been shown that dietary antioxidants or other supplements can modulate levels of AFB1 and oxidative stress biomarkers (41,42). One limitation of the present data is that, we did not analyze dietary antioxidants or other covariants that might alter individual oxidative stress status, although questionnaire data excluded the possibility of green tea consumption and the ~50 km geographic distance between Fusui and Nanning might not result in large differences in vegetable or fruit consumption. It therefore merits further investigation whether these covariants in the diet could also potentially contribute to the association between AFB1 exposure and oxidative stress, justifying a chemoprevention strategy among this population (42).

Since liver is the main target organ of AFB1, one may question why there is an association between AFB1 exposure and oxidative DNA damage in peripheral leukocytes. There may be several explanations. First, it is well recognized that the toxic effect of AFB1 is mainly due to its biotransformation in the liver to a highly reactive intermediate AFB1 8,9-epoxide metabolites (43) by microsomal cytochrome p450s, such as CYP1A2, 3A4 and 3A5. Although it is still unknown whether microsomal cytochrome p450s exert the same role in leukocytes as in liver parenchyma cells, CYP1A2 and CYP3A4 activity and messenger RNA content were measurable in peripheral blood lymphocytes after drug administration (44). The amount of CYP3A4 in human peripheral blood lymphocytes remains an open question (45), but CYP3A5 is capable of catalyzing AFB1 activation (46) and is expressed in peripheral blood cells (47). Both CYP3A5 and CYP3A4 demonstrated comparable capacities for AFB1 bioactivation (48). Second, leukocytes circulating through or released from liver might reflect events happening inside the liver (49). Finally, there is evidence that the short-live electrophiles generated from metabolism in liver cause DNA damage to other organs (50).

AFB1 and oxidative protein damage
PCC (aldehyde or ketone) is a widely used marker for the presence of oxidative stress in physiological and pathological conditions (51). Our study showed, for the first time, a significant association between protein carbonyl levels and aflatoxin exposure, measured as both AAA and urinary AFB1, and between protein damage levels and biochemical alterations in liver, the target organ for aflatoxin biotransformation and its carcinogenic effects. These data suggest that oxidative protein damage in the Guangxi adolescents may arise mainly from aflatoxin exposure. Moreover, the oxidative modification of proteins might lead to accelerated protein degradation and increased catabolism or suppress protein synthesis in the liver among subjects with high levels of oxidative stress (52), thus exposing these adolescents to a particular pathophysiological situation. For example, it has been noted that low doses of AFB1 depress growth and alter many aspects of humoral and cellular immunity in piglets, such as a decrease of the total number of WBCs and complement titers and a decrease of pro-inflammatory Interleukin-1ß (IL-1beta) and Tumour necrosis factor-{alpha} (TNF-alpha) and an increase in anti-inflammatory (IL-10) cytokine messenger RNA expression (53,54). In a cohort of 472 Gambian children (6–9 years), secretory IgA in saliva was markedly lower in children with detectable AAA compared with those with non-detectable levels (55). Further investigation into whether this impaired immunity caused by AFB1 and/or AFB1-relevant oxidative stress could increase susceptibility to HBV infection will help in the understanding of the synergistic effect of HBV and AFB1 in inducing HCC.

Oxidative stress and DNA repair
Consistent with the significantly higher level of plasma AAA and PCC in Fusui, there were significantly higher levels of hOGG1 in leukocytes from children from Fusui than Nanning. The correlation network among AFB1 exposure, both 8-OHdG in urine and leukocyte DNA and protein carbonyl levels in plasma, and hOGG1 expression, suggests that AFB1 exposure may up-regulate DNA repair in this population. This finding supports the idea that normal cells exert their defensive mechanism against ubiquitous oxidative DNA damage (24), and hOGG1 works as a housekeeping gene, ubiquitously expressed during the cell cycle (56). 8-OHdG repair activity was increased in leukocytes of smokers compared with non-smokers (57) and in lung cells exposed to asbestos (58). It has also been suggested that quantitative assessment of hOGG1 expression in peripheral blood cells can provide information on exposure to environmental carcinogens (59). The present study clearly demonstrated that hOGG1 in leukocytes mirrors oxidative status in a population at high risk of HCC, suggesting that hOGG1 could be a useful biomarker for monitoring of oxidative stress, in addition to 8-OHdG detection.

Methodology—consistency between 8-OHdG assays
Leukocyte DNA 8-OHdG (HPLC–ECD versus FCM).
There are inherent difficulties in the measurement of 8-OHdG due to the potential for oxidation of guanosine during sample preparation (60). In the present study, the correlation between HPLC and FCM values was significant but weak (r = 0.395, P < 0.001). 8-OHdG was stained by Biotrin OxyDNA Assay Kit (Fluorescein isothiocyanate-conjugated probe, Biotrin International Ltd, Dublin, Ireland) according to the manufacturer's protocol with minor modification (61). The probe used in the FCM assay has been shown by the manufacturer to cross-react with 8-oxo (8-OH) adenine/adenosine. Thus, the FCM assay might overestimate 8-OHdG levels compared with HPLC. However, there might be leakage of mitochondrial DNA during the FCM staining. Therefore, it is unclear whether the FCM assay over- or underestimates true 8-OHdG levels.

Urinary 8-OHdG (HPLC–ECD versus ELISA).
It has been noted that crude urine samples contain cross-reacting substances, modified forms of 8-OHdG and other structurally related compounds, reacting with the 8-OHdG antibody (62). In the present study, when crude urine was applied directly to ELISA, the r value (0.334) of HPLC versus ELISA was comparable with those found by Prieme et al. (63) and Shimoi et al. (62) (0.42–0.46). In Yoshida et al. (64) and our previous report (26), using two different 8-OHdG antibodies (N45.1 and 1F7) achieved higher correlations (0.87–0.88) between HPLC and ELISA data with a pre-purification step by either HPLC or immunoaffinity, respectively, prior to the ELISA. But the 8-OHdG levels determined by ELISA, after immunoaffinity purification, using 1F7 were ~6-fold higher than those determined by HPLC (26). In the present study, there is also a better correlation with other biomarkers for the HPLC data than the ELISA data. The ease and economy of immuno-based platforms suggests that they may be appropriate in studies comparing relative urinary 8-OHdG values, whereas their weak correlations with HPLC data suggests that HPLC might be more appropriate for absolute quantification or comparisons between different laboratories.

Methodology—urinary creatinine issues
Barr et al. (65) reported that the significant predictors of urinary creatinine concentrations include age, sex, race/ethnicity, body mass index and fat-free mass, and the unintentional adjustment for these covariates when urinary metabolites are expressed per gram creatinine can have profound effects on the interpretation of data. We found strong associations between creatinine (plasma or urine) and biomarkers (data not shown). There were significant correlations between urinary/plasma creatinine and urinary/plasma biomarkers and between urinary/plasma creatinine and anthropologic/liver function indices, suggesting that the turnover of these biomarkers in vivo might associate with the metabolism of creatinine and in consequence affect the interpretation of the relation among these biomarkers if they are adjusted unintentionally by creatinine. For example, the excretion of AFB1 by the kidney may not mimic that of creatinine, which is largely by passive filtration and partly by active secretion. AFB1 is reported to be readily excreted as an urinary metabolite (66), and urinary AFB1 concentrations were 70 times higher than AFB1 concentration in plasma 5 h after gastric instillation of the compound, suggesting an efficient excretion of AFB1 (67). Therefore, in order to explore associations between biomarkers in urine and plasma, in addition to adjustment for urinary creatinine, it might be necessary to introduce anthropologic or physiologic factors to minimize the bias caused by creatinine metabolism. In this case, the biomarkers could be expressed as unit per milligram creatinine per square meter BSA or unit per milligram creatinine per kilogram BW. A similar approach that adjustment (per kilogram BW) to normalize the influence of individual body mass index or BW variations in 24 h urinary excretion of 8-OHdG has been reported (27–30).

There are a number of limitations in this study that must be addressed. First, the present association study was done only on samples collected in a narrow time-slot yet seasonal variation of levels of both plasma AAA (68,69) and urinary 8-OHdG (70) in several populations has been reported. In this study, the significant difference in AAA but not urinary AFB1 levels between the two regions might suggests the influence of time frame of sample collection. It is therefore necessary to follow-up these subjects to test if a fluctuation in oxidative stress biomarkers mimics that of AFB1 biomarkers. Second, a synergistic interaction between AFB1 and HBV in hepatocarcinogenesis has long been noted (71). The exclusion of HBV infectants from this study simplified our analysis, however, we do not know if there is a synergistic effect between AFB1 and HBV in inducing oxidative stress in this population. Third, as addressed above, we did not analyze the dietary antioxidants or other covariants that might alter an individual's oxidative stress status. Despite the above confounding factors, we present here a comprehensive profile of biomarkers of oxidative stress in adolescents exposed to environmental carcinogens, highlighting the significant contribution from aflatoxins to macromolecular damage in this population. The clear association between oxidative biomarkers and exposure to AFB1 in this population suggests the necessity of interventions targeted to younger generations to reduce their exposure as well as to minimize oxidative damage, in order to substantially reduce the risk of HCC in this area.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Nature Science Foundation of China (NSFC 30460143, 30560133); China Postdoctoral Science Foundation (2004035617); Guangxi Nature Sciences (GKJ0229026, 0236030); Guangxi Educational Committee (GZBH 2000-272); Guangxi Health Ministry Medicine (Z200146, 2001087); Singapore Science (R-186-000-044-213); USA National Institutes of Health (ES05116 and ES09089).


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We thank Dr Xue Qin, Dr Shan Li (Laboratory center, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Province, China) and Mr Her Yam Ong (Department of Community, Occupational and Family Medicine, National University of Singapore, Singapore) for laboratory assistance. We thank Dr Cheryl Ann Winkler (Laboratory of Genomic Diversity, National Cancer Institute at Frederick, USA) for critical review of the manuscript.

Conflict of interest statement: None declared.


   References
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 Abstract
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 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received April 25, 2007; revised July 24, 2007; accepted August 7, 2007.


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