Abstract
We previously reported that global DNA hypomethylation, measured as Sat2 methylation in white blood cells (WBC), and aflatoxin B1 (AFB1) exposure were associated with increased hepatocellular carcinoma risk. In this study, we assessed the association between AFB1 exposure and global DNA methylation. We measured LINE-1 and Sat2 methylation in WBC DNA samples from 1140 cancer free participants of the Cancer Screening Program (CSP) cohort. Blood and urine samples were used to determine the level of AFB1-albumin (AFB1-Alb) adducts and urinary AFB1 metabolites. In continuous models, we found reverse associations of urinary AFB1 with LINE-1 and Sat2 methylation. The odds ratio (OR) per 1 unit decrease were 1.12 (95%CI = 1.03–1.22) for LINE-1 and 1.48 (95%CI = 1.10–2.00) for Sat2 methylation. When compared with subjects in the highest quartile of LINE-1, we found that individuals in the 2nd and 3rd quartiles were less likely to have detectable AFB1-Alb adducts, with ORs (95%CI) of 0.61 (0.40–0.93), 0.61 (0.40-.94), and 1.09 (0.69–1.72), respectively. The OR for detectable AFB1-Alb was 1.81 (95%CI = 1.15–2.85) for subjects in the lowest quartile of Sat2 methylation. The OR for detection of urinary AFB1 for those with LINE-1 methylation in the lowest quartile compared with those in the highest quartile was 1.87 (95%CI = 1.15–3.04). The corresponding OR was 1.75 (95%CI = 1.08–2.82) for subjects in the lowest quartile of Sat2 methylation. The association between AFB1 exposure and global DNA methylation may have implications for the epigenetic effect of AFB1 on hepatocellular carcinoma development and also suggests that changes in DNA methylation may represent an epigenetic biomarker of dietary AFB1 exposure.
Introduction
Aflatoxins are naturally occurring mycotoxins produced by only a few Aspergillus species of which A. flavus and A. parasiticus are the most important; they live in hot and humid conditions. Aflatoxins commonly contaminate foods such as peanuts, grain and corn. Aflatoxin B1 (AFB1), the most potent hepatocarcinogen, can induce principally G→T mutations, including a p53 codon 249 hotspot mutation.Citation1-Citation3 Once ingested, AFB1 is metabolized by the cytochrome P-450 system at the 8,9-vinyl bond to produce an unstable reactive intermediate, AFB1-8,9-epoxide.Citation4 This intermediate can bind covalently to DNA,Citation5 forming AFB1-guanine adducts, and to protein, forming AFB1-albumin and other protein adducts.Citation6,Citation7 Much epidemiological evidence suggests that dietary exposure to AFB1 is an important contributor to the high incidence of hepatocellular carcinoma (HCC) in Eastern and South-Eastern Asia, including Taiwan (reviewed in ref. Citation8). We previously applied two biological markers of AFB1 exposure, AFB1-albumin (AFB1-Alb) adducts and urinary AFB1 metabolites, to understand the role of AFB1 exposure on risk of HCC.Citation9-Citation12 Using data on the analyses of urine and albumin samples banked up to 12 y before diagnosis in the Cancer Screening Program (CSP) in Taiwan, our results suggested that high dietary exposed to AFB1 was associated with 1.5–1.8 times the risk of HCC compared with low exposure.Citation12 Other prospective studies also showed strong associations between biological markers of AFB1 exposure in serum or urine and risk of subsequent HCC.Citation13-Citation16
DNA methylation may play an important role in causing disease by silencing genes through hypermethylation or activating genes through hypomethylation.Citation17 In addition to gene- specific DNA methylation, lower genome-wide aberrant DNA methylation (also referred to as global methylation) in regions that are normally methylated, such as repeats or transposable elements, can lead to genomic instability and altered gene transcription, impacting normal growth and development.Citation18 DNA methylation typically occurs in CpG dinucleotides and up to 80% of CpG dinucleotides occurs in repetitive sequences. There are different types of repetitive sequences scattered throughout the genome (e.g., satellite repeats, SINE [short interspersed nuclear element], and LINES [long interspersed nuclear elements]).Citation19 Changes in DNA methylation patterns are one of the most common molecular alterations in HCC tumor tissues.Citation20-Citation27 Global hypomethylation is associated with genomic instability and an increased number of mutational events (reviewed in ref. Citation28). We recently conducted a prospective case-control study nested within the CSP cohort where there is a high incidence of HCC and high AFB1 exposure, and found that global hypomethylation in baseline white blood cell (WBC) DNA was significantly associated with increased HCC risk later in life.Citation29 Whether dietary exposure to AFB1 is associated with WBC DNA methylation change has not been evaluated. Using biospecimens and data from 1140 cancer free participants in the CSP cohort, we tested the hypothesis that AFB1 exposure is associated with a global decrease in methylation level in WBC DNA.
Results
shows the levels of LINE-1 and Sat2 methylation by subjects’ characteristics. Males had statistically significant lower Sat2 methylation compared with females (4.6 ± 0.6 vs 4.8 ± 0.5 for logSat2; p = 0.0002). There was no difference in LINE-1 and Sat2 methylation in relation with smoking, alcohol or anti-HCV status. Individuals positive for HBsAg showed statistically higher Sat2 methylation than negative individuals (4.7 ± 0.6 vs 4.6 ± 0.6 for logSat2, p = 0.001).
Table 1. Mean levels of LINE-1 and Sat2 by subjects’ characteristics
shows the distributions of biomarkers of AFB1 by subject’s characteristics. Both older age and being positive for HBsAg were associated with high AFB1-Alb adducts but not urinary AFB1. Male gender and anti-HCV status were associated with both biomarkers of AFB1. Level of urinary AFB1 was associated with smoking and alcohol drinking.
Table 2. Distribution of subject’s characteristics by levels of biomarkers of AFB1 exposure
The odds ratios (ORs) from logistic regression models of the association between DNA methylation and detection of AFB1-Alb adducts are presented in . We did not find any association between detection of AFB1-Alb adducts and LINE-1 or Sat2 methylation as continuous variables. When LINE-1 methylation levels were stratified into quartiles based on all values, we observed a U shaped association with AFB1-Alb adducts; the adjusted ORs for detection of AFB1-Alb adducts were 0.61 (95%CI = 0.40–0.93), 0.61 (95% CI = 0.40–0.94) and 1.09 (95%CI = 0.69–1.72) for subjects with adducts in the 3rd, 2nd, and 1st quartile of LINE-1, respectively, compared with those in the highest quartile. The ORs were 1.15 (95% CI = 0.76–1.76), 1.03 (95% CI = 0.68–1.56) and 1.81 (95% CI = 1.15–2.85) for subjects in the 3rd, 2nd and lowest quartile of Sat2 methylation, respectively, compared with subjects in the highest.
Table 3. The association of global DNA methylation with detection of AFB1-albumin adducts
The association of DNA methylation with detection of urinary AFB1 is given in . We observed evidence of a reverse association between urinary AFB1 and methylation in LINE-1 and Sat2. The association using a continuous measure was statistically significant (OR per 1 unit decrease in LINE-1 methylation = 1.12, 95% CI = 1.03–1.22). When LINE-1 methylation was modeled in quartiles, we found individuals with detectable urinary AFB1 were more likely to have lower levels of LINE-1 methylation. The adjusted OR for those in the second and lowest quartile of LINE-1 methylation compared with those in the highest quartile were 1.70 (95%CI = 1.07–2.69) and 1.87 (95%CI = 1.15–3.04), respectively. We also found an association between Sat2 methylation and detection of urinary AFB1 as a continuous or categorical variable; the OR per 1 unit decrease in natural log Sat2 methylation was 1.48 (95%CI = 1.10–2.00). The ORs were 1.33 (0.85–2.07), 1.26 (0.80–1.97) and 1.75 (1.08–2.82) for those in the 3rd, 2nd, and lowest quartiles, compared with subjects in the highest quartile.
Table 4. The association of global DNA methylation with of detection of urinary AFB1 metabolites
Discussion
We previously reported that global DNA hypomethylation in WBC DNA was significantly associated with increased HCC risk later in life.Citation29 In this study, we found that decreased LINE-1 or Sat2 methylation was associated with dietary exposed to AFB1. Those in the higher quartiles of AFB1-Alb adducts had lower LINE-1 and Sat2 methylation than those in the first quartile. Compared with individuals with undetectable AFB1 biomarkers, those with detectable AFB1-Alb adducts or urinary AFB1 metabolites were more likely to be in the lowest quartile of LINE-1 or Sat2 methylation. Our results provide data in humans supporting the hypothesis that exposure to AFB1 contributes to decreased global DNA methylation and that AFB1 may play a role in HCC by altering global DNA methylation status.
Dietary exposure to AFB1 is one of the major risk factors for HCC in regions of high incidence. Despite the fact that most prospective studies, including ours, report strong associations between biological markers of AFB1 exposure in serum or urine and risk of subsequent HCC (reviewed inCitation8), the underlying mechanisms leading to development of HCC are not fully understood. The biotransformation of AFB1 to the highly reactive exo-8,9-epoxide is the most well-known toxic and carcinogenic pathway.Citation30 In addition to the formation of adducts, it is believed that AFB1 acts as a carcinogen by mechanisms that include the formation of reactive oxygen species leading to increased hepatic oxidative damage.Citation31,Citation32 In our previous studies, we found that AFB1 exposure was positively associated with level of oxidative DNA damage in humans as measured by urinary 8-oxo-7,8-dihydro-guanine (8-oxoG)Citation33 as well as urinary 15-F2t-isoprostanes, a marker of lipid peroxidation.Citation34 Oxidative DNA damage such as 8-oxoG can interfere with the ability of methyltransferases to interact with DNA,Citation35,Citation36 thus resulting in global decreased DNA methylation. Long-term exposure to oxidative stress has been shown to increase oxidative damage to methylated cytosine residues and depletion in the level of 5-methylcytosine in repeated elements.Citation36,Citation37
The effects of changes in epigenetic marks can be as profound as DNA sequence mutations and may increase susceptibility to disease including cancer.Citation38 Previous studies indicated that environmental exposures may not cause immediate effects, but lead to changes in the epigenome that “prime” an individual for later pathogenesis,Citation39 i.e., increased individual susceptibility. Thus, epigenomic alteration provides a molecular mechanism for the long-term effects of carcinogenic exposures on cancer risks. Animal and a few human studies have demonstrated that dietary factors are one of the most important factors modifying DNA methylation resulting in phenotype changes.Citation40-Citation43 This evidence, including ours, supports the hypothesis that certain dietary factors may contribute directly to “methylation equilibrium status” by preventing or encouraging either promoter hyper or global hypomethylation and thus have long-term effects on cancer development.Citation44,Citation45
Significant geographic variation in the methylation status of various CpG islands was observed when the methylation status of 12 CpG islands in 85 HCC tumors from various geographic locations was investigated,Citation46 indicating environmental factors may induce DNA methylation aberrations in HCC tumors. We previously measured promoter methylation in candidate genes in 83 HCC tumors and examined its relationship to AFB1 exposure.Citation21-Citation24 We found hypermethylation in promoter regions of tumor suppressor genes including p16INK4A and RASSF1A, correlated with AFB1-DNA adducts in liver tissues and AFB1-Alb adducts.Citation21-Citation24 The association of RASSF1A methylation and AFB1-DNA adducts was also reported in another study.Citation47 These data also suggest that exposure to dietary AFB1 may alter methylation status of key genes involved in development of HCC.
In agreement with our previous study,Citation29 we found individuals positive for HBsAg had statistically significant higher Sat2 methylation, compared with those negative for HBsAg and individuals negative for HBsAg and with detectable AFB1 exposure had the lowest Sat2 methylation compared with other groups. An in vitro study demonstrated that exposure to HBV leads to upregulation of host DNA methyltransferase genes, resulting in increased methylation in host and viral DNA.Citation48 To better understand the role of HBV infection and the combined effect of HBV with AFB1 exposure on HCC risk, it is important to characterize genome-wide DNA methylation among individuals with HBV infection alone and individuals with both HBV infection and AFB1 exposure.
Men have a higher incidence of HCC than women (reviewed in ref. Citation49). Many studies have found that LINE-1 methylation was higher in males than in females (reviewed in ref. Citation50). We found males had lower Sat2 methylation than females. The conflicting data might be due to differences in the distribution of other risk factors including diet that differ by gender in the Taiwanese population. In our present study population, males had higher value of urinary AFB1 metabolites (60.5 ± 44.9 fmol/mL for males verses 38.9 ± 43.1 for females, p < 0.0001). In general, AFB1-Alb adducts have been recognized as long-term markers of AFB1 exposure. Because albumin adducts are as long lived as albumin, which has a half-life of 21 d in humans, they provide information on accumulated exposure over a period of 2 to 3 mo.Citation7,Citation51 The long-term stability of AFB1-Alb adducts has been confirmed,Citation52 as well as their significant correlation with AFB1 intake.Citation53 Measurement of the adduct levels in urine, however, provides a noninvasive means of estimating the levels of AFB1 exposure, with a correlation of 0.65 found between total dietary AFB1 intake and urinary AFB1 extraction.Citation54 We previously reported that there is a weak linear correlation between levels of AFB1-Alb adducts and urinary AFB1 metabolites.Citation12 Measuring AFB1-Alb adducts in blood collected at 2 visits on average of 1.6 y apart, we previously found substantial intra-individual variability in AFB1 exposure in Taiwan, which was likely due to dietary influences.Citation55
Our finding of an association of biomarkers of AFB1 with global DNA hypomethylation in the WBC DNA must be interpreted with caution. First, the putative causal role of AFB1 exposure in global decreased DNA methylation could not be verified. Biomarkers of AFB1 and of global DNA methylation were only measured at baseline, making temporal separation of cause and effect difficult. In addition, we previously reported that changes in WBC DNA methylation over time are highly associated with baseline values of the assay and vary by assay type.Citation56 A longitudinal rather than a cross-sectional study should be conducted to ascertain the causal association between AFB1 exposure and global DNA hypomethylation. Nevertheless, the association indicates the presence of AFB1 biomarkers in persons with lower levels of DNA methylation. Further investigations, incorporating prospective and dietary intervention studies, are required to confirm AFB1-related HCC via altering DNA methylation status. Second, studies have shown that global DNA methylation differs by blood cell type.Citation57,Citation58 However, we measured LINE-1 and Sat2 by MethyLight in 48 women with DNA available from four different sources including white blood cells, granulocytes, mononuclear cells and lymphoblastoid cell lines. We found methylation levels were not significantly different among four cell types.Citation59 It is unclear if the associations we found in the present study were due to the differences in the distributions of cell types among people with different AFB1 exposure. Currently only one animal study showed percentage changes in T cells and B cells with aflatoxin dosing.Citation60 Studies are needed to show that AFB1 exposure does not change blood cell type human populations. Finally, although we observed a significant association of global DNA hypomethylation with AFB1 exposure, the magnitude of change in the level of DNA methylation is small and the biological significance is unclear.
Despite these limitations, this study has numerous strengths. First, we applied antibody-based methods for measurement of AFB1 exposure including AFB1-albumin and urinary AFB1 metabolites, which provide more accurate assessment of AFB1 exposure at the individual level than food questionnaires. Second, this study design allowed us to investigate the associations of methylation levels of different types of DNA repetitive elements with AFB1 exposure in a population with high AFB1 exposure and high prevalence of HCC, providing potential biological mechanisms for the process of hepatocarcinogenesis.
In summary, we found an inverse association between biomarkers of AFB1 and biomarkers of global DNA methylation. These results strongly suggest that AFB1 exposure may result in a decrease in DNA methylation globally. Our results provide information on the application of biomarkers in human populations at high-risk for cancer and that AFB1-induced global DNA hypomethylation may, in addition to the formation of AFB1-DNA adducts, have an important role in AFB1 carcinogenicity.
Materials and Methods
Study cohort
Subjects are from the CSP cohort recruited in Taiwan. This study was approved by Columbia University’s Institutional Review Board as well as the Research Ethics Committee of the College of Public Health, National Taiwan University. Written informed consent was obtained from all subjects and strict quality controls and safeguards were used to protect confidentiality. The cohort characteristics have been described in detail previously.Citation9,Citation12,Citation29,Citation33,Citation34 Briefly, this cohort was originally set up for the evaluation of cancer screening efficacy. Study subjects were voluntary participants in a free cancer screening program implemented in seven urban townships in Taiwan. Individuals who were between 30 and 65 y old were recruited between July 1990 and June 1992 with a total of 12,020 males and 11,923 females. All participants were administrated a structured questionnaire to obtain epidemiological information on socio-demographic characteristics, habits of alcohol intake and cigarette smoking, health history and family history of cancer. Habitual cigarette smoking was defined as having smoked > 4 d/week for at least six months. Information about duration and intensity was also obtained. Habitual alcohol intake was defined as drinking alcohol containing products > 4 d/week for at least six months. For each subject, 15 ml of blood and spot urine were collected, and transported on dry ice to a central laboratory at the National Taiwan University. WBC, plasma and red blood cells were separated and all biospecimens were stored at -70°C. Hepatitis B virus surface antigen (HBsAg), was tested by radioimmunoassay (Abbott Laboratories); antibody against hepatitis C virus (anti-HCV) was tested by enzyme immunoassay using commercial kits (Abbott Laboratories).
Currently, there is passive follow up of the cohort. New cases are detected by computerized data linkage with profiles on the national cancer registry and death certification systems in Taiwan. The overall follow-up rate is 98%. Through June 2008, 305 cases were identified. We randomly selected controls from cohort subjects who were not affected with HCC through the follow-up period by matching to each case by age (± 5 y), gender, residential township and date of recruitment (± 3 mo). We applied a nested case-control study design to examine the etiology of AFB1 exposure by measuring biomarker of AFB1 on HCC risk.Citation12 Baseline WBCs and urine were shipped to Columbia University on dry ice for determination of biomarkers of AFB1 exposure and global DNA methylation.
Study subjects
For this study, we used information from 1140 cancer free participants in the CSP cohort for whom we previously measured biomarkers of AFB1 exposure including AFB1-Alb adducts and AFB1 metabolitesCitation9,Citation12 and biomarkers of global DNA methylation including LINE-1 and Sat2 in baseline samples.Citation29
AFB1-albumin adducts in blood and AFB1 metabolites in urine
Plasma samples were assayed for albumin adducts by enzyme-linked immunosorbent assay (ELISA) as previously described.Citation9,Citation12 Briefly, 50 µl of albumin extracts, equivalent to 200 µg albumin, were added to plates previously coated with 3 ng of AFB1 epoxide-modified human serum albumin (HSA). Polyclonal antiserum #7 was used at 1:2x105 dilution and the secondary antiserum, goat anti-rabbit IgG-alkaline phosphatase conjugate, was used at 1:750 dilution. Concentrations of AFB1-Alb adducts were determined using a standard curve of serially diluted AFB1 epoxide-modified HSA that had been enzymatically digested. Samples with less than 20% inhibition were considered undetectable and assigned a value of 0.01 fmol/µg. Two controls samples were analyzed with each batch of test samples, a pooled sample of plasma from non-smoking US subjects and a positive control of serum from a rat treated with 1.5 mg AFB1. The coefficient of variation (CV) of the mean of the two controls was 20% (n = 13).
Urines were assayed essentially as described previously.Citation9,Citation12 Seppak urine extracts (50 µl) were added to plates previously coated with 3 ng of AFB1 epoxide-modified bovine serum albumin. AF8E11 was used at 1:1500 dilution and the secondary antiserum, goat anti-mouse alkaline phosphatase was used at 1:1000 dilution. AF8E11 mainly reacts with AFB1, but there is significant cross-reactivity with a number of aflatoxin derivatives, including AFB2, AFM1, AFG1, and AFP1Citation61. Concentrations of urinary metabolites were determined using a standard curve of serially diluted AFB1. Samples with less than 20% inhibition were considered undetectable and assigned a value of 1 fmol/ml. A pooled sample of urine from 5 controls was used as a quality control and analyzed with each batch of test samples. The CV was 10% (n = 5).
DNA extraction and bisulfite treatment
Genomic DNA was extracted from WBCs by a salting out procedure. Cells were lysed with SDS in a nuclei lysis buffer and treated with RNase A (final 133 µg/mL) and RNase T1 (final 20 units/mL) to remove RNA. Proteins were co-precipitated with NaCl (330 µL of saturated NaCl added per 1mL solution) by centrifugation. Genomic DNA was recovered from the supernatant by precipitation with 100% ethanol, washed in 70% ethanol, and dissolved in the Tris-EDTA buffer.
Aliquots of DNA (500ng) were bisulfite-treated with the EZ DNA methylation kit (Zymo Research). The DNA was resuspended in 20 µL of distilled water and stored at -20°C until use.
MethyLight assay
We used the sequences of probe and forward and reverse primers of Sat2-M1 described in Weisenberger et al.Citation62 PCR was performed in a 10 µl reaction volume with 0.3 µM forward and reverse PCR primers, 0.1 µL probe, 3.5 µM MgCl2, using the following PCR program: 95°C for 10 min, then 55 cycles of 95°C for 15 sec, followed by 60°C for 1 min. Assays were run on an ABI Prism 7900 Sequence Detection System (LifeTechnologies). The MethyLight data were expressed as percent of methylated reference (PMR) values and are the mean of duplicates.
PMR = 100% * 2 exp -{Delta Ct (target gene in sample - control gene in sample) - Delta Ct (100% methylated target in reference sample - control gene in reference sample)} Ten percent of subjects were assayed in duplicate, and the inter assay CV for the Sat2 MethyLight assay was 1.0%.
Pyrosequencing assay
Pyrosequencing for LINE-1 methylation levels was performed using PCR and sequencing primers as previously described, with minor modifications to the original protocol.Citation29,Citation63 Briefly, PCR was performed in a 25 µL reaction mix containing 50 ng bisulfite-converted DNA, 1× Pyromark PCR Master Mix (Qiagen), 1× Coral Load Concentrate (Qiagen), and 0.2 µM forward and reverse primers, using the following PCR program: 95°C for 15 min, then 44 cycles of 95°C for 30 sec followed by 56°C for 30 sec and 72°C for 30 sec, with a final extension at 72°C for 10 min. Following amplification, the biotinylated PCR products were purified and incubated with the sequencing primer designed to bind adjacent to the CpG sites of interest. Pyrosequencing was conducted using a PyroMark Q24 instrument (Qiagen), with subsequent quantitation of methylation levels determined with the PyroMark Q24 1.010 software. Percent methylation within a sample was subsequently determined by averaging across all three interrogated CpG sites in the analysis. Ten percent of subjects were assayed in duplicate, and the inter assay CV for the LINE-1 pyrosequencing assay was 0.5%.
The laboratory investigator who performed all assays was blinded to epidemiologic data.
Statistical methods
We used one-way ANOVA to test if there is any difference in methylation among groups and performed the Tukey Studentized Range comparisons with Bonferroni correction to determine which groups are different. Because Sat2 methylation was not normally distributed, all statistical analyses used data after natural log transformation. To estimate associations with AFB1 exposure, we used logistic regression models to calculate odds ratios (ORs) and 95% confidence intervals (Cls). We modeled the associations adjusting for age (years, continuous), sex, HBsAg (Yes vs. No), Anti-HCV (Yes vs. No), smoking (Ever vs. Never), and alcohol consumption (Ever vs. Never) in models. Markers of global DNA methylation were assessed both as continuous measures and as quartiles, based on the distribution among all subjects to accommodate possible nonlinear associations. All analyses were performed with SAS software 9.0 (SAS Institute). All statistical tests were based on two-tailed probability.
| Abbreviations: | ||
| 8-oxodG | = | 8-oxodeoxyguanosine |
| AFB1 | = | aflatoxin B1 |
| Anti-HCV | = | antibodies to hepatitis C virus |
| BMI | = | body mass index |
| CSP | = | cancer screening program |
| ELISA | = | enzyme-linked immunosorbent assay |
| HBsAg | = | hepatitis B virus surface antigen |
| HBV | = | hepatitis B virus |
| HCC | = | hepatocellular carcinoma |
| WBC | = | white blood cell |
Acknowledgments
This work was supported by National Institutes of Health grants RO1ES005116, P30ES009089, and P30CA013696
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
References
- Bressac B, Kew M, Wands J, Ozturk M. Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature 1991; 350:429 - 31; http://dx.doi.org/10.1038/350429a0; PMID: 1672732
- Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC. Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature 1991; 350:427 - 8; http://dx.doi.org/10.1038/350427a0; PMID: 1849234
- Lunn RM, Zhang Y-J, Wang L-Y, Chen CJ, Lee PH, Lee CS, et al. p53 mutations, chronic hepatitis B virus infection, and aflatoxin exposure in hepatocellular carcinoma in Taiwan. Cancer Res 1997; 57:3471 - 7; PMID: 9270015
- Forrester LM, Neal GE, Judah DJ, Glancey MJ, Wolf CR. Evidence for involvement of multiple forms of cytochrome P-450 in aflatoxin B1 metabolism in human liver. Proc Natl Acad Sci U S A 1990; 87:8306 - 10; http://dx.doi.org/10.1073/pnas.87.21.8306; PMID: 2122459
- Essigmann JM, Croy RG, Nadzan AM, Busby WF Jr., Reinhold VN, Büchi G, et al. Structural identification of the major DNA adduct formed by aflatoxin B1 in vitro. Proc Natl Acad Sci U S A 1977; 74:1870 - 4; http://dx.doi.org/10.1073/pnas.74.5.1870; PMID: 266709
- Gallagher EP, Wienkers LC, Stapleton PL, Kunze KL, Eaton DL. Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1. Cancer Res 1994; 54:101 - 8; PMID: 8261428
- Sabbioni G, Skipper PL, Büchi G, Tannenbaum SR. Isolation and characterization of the major serum albumin adduct formed by aflatoxin B1 in vivo in rats. Carcinogenesis 1987; 8:819 - 24; http://dx.doi.org/10.1093/carcin/8.6.819; PMID: 3111739
- Wu HC, Santella R. The role of aflatoxins in hepatocellular carcinoma. Hepat Mon 2012; 12:10 HCC e7238; http://dx.doi.org/10.5812/hepatmon.7238; PMID: 23162603
- Wang LY, Hatch MC, Chen CJ, Levin B, You SL, Lu SN, et al. Aflatoxin exposure and risk of hepatocellular carcinoma in Taiwan. Int J Cancer 1996; 67:620 - 5; http://dx.doi.org/10.1002/(SICI)1097-0215(19960904)67:5<620::AID-IJC5>3.0.CO;2-W; PMID: 8782648
- Chen CJ, Wang LY, Lu SN, Wu MH, You SL, Zhang YJ, et al. Elevated aflatoxin exposure and increased risk of hepatocellular carcinoma. Hepatology 1996; 24:38 - 42; http://dx.doi.org/10.1002/hep.510240108; PMID: 8707279
- Sun Z, Lu P, Gail MH, Pee D, Zhang Q, Ming L, et al. Increased risk of hepatocellular carcinoma in male hepatitis B surface antigen carriers with chronic hepatitis who have detectable urinary aflatoxin metabolite M1. Hepatology 1999; 30:379 - 83; http://dx.doi.org/10.1002/hep.510300204; PMID: 10421643
- Wu H-C, Wang Q, Yang H-I, Ahsan H, Tsai WY, Wang LY, et al. Aflatoxin B1 exposure, hepatitis B virus infection, and hepatocellular carcinoma in Taiwan. Cancer Epidemiol Biomarkers Prev 2009; 18:846 - 53; http://dx.doi.org/10.1158/1055-9965.EPI-08-0697; PMID: 19273485
- Ross RK, Yuan JM, Yu MC, Wogan GN, Qian GS, Tu JT, et al. Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma. Lancet 1992; 339:943 - 6; http://dx.doi.org/10.1016/0140-6736(92)91528-G; PMID: 1348796
- Qian GS, Ross RK, Yu MC, Yuan JM, Gao YT, Henderson BE, et al. A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People’s Republic of China. Cancer Epidemiol Biomarkers Prev 1994; 3:3 - 10; PMID: 8118382
- Chen CJ, Yu MW, Liaw YF, Wang LW, Chiamprasert S, Matin F, et al. Chronic hepatitis B carriers with null genotypes of glutathione S-transferase M1 and T1 polymorphisms who are exposed to aflatoxin are at increased risk of hepatocellular carcinoma. Am J Hum Genet 1996; 59:128 - 34; PMID: 8659516
- Yu M-W, Lien J-P, Chiu Y-H, Santella RM, Liaw Y-F, Chen C-J. Effect of aflatoxin metabolism and DNA adduct formation on hepatocellular carcinoma among chronic hepatitis B carriers in Taiwan. J Hepatol 1997; 27:320 - 30; http://dx.doi.org/10.1016/S0168-8278(97)80178-X; PMID: 9288607
- Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002; 3:415 - 28; PMID: 12042769
- Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene 2002; 21:5400 - 13; http://dx.doi.org/10.1038/sj.onc.1205651; PMID: 12154403
- Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al, International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 2001; 409:860 - 921; http://dx.doi.org/10.1038/35057062; PMID: 11237011
- Shen J, Wang S, Zhang Y-J, Kappil M, Wu HC, Kibriya MG, et al. Genome-wide DNA methylation profiles in hepatocellular carcinoma. Hepatology 2012; 55:1799 - 808; http://dx.doi.org/10.1002/hep.25569; PMID: 22234943
- Zhang Y-J, Ahsan H, Chen Y, Lunn RM, Wang LY, Chen SY, et al. High frequency of promoter hypermethylation of RASSF1A and p16 and its relationship to aflatoxin B1-DNA adduct levels in human hepatocellular carcinoma. Mol Carcinog 2002; 35:85 - 92; http://dx.doi.org/10.1002/mc.10076; PMID: 12325038
- Zhang Y-J, Rossner P Jr., Chen Y, Agrawal M, Wang Q, Wang L, et al. Aflatoxin B1 and polycyclic aromatic hydrocarbon adducts, p53 mutations and p16 methylation in liver tissue and plasma of hepatocellular carcinoma patients. Int J Cancer 2006; 119:985 - 91; http://dx.doi.org/10.1002/ijc.21699; PMID: 16570275
- Zhang YJ, Chen Y, Ahsan H, Lunn RM, Chen SY, Lee PH, et al. Silencing of glutathione S-transferase P1 by promoter hypermethylation and its relationship to environmental chemical carcinogens in hepatocellular carcinoma. Cancer Lett 2005; 221:135 - 43; http://dx.doi.org/10.1016/j.canlet.2004.08.028; PMID: 15808399
- Zhang Y-J, Chen Y, Ahsan H, Lunn RM, Lee PH, Chen CJ, et al. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation and its relationship to aflatoxin B1-DNA adducts and p53 mutation in hepatocellular carcinoma. Int J Cancer 2003; 103:440 - 4; http://dx.doi.org/10.1002/ijc.10852; PMID: 12478658
- Shen J, Wang S, Zhang YJ, Wu HC, Kibriya MG, Jasmine F, et al. Exploring genome-wide DNA methylation profiles altered in hepatocellular carcinoma using Infinium HumanMethylation 450 BeadChips. Epigenetics 2013; 8:34 - 43; http://dx.doi.org/10.4161/epi.23062; PMID: 23208076
- Lambert M-P, Paliwal A, Vaissière T, Chemin I, Zoulim F, Tommasino M, et al. Aberrant DNA methylation distinguishes hepatocellular carcinoma associated with HBV and HCV infection and alcohol intake. J Hepatol 2011; 54:705 - 15; http://dx.doi.org/10.1016/j.jhep.2010.07.027; PMID: 21146512
- Hernandez-Vargas H, Lambert MP, Le Calvez-Kelm F, Gouysse G, McKay-Chopin S, Tavtigian SV, et al. Hepatocellular carcinoma displays distinct DNA methylation signatures with potential as clinical predictors. PLoS One 2010; 5:e9749; http://dx.doi.org/10.1371/journal.pone.0009749; PMID: 20305825
- Wilson AS, Power BE, Molloy PL. DNA hypomethylation and human diseases. Biochim Biophys Acta 2007; 1775:138 - 62; PMID: 17045745
- Wu H-C, Wang Q, Yang H-I, Tsai W-Y, Chen C-J, Santella RM. Global DNA methylation levels in white blood cells as a biomarker for hepatocellular carcinoma risk: a nested case-control study. Carcinogenesis 2012; 33:1340 - 5; http://dx.doi.org/10.1093/carcin/bgs160; PMID: 22581841
- Bailey EA, Iyer RS, Stone MP, Harris TM, Essigmann JM. Mutational properties of the primary aflatoxin B1-DNA adduct. Proc Natl Acad Sci U S A 1996; 93:1535 - 9; http://dx.doi.org/10.1073/pnas.93.4.1535; PMID: 8643667
- Shen H-M, Ong C-N, Lee B-L, Shi C-Y. Aflatoxin B1-induced 8-hydroxydeoxyguanosine formation in rat hepatic DNA. Carcinogenesis 1995; 16:419 - 22; http://dx.doi.org/10.1093/carcin/16.2.419; PMID: 7859375
- Shen H-M, Shi C-Y, Shen Y, Ong C-N. Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B1. Free Radic Biol Med 1996; 21:139 - 46; http://dx.doi.org/10.1016/0891-5849(96)00019-6; PMID: 8818628
- Wu H-C, Wang Q, Wang L-W, Yang HI, Ahsan H, Tsai WY, et al. Urinary 8-oxodeoxyguanosine, aflatoxin B1 exposure and hepatitis B virus infection and hepatocellular carcinoma in Taiwan. Carcinogenesis 2007; 28:995 - 9; http://dx.doi.org/10.1093/carcin/bgl234; PMID: 17127712
- Wu H-C, Wang Q, Yang H-I, Ahsan H, Tsai WY, Wang LY, et al. Urinary 15-F2t-isoprostane, aflatoxin B1 exposure and hepatitis B virus infection and hepatocellular carcinoma in Taiwan. Carcinogenesis 2008; 29:971 - 6; http://dx.doi.org/10.1093/carcin/bgn057; PMID: 18310087
- Turk PW, Laayoun A, Smith SS, Weitzman SA. DNA adduct 8-hydroxyl-2′-deoxyguanosine (8-hydroxyguanine) affects function of human DNA methyltransferase. Carcinogenesis 1995; 16:1253 - 5; http://dx.doi.org/10.1093/carcin/16.5.1253; PMID: 7767994
- Valinluck V, Tsai H-H, Rogstad DK, Burdzy A, Bird A, Sowers LC. Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acids Res 2004; 32:4100 - 8; http://dx.doi.org/10.1093/nar/gkh739; PMID: 15302911
- Pogribny IP, Tryndyak VP, Woods CG, Witt SE, Rusyn I. Epigenetic effects of the continuous exposure to peroxisome proliferator WY-14,643 in mouse liver are dependent upon peroxisome proliferator activated receptor α. Mutat Res 2007; 625:62 - 71; http://dx.doi.org/10.1016/j.mrfmmm.2007.05.004; PMID: 17586532
- Jones PA, Baylin SB. The epigenomics of cancer. Cell 2007; 128:683 - 92; http://dx.doi.org/10.1016/j.cell.2007.01.029; PMID: 17320506
- Lahiri DK. An integrated approach to genome studies. Science 2011; 331:147; http://dx.doi.org/10.1126/science.331.6014.147-a; PMID: 21233370
- Cooney CA, Dave AA, Wolff GL. Maternal methyl supplements in mice affect epigenetic variation and DNA methylation of offspring. J Nutr 2002; 132:Suppl 2393S - 400S; PMID: 12163699
- Jousse C, Parry L, Lambert-Langlais S, Maurin AC, Averous J, Bruhat A, et al. Perinatal undernutrition affects the methylation and expression of the leptin gene in adults: implication for the understanding of metabolic syndrome. FASEB J 2011; 25:3271 - 8; http://dx.doi.org/10.1096/fj.11-181792; PMID: 21670064
- Tobi EW, Lumey LH, Talens RP, Kremer D, Putter H, Stein AD, et al. DNA methylation differences after exposure to prenatal famine are common and timing- and sex-specific. Hum Mol Genet 2009; 18:4046 - 53; http://dx.doi.org/10.1093/hmg/ddp353; PMID: 19656776
- Heijmans BT, Tobi EW, Stein AD, Putter H, Blauw GJ, Susser ES, et al. Persistent epigenetic differences associated with prenatal exposure to famine in humans. Proc Natl Acad Sci U S A 2008; 105:17046 - 9; http://dx.doi.org/10.1073/pnas.0806560105; PMID: 18955703
- Arasaradnam RP, Commane DM, Bradburn D, Mathers JC. A review of dietary factors and its influence on DNA methylation in colorectal carcinogenesis. Epigenetics 2008; 3:193 - 8; http://dx.doi.org/10.4161/epi.3.4.6508; PMID: 18682688
- Mathers JC. Pulses and carcinogenesis: potential for the prevention of colon, breast and other cancers. Br J Nutr 2002; 88:Suppl 3 S273 - 9; http://dx.doi.org/10.1079/BJN2002717; PMID: 12498627
- Shen L, Ahuja N, Shen Y, Habib NA, Toyota M, Rashid A, et al. DNA methylation and environmental exposures in human hepatocellular carcinoma. J Natl Cancer Inst 2002; 94:755 - 61; http://dx.doi.org/10.1093/jnci/94.10.755; PMID: 12011226
- Feng Y, Xue W, Li P, et al. RASSF1A Hypermethylation is Associated with Aflatioxin B1 and Polycyclic Aromatic Hydrocarbon Exposure in Hepatocellular Carcinoma. Hepatogastroenterology 2011; 59:118 - 9
- Vivekanandan P, Daniel HD-J, Kannangai R, Martinez-Murillo F, Torbenson M, Hepatitis B. Hepatitis B virus replication induces methylation of both host and viral DNA. J Virol 2010; 84:4321 - 9; http://dx.doi.org/10.1128/JVI.02280-09; PMID: 20147412
- Yeh SH, Chen PJ. Gender disparity of hepatocellular carcinoma: the roles of sex hormones. Oncology 2010; 78:Suppl 1 172 - 9; http://dx.doi.org/10.1159/000315247; PMID: 20616601
- Terry MB, Delgado-Cruzata L, Vin-Raviv N, Wu HC, Santella RM. DNA methylation in white blood cells: association with risk factors in epidemiologic studies. Epigenetics 2011; 6:828 - 37; http://dx.doi.org/10.4161/epi.6.7.16500; PMID: 21636973
- Makarananda K, Pengpan U, Srisakulthong M, Yoovathaworn K, Sriwatanakul K. Monitoring of aflatoxin exposure by biomarkers. J Toxicol Sci 1998; 23:Suppl 2 155 - 9; http://dx.doi.org/10.2131/jts.23.SupplementII_155; PMID: 9760454
- Scholl PF, Groopman JD. Long-term stability of human aflatoxin B1 albumin adducts assessed by isotope dilution mass spectrometry and high-performance liquid chromatography-fluorescence. Cancer Epidemiol Biomarkers Prev 2008; 17:1436 - 9; http://dx.doi.org/10.1158/1055-9965.EPI-07-2926; PMID: 18559559
- Gan L-S, Skipper PL, Peng XC, Groopman JD, Chen JS, Wogan GN, et al. Serum albumin adducts in the molecular epidemiology of aflatoxin carcinogenesis: correlation with aflatoxin B1 intake and urinary excretion of aflatoxin M1. Carcinogenesis 1988; 9:1323 - 5; http://dx.doi.org/10.1093/carcin/9.7.1323; PMID: 3133131
- Zhu JQ, Zhang LS, Hu X, Xiao Y, Chen JS, Xu YC, et al. Correlation of dietary aflatoxin B1 levels with excretion of aflatoxin M1 in human urine. Cancer Res 1987; 47:1848 - 52; PMID: 3102051
- Ahsan H, Wang L-Y, Chen C-J, Tsai W-Y, Santella RM. Variability in aflatoxin-albumin adduct levels and effects of hepatitis B and C virus infection and glutathione S-transferase M1 and T1 genotype. Environ Health Perspect 2001; 109:833 - 7; http://dx.doi.org/10.1289/ehp.01109833; PMID: 11564620
- Wu H-C, Wang Q, Delgado-Cruzata L, Santella RM, Terry MB. Genomic methylation changes over time in peripheral blood mononuclear cell DNA: differences by assay type and baseline values. Cancer Epidemiol Biomarkers Prev 2012; 21:1314 - 8; http://dx.doi.org/10.1158/1055-9965.EPI-12-0300; PMID: 22665578
- Reinius LE, Acevedo N, Joerink M, Pershagen G, Dahlén SE, Greco D, et al. Differential DNA methylation in purified human blood cells: implications for cell lineage and studies on disease susceptibility. PLoS One 2012; 7:e41361; http://dx.doi.org/10.1371/journal.pone.0041361; PMID: 22848472
- Deaton AM, Webb S, Kerr ARW, Illingworth RS, Guy J, Andrews R, et al. Cell type-specific DNA methylation at intragenic CpG islands in the immune system. Genome Res 2011; 21:1074 - 86; http://dx.doi.org/10.1101/gr.118703.110; PMID: 21628449
- Wu H-C, Delgado-Cruzata L, Flom JD, Kappil M, Ferris JS, Liao Y, et al. Global methylation profiles in DNA from different blood cell types. Epigenetics 2011; 6:76 - 85; http://dx.doi.org/10.4161/epi.6.1.13391; PMID: 20890131
- Hinton DM, Myers MJ, Raybourne RA, Francke-Carroll S, Sotomayor RE, Shaddock J, et al. Immunotoxicity of aflatoxin B1 in rats: effects on lymphocytes and the inflammatory response in a chronic intermittent dosing study. Toxicol Sci 2003; 73:362 - 77; http://dx.doi.org/10.1093/toxsci/kfg074; PMID: 12700391
- Hatch MC, Chen C-J, Levin B, Ji BT, Yang GY, Hsu SW, et al. Urinary aflatoxin levels, hepatitis-B virus infection and hepatocellular carcinoma in Taiwan. Int J Cancer 1993; 54:931 - 4; http://dx.doi.org/10.1002/ijc.2910540611; PMID: 8392983
- Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E, et al. Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res 2005; 33:6823 - 36; http://dx.doi.org/10.1093/nar/gki987; PMID: 16326863
- Yang AS, Estécio MRH, Doshi K, Kondo Y, Tajara EH, Issa JPJ. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 2004; 32:e38; http://dx.doi.org/10.1093/nar/gnh032; PMID: 14973332