Search

During the 60 years since the first scientific reports about a relation between aflatoxin exposure and adverse health consequences, both in animals and humans, there has been a remarkable number of basic, clinical and population science studies characterising the impact of this mycotoxin on diseases such as liver cancer. Many of these human investigations to date have focused on populations residing in Asia and Africa due to the high incidence of liver cancer and high exposures to aflatoxin. These studies formed the basis for the International Agency for Research on Cancer to classify the aflatoxins as Group 1 known human carcinogens. In addition, aflatoxin contamination levels have been used in international commodity trade to set the price of various staples such as maize and groundnuts. While there have been many case-control and prospective cohort studies of liver cancer risk over the years there have been remarkably few investigations focused on liver cancer in Latin America. Our interdisciplinary and multiple institutional collaborative has been developing a long-term strategy to characterise the role of aflatoxin and other mycotoxins as health risk factors in Guatemala and neighbouring countries. This paper summarises a number of the investigations to date and provides a roadmap of our strategies for the near term to discern the emergent aetiology of liver cancer in this region. With these data in hand public health-based prevention strategies could be strategically implemented and conducted to lower the impact of these mycotoxins on human health.

Significance Recently, investigators have shown that only a few driver gene mutational events appear to be needed for cancer to occur. However, the reason that some mutational events precede others in the same cancer and the explanation for tissue-specific differences in this timing, remain mysterious. We here combine mathematical modeling with epidemiologic studies and sequencing data to address these questions. We suggest that the first driver event in cancers generally occurred at early ages and provide estimates for the fitness of different types of drivers during tumor evolution, showing how they vary with the tissue of origin., Cancer is driven by the sequential accumulation of genetic and epigenetic changes in oncogenes and tumor suppressor genes. The timing of these events is not well understood. Moreover, it is currently unknown why the same driver gene change appears as an early event in some cancer types and as a later event, or not at all, in others. These questions have become even more topical with the recent progress brought by genome-wide sequencing studies of cancer. Focusing on mutational events, we provide a mathematical model of the full process of tumor evolution that includes different types of fitness advantages for driver genes and carrying-capacity considerations. The model is able to recapitulate a substantial proportion of the observed cancer incidence in several cancer types (colorectal, pancreatic, and leukemia) and inherited conditions (Lynch and familial adenomatous polyposis), by changing only 2 tissue-specific parameters: the number of stem cells in a tissue and its cell division frequency. The model sheds light on the evolutionary dynamics of cancer by suggesting a generalized early onset of tumorigenesis followed by slow mutational waves, in contrast to previous conclusions. Formulas and estimates are provided for the fitness increases induced by driver mutations, often much larger than previously described, and highly tissue dependent. Our results suggest a mechanistic explanation for why the selective fitness advantage introduced by specific driver genes is tissue dependent.

Objective Persistent organic pollutants (POPs) are lipophilic environmental toxicants that accumulate in adipose tissue. Weight loss leads to mobilization and increased redistribution of these toxicants. Many are obesogens and endocrine disruptors. Increased exposure could pose long-term health risks. The study objective was to measure the changes in serum concentrations of lipophilic POPs during significant weight loss. Methods This study enrolled 27 patients at a university hospital in a longitudinal, 6-month, observational study examining changes in POP blood levels after bariatric surgery. The primary outcome was the changes in the concentrations of 24 polychlorinated biphenyls (PCBs), 9 organochlorine pesticides (OCPs), 11 polybrominated diphenyl ethers, 2,2',4,4',5,5'-hexabromobiphenyl, and 4 perfluorochemicals (PFCs). Results Older adults (those born before 1976) had baseline levels of PCBs, OCPs, and PFCs that were two- to fivefold higher than younger adults (those born after 1976). Older adults had greater increases in PCBs, OCPs, and polybrominated diphenyl ethers associated with weight loss. Conversely, younger adults had greater increases in PFCs associated with weight loss. On average, blood POP levels increased as weight loss occurred. Conclusions Although weight loss is considered beneficial, the release and redistribution of POPs to other lipid-rich organs such as the brain, kidneys, and liver warrant further investigation. Interventions should be considered to limit organ exposure to POPs when weight loss interventions are planned.

Exploration of animal models leads to discoveries that can reveal candidate biomarkers for translation to human populations. Herein, a model of hepatocarcinogenesis and protection was used in which rats treated with aflatoxin (AFB1 ) daily for 28 days (200 µg/kg BW) developed tumors compared with rats completely protected from tumors by concurrent administration of the chemoprotective agent, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Differential expression of miRNAs in tumors (AFB1 ) and nontumor (AFB1 + CDDO-Im) bearing livers and their levels in sera over the life-course of the animals was determined. miRNA transcriptome analysis identified 17 miRNAs significantly upregulated at greater than five-fold in the tumors. The ten most dysregulated miRNAs judged by fold-change and biological significance were selected for further study, including liver-specific miR-122-5p. Validation of sequencing results by real-time PCR confirmed the upregulation of the majority of these miRNAs in tumors, including miR-182, as well as miR-224-5p as the most dysregulated of these miRNAs (over 400-fold). The longitudinal analysis of levels of miR-182 in sera demonstrated significant and persistent increases (5.13-fold; 95% CI: 4.59-5.70). The increase in miR-182 was detected months before any clinical symptoms were present in the animals. By the terminal time point of the study, in addition to elevated levels of serum miR-182, serum miR-122-5p was also found to be increased (>1.5-fold) in animals that developed hepatocarcinomas. Thus, using the data from an unbiased discovery approach of the tissue findings, serum miR-182 was found to track across the complex, multistage process of hepatocarcinogenesis opening an opportunity for translation to human populations.

Background: To evaluate the association of liver fibrosis scores with PSA level among U.S. adult men overall and by race/ethnicity. Methods: Data from the National Health and Nutrition Examination Survey (NHANES), 2001–2010, were used. Males ages ≥40 years without a prostate cancer diagnosis and who had serum PSA, liver enzymes, albumin, and platelet counts measured as part of NHANES protocol were included. Liver fibrosis was measured using three scores: aspartate aminotransferase to platelet ratio index (APRI), fibrosis 4 index (FIB-4), and NAFLD fibrosis score (NFS). We assessed overall and race/ethnicity-stratified geometric mean PSA by fibrosis score using predictive margins by linear regression, and the association of abnormal fibrosis scores (APRI > 1, FIB-4 > 2.67, NFS > 0.676) and elevated PSA (>4 ng/mL) by logistic regression. Results: A total of 6,705 men were included. Abnormal liver fibrosis scores were present in 2.1% (APRI), 3.6% (FIB-4), and 5.6% (NFS). Men with higher fibrosis scores had lower geometric mean PSA (all Ptrend < 0.02). Men with abnormal APRI had a lower odds of PSA > 4 ng/mL [adjusted OR (aOR) = 0.33; 95% confidence interval (CI), 0.11–0.96]. Compared with men with 0 abnormal scores, those with 2 or 3 abnormal fibrosis scores had a lower odds of PSA > 4 ng/mL (aOR = 0.55; 95% CI, 0.33–0.91). The patterns were similar by race/ethnicity. Conclusions: Men of all race/ethnicities with higher liver fibrosis scores had lower serum PSA, and men with advanced fibrosis scores had a lower odds of an elevated PSA. Impact: These findings support further research to inform the likelihood of delay in prostate cancer detection in men with abnormal liver function.

Qidong (Jiangsu, China) has been of interest to cancer epidemiologists and biologists because, until recently, it was an endemic area for liver cancer, having amongst the highest incidence rates in the world. The establishment of the Qidong Cancer Registry together with the Qidong Liver Cancer Institute in 1972 has charted the patterns of liver cancer incidence and mortality in a stable population throughout a period of enormous economic, social, and environmental changes as well as of improvements in health care delivery. Updated incidence trends in Qidong are described. Notably, the China age-standardized incidence rate for liver cancer has dropped by over 50% in the past several decades. Molecular epidemiologic and genomic deep sequencing studies have affirmed that infection with hepatitis B virus as well as dietary exposure to aflatoxins through contamination of dietary staples such as corn, and to microcystins – blue-green algal toxins found in ditch and pond water – were likely important etiologic factors that account for the high incidence of liver cancer in this region. Public health initiatives to facilitate universal vaccination of newborns against HBV and to improve drinking water sources in this rural area, as well as economic and social mandates serendipitously facilitating dietary diversity, have led to precipitous declines in exposures to these etiologic factors, concomitantly driving substantive declines in the liver cancer incidence seen now in Qidong. In this regard, Qidong serves as a template for the global impact that a package of intervention strategies may exert on cancer burden.

Background There are no data on the prevalence of non-alcoholic fatty liver disease (NAFLD) in general population samples in Guatemala or in other Central American countries. The prevalence and distribution of NAFLD and its associated risk factors were evaluated in a population-based sample of adults in Guatemala. Methods Cross-sectional study of 411 men and women 40 years of age or older residing in urban and rural areas of Guatemala. Metabolic outcomes included obesity, central obesity, hypercholesterolemia, diabetes, and metabolic syndrome (MetS). Liver disease outcomes included elevated liver enzymes, elevated Fatty Liver Index (FLI), and elevated FIB-4 score. Results The overall prevalence of obesity, central obesity, diabetes, and MetS were 30.9, 74.3, 21.6, and 64.2%, respectively. The fully-adjusted prevalence ratios (95% CI) for obesity, central obesity, diabetes, and MetS comparing women to men were 2.83 (1.86–4.30), 1.72 (1.46–2.02), 1.18 (1.03–1.34), and 1.87 (1.53–2.29), respectively. The overall prevalence of elevated liver enzymes (ALT or AST), elevated FLI, and elevated FIB-4 scores were 38.4, 60.1, and 4.1%, respectively. The fully-adjusted prevalence ratios (95% CI) for elevated liver enzymes (either ALT or AST) and elevated FLI score comparing women to men were 2.99 (1.84–4.86) and 1.47 (1.18–1.84), respectively. Conclusions The prevalence of metabolic abnormalities and liver outcomes in this general population study was very high. The prevalence of metabolic and liver abnormalities was particularly high among women, an observation that could explain the atypical 1:1 male to female ratio of liver cancer in Guatemala.

Dietary exposure to aflatoxin B(1) (AFB(1)) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB(1) exposure leads to the formation of AFB(1)-N(7)-guanine (AFB(1)-N(7)-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B(1) (AFB(1)-FapyGua) in DNA. These adducts lead to G → T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB(1) as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB(1)-N(7)-Gua and the two diastereomers of AFB(1)-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB(1)-treated mice. Levels varying from 1.5 to 45 lesions/10(6) DNA bases in AFB(1)-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB(1) treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB(1) treatment of mice. On the other hand, significantly increased levels of (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines were observed in liver DNA of AFB(1)-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB(1)-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB(1) exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.

Background and aims: Liver cancer is one of the most dominant malignant tumors in the world. The trends of liver cancer mortality over the past six decades have been tracked in the epidemic region of Qidong, China. Using epidemiological tools, we explore the dynamic changes in age-standardized rates to characterize important aspects of liver cancer etiology and prevention. Methods Mortality data of liver cancer in Qidong from 1958 to 1971 (death retrospective survey) and from 1972 to 2017 (cancer registration) were tabulated for the crude rate (CR), and age-standardized rate and age-birth cohorts. The average annual percentage change was calculated by the Joinpoint Regression Program. Results The natural death rate during 1958–2017 decreased from 9‰ to 5.4‰ and then increased to 8‰ as the population aged; cancer mortality rates rose continuously from 57/105 to 240/105. Liver cancer mortality increased from 20/105 to 80/105, and then dropped to less than 52/105 in 2017. Liver cancer deaths in 1972–2017 accounted for 30.53% of all cancers, with a CR of 60.48/105, age-standardized rate China (ASRC) of 34.78/105, and ASRW (world) of 45.71/105. Other key features were the CR for males and females of 91.86/105 and 29.92/105, respectively, with a sex ratio of 3.07:1. Period analysis showed that the ASRs for mortality of the age groups under 54 years old had a significant decreasing trend. Importantly, birth cohort analysis showed that the mortality rate of liver cancer in 40–44, 35–39, 30–34, 25–29, 20–24, 15–19 years cohort decreased considerably, but the rates in 70–74, and 75+ increased. Conclusions The crude mortality rate of liver cancer in Qidong has experienced trends from lower to higher levels, and from continued increase at a high plateau to most recently a gradual decline, and a change greatest in younger people. Many years of comprehensive prevention and intervention measures have influenced the decline of the liver cancer epidemic in this area. The reduction of intake levels of aflatoxin might be one of the most significant factors as evidenced by the dramatic decline of exposure biomarkers in this population during the past three decades.

BACKGROUND & AIMS This study aimed to analyse the association of sex hormone levels with liver enzyme levels and non-alcoholic fatty liver disease (NAFLD) in a nationally representative sample of men. METHODS 919 men from the US National Health and Nutrition Examination Study (NHANES) III were included in this cross-sectional analysis of data from 1988-1991. We used existing data on serum total and free testosterone, total and free estradiol, androstanediol glucuronide (AAG), sex steroid binding globulin (SHBG), and estimated their associations with aspartate aminotransferase (AST), and alanine aminotransferase (ALT) and NAFLD, as determined using ultrasound, after adjusting for possible confounders including age, race, smoking, alcohol, physical activity, waist circumference and steroid hormones. RESULTS Lower total testosterone and higher free estradiol were associated with higher odds of NAFLD after adjusting for confounders including the other sex hormones. Lower total testosterone was associated with higher odds of elevated AST, but not ALT. Free testosterone, total estradiol, SHBG and AAG were not associated with NAFLD or liver enzymes. CONCLUSIONS This study supports an inverse association between total testosterone concentration and NAFLD in men independent of other sex hormones (SHBG, AAG, estradiol) and known risk factors such as obesity, age and lifestyle. Exploration of whether total testosterone might be a non-invasive marker for NAFLD diagnosis is warranted.

Collectively, liver cancer accounts for 8.2% of all reported cancer deaths in men and women, and it is the third/fourth most common cause of cancer mortality worldwide. Currently, there are more than 840,000 new cases of this nearly always fatal cancer each year. The combined age-standardized rate of mortality from liver cancer for men and women worldwide was 8.5 per 100,000 in 2018. The countries that traditionally are considered as part of the Middle East span two World Health Organization (WHO) regions: Eastern Mediterranean and Europe. Of these countries, Egypt has the second highest age-standardized rate of mortality from liver cancer, 49.0 and 16.7 per 100,000 for men and women, respectively, in the world. Indeed, this mortality rate is only exceeded globally by Mongolia. Further, liver cancer has a poor prognosis, less than a 15% 5-year survival. The etiologies of liver cancer diagnosed today are well understood, and with the emergence of fatty liver disease as a risk factor, this cancer is especially emergent in the countries of the Middle East where a majority of adults are now obese and the rates are rising.

ObjectiveIn Guatemala, cirrhosis is among the 10 leading causes of death, and mortality rates have increased lately. The reasons for this heavy burden of disease are not clear as the prevalence of prominent risk factors, such as hepatitis B virus, hepatitis C virus and heavy alcohol consumption, appears to be low. Aflatoxin B1 (AFB1) exposure, however, appears to be high, and thus could be associated with the high burden of cirrhosis. Whether AFB1 increases the risk of cirrhosis in the absence of viral infection, however, is not clear.DesignCirrhosis cases (n=100) from two major referral hospitals in Guatemala City were compared with controls (n=200) from a cross-sectional study. Logistic regression was used to estimate the ORs and 95% CIs of cirrhosis and quintiles of AFB1 in crude and adjusted models. A sex-stratified analysis was also conducted.ResultsThe median AFB1 level was significantly higher among the cases (11.4 pg/mg) than controls (5.11 pg/mg). In logistic regression analyses, higher levels of AFB1 was associated with cirrhosis (quintile 5 vs quintile 1, OR: 11.55; 95% CI 4.05 to 32.89). No attenuation was observed with adjustment by sex, ethnicity, hepatitis B virus status, and heavy alcohol consumption. A significantly increasing trend in association was observed in both models (p trend 1 association was more prominent among men.ConclusionsThe current study found a significant positive association between AFB1 exposure and cirrhosis. Mitigation of AFB1 exposure and a better understanding of additional risk factors may be important to reduce the burden of cirrhosis in Guatemala.

Background Previous studies have suggested an association between Helicobacter pylori (H pylori) and nonalcoholic fatty liver disease (NAFLD). The aim of the current study was to examine the association in Guatemala, a region with elevated prevalences of both H pylori and NAFLD. Associations between H pylori and other metabolic conditions were also examined, as were associations between H hepaticus and H bilis and the metabolic conditions. Materials & methods The analysis included 424 participants from a cross-sectional study in Guatemala. H pylori seropositivity was defined as positivity for ≥ 4 antigens. Seropositivities for H bilis and H hepaticus were defined as positivity for ≥ 2 antigens. NAFLD was estimated using the Fatty Liver Index and the Hepatic Steatosis Index. Other conditions examined were obesity, central obesity, hypercholesterolemia, low HDL, diabetes and metabolic syndrome (MetSyn). Prevalence odds ratios (POR) and 95% confidence intervals (CIs) were estimated. Results No overall associations between H pylori, H hepaticus, or H bilis and NAFLD or related metabolic conditions were found. Seropositivity for H pylori antigens CagA and VacA and H hepaticus antigen HH0713 was each significantly associated with NAFLD, however. In addition, associations were observed between the H pylori antigens HyuA, HP1564, and UreA and specified metabolic conditions. Conclusions While no overall associations between H pylori or Helicobacter species with NAFLD or related conditions were observed, some selected Helicobacter spp. antigens were associated with NAFLD. Further research is warranted to examine whether H. species are associated with any metabolic condition.

Background and aims Guatemala has the highest incidence of hepatocellular carcinoma (HCC) in the Western hemisphere. The major risk factors in Guatemala are not well characterized, but the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) appears to be low, while the prevalence of aflatoxin (AFB1) exposure appears to be high. To examine whether AFB1 may contribute to the elevated incidence of HCC in Guatemala, this study examined the frequency of the AFB1‐signature mutation in the TP53 gene (R249S) as well as other somatic mutations. In addition, we assessed whether the frequency of the TP53 mutation differed by sex. Methods Formalin‐fixed, paraffin‐embedded (FFPE) HCC tissues were obtained from three hospitals in Guatemala City between 2016 and 2017. In addition, tumor tissues preserved in RNAlater were also obtained. Sociodemographic and clinical information including HBV and HCV status were collected. Targeted sequencing of TP53 was performed in the FFPE samples, and a panel of 253 cancer‐related genes was sequenced in the RNAlater samples. Results Ninety‐one FFPE tissues were examined, from 52 men and 39 women. Median (IQR) age at diagnosis was 62 (51‐70). Among those with known HBV and HCV status, two were HBV+ and three were HCV+. Overall, 47% of the HCCs had a TP53 mutation. The AFB1‐signature R249S mutation was present in 24%. No overlap between the R249S mutation and HBV+ was observed in this cohort. Among 18 RNAlater samples examined, 44% had any TP53 mutation and 33% had the R249S mutation. Other somatic mutations were identified in known HCC driver genes. Conclusions The presence of the TP53 R249S mutation in the samples studied suggests that AFB1 may contribute to the high incidence of HCC in Guatemala. The proportion of HBV+ tumors was low, suggesting that AFB1 may be associated with HCC in the absence of concomitant HBV infection. Further investigation of AFB1 and other risk factors for HCC in Guatemala is warranted.

Background: There was rare data to describe decades’ long natural history or changes of a cancer. We describe and explore the characteristics and trend of liver cancer mortality in Qidong, China over the past 60 years. Methods: Mortality data of liver cancer in Qidong from 1958 to 1971 (death retrospective survey) and from 1972 to 2017 (cancer registration) were tabulated for the crude rate (CR), age-standardized rate (by China’s population, ASRC and by Worlld population, ASRW) and age-birth cohorts. The average annual percentage change (AAPC) was calculated by the Joinpoint Regression Program. Findings: The natural death rate during 1958-2017 decreased from 9‰ to 5.4‰ and then increased to 8‰; Cancer mortality rate rose continuously from 57/10 5 to 240/10 5 . Liver cancer mortality increased from 20/10 5 to 80/105, and then dropped to less than 52/105 in 2017. Liver cancer deaths in 1972-2017 accounted for 30.53% of all cancers, with CR of 60.48/105, ASRC of 34.78/105, and ASRW of 45.71/105. The truncated rate of 35-64 was 104.06/105, cumulative mortality rate of 0-74 years, and cumulative risk rate were 4.79% and 4.68%, respectively. The CR for males and females were 91.86/105 and 29.92/105, respectively, with a sex ratio of 3.07:1. From 1972 to 2017, the AAPC of the ASRW derived by the Joinpoint analysis was -1.3%, in which -1.5% for men and -1.0% for women. The AAPC of joinpoints was -1.8% for both sexes, and -1.8% for males, -1.4% for females, respectively. Period analysis shows that the age-specific mortality rates of the age groups under 54 years old had a significant decreasing trend. Birth cohort analysis showed that the mortality rate of liver cancer in 40-44, 35-39, 30-34, 25-29, 20-24, 15-19 years cohort decreased, but the rates in 70-74, and 75+ were increased. Interpretation: The crude mortality rate of liver cancer in Qidong has experienced trends from lower to higher levels, and from continued increase at a high plateau to most recently a gradual decline, and change greatest in younger people. Many years of comprehensive prevention and intervention measures may have influenced the decrease of the liver cancer epidemic in this area. The reduction of intake levels of aflatoxin might be one of the most significant factors as evidenced by the decline of exposure biomarkers in this population. Funding Statement: The US National Institutes of Health through grants R01 CA196610 and R35 CA197222, the Chinese National Key Projects (2008ZX10002-015, 2008ZX10002-017, 2012ZX10002009), the Scientifc Research Projet of “333 Project” in Jiangsu (BRA2019030), and the Nantong Science and Techonology Project (MS22019008). Declaration of Interests: The authors declare no competing interests related to this study.

Aflatoxin B1 (AFB1) is a potent human and animal hepatocarcinogen. To investigate the effects of aflatoxin on miRNA expression during the initiation phase of carcinogenesis, next-generation sequencing was used to analyze liver tissues from F344 rats exposed to 200 µg/kg per day AFB1 for 4 weeks. A panel of miRNAs was identified that was upregulated with AFB1 treatment compared to controls: rno-miR-434-3p, rno-miR-411-5p, rno-miR-221-3p, rno-miR-127-3p, rno-miR-205, rno-miR-429, rno-miR-34a-5p, rno-miR-181c-3p, rno-miR-200b-3p, and rno-miR-541-5p. Analysis of rat livers exposed to AFB1 plus the chemopreventive triterpenoid CDDO-Im revealed a striking abrogation of this upregulation. These changes were validated by real-time PCR. We also explored the temporal variation in expression of the candidate miRNAs during the 4-week dosing period. Most of the candidate miRNAs were upregulated at week 1 and increased for the duration of AFB1 dosing over the 4-week period. Treatment with CDDO-Im ameliorated these effects at all time points. All candidate miRNAs were detectable in serum from aflatoxin treated animals; however, there was no significant difference in expression for 7 of the 11 miRNAs examined. Exposure to AFB1 upregulated miR-122-5p (5 fold), 34a-5p (13 fold), and 181c-3p (170 fold) compared with controls. The findings from this study give insight into epigenetic changes induced by aflatoxin taking place during the initial step of carcinogenesis. This article is protected by copyright. All rights reserved

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB1) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB1-induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB1 show significant increases in the levels of AFB1-Fapy-dG in Neil1-/- vs. wild-type liver DNA. Further, Neil1-/- mice are highly susceptible to AFB1-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1-/- The magnitude of this effect in Neil1-/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB1-associated HCCs.

Hepatocellular carcinoma (HCC) has emerged as a major cause of cancer deaths globally. The landscape of systemic therapy has recently changed, with six additional systemic agents either approved or awaiting approval for advanced stage HCC. While these agents have the potential to improve outcomes, a survival increase of 2–5 months remains poor and falls short of what has been achieved in many other solid tumor types. The roles of genomics, underlying cirrhosis, and optimal use of treatment strategies that include radiation, liver transplantation, and surgery remain unanswered. Here, we discuss new treatment opportunities, controversies, and future directions in managing HCC.

Background Cervical cancer is common in Kenyan women. Cofactors in addition to infection with oncogenic human papillomavirus (HPV) are likely to be important in causing cervical cancer, because only a small percentage of HPV-infected women will develop this malignancy. Kenyan women are exposed to dietary aflatoxin, a potent carcinogen and immunosuppressive agent, which may be such a cofactor. Methods Demographics, behavioral data, plasma, and cervical swabs were collected from 88 human immunodeficiency virus-uninfected Kenyan women without cervical dysplasia. Human papillomavirus detection was compared between women with or without plasma aflatoxin B1-lysine (AFB1-lys) and evaluated in relation to AFB1-lys concentration. Results Valid HPV testing results were available for 86 women (mean age 34.0 years); 49 women (57.0%) had AFB1-lys detected and 37 (43.0%) had none. The AFB1-lys detection was not associated with age, being married, having more than secondary school education, home ownership, living at a walking distance to healthcare ≥60 minutes, number of lifetime sex partners, or age of first sex. The AFB1-lys detection and plasma concentrations were associated with detection of oncogenic HPV types. Conclusions The AFB1-lys positivity and higher plasma AFB1-lys concentrations were associated with higher risk of oncogenic HPV detection in cervical samples from Kenya women. Further studies are needed to determine whether aflatoxin interacts with HPV in a synergistic manner to increase the risk of cervical cancer.

Maternal systemic inflammation during pregnancy may restrict embryo−fetal growth, but the extent of this effect remains poorly established in undernourished populations. In a cohort of 653 maternal−newborn dyads participating in a multi-armed, micronutrient supplementation trial in southern Nepal, we investigated associations between maternal inflammation, assessed by serumα1-acid glycoprotein and C-reactive protein, in the first and third trimesters of pregnancy, and newborn weight, length and head and chest circumferences. Median (IQR) maternal concentrations inα1-acid glycoprotein and C-reactive protein in the first and third trimesters were 0.65 (0.53–0.76) and 0.40 (0.33–0.50) g/l, and 0.56 (0.25–1.54) and 1.07 (0.43–2.32) mg/l, respectively.α1-acid glycoprotein was inversely associated with birth size: weight, length, head circumference and chest circumference were lower by 116 g (P= 2.3 × 10−6), and 0.45 (P= 3.1 × 10−5), 0.18 (P= 0.0191) and 0.48 (P= 1.7 × 10−7) cm, respectively, per 50% increase inα1-acid glycoprotein averaged across both trimesters. Adjustment for maternal age, parity, gestational age, nutritional and socio-economic status and daily micronutrient supplementation failed to alter any association. Serum C-reactive protein concentration was largely unassociated with newborn size. In rural Nepal, birth size was inversely associated with low-grade, chronic inflammation during pregnancy as indicated by serumα1-acid glycoprotein.

Intrahepatic cholangiocarcinoma (iCCA) has over the last 10-20 years become the focus of increasing concern, largely due to its rising incidence and high mortality rates worldwide. The significant increase in mortality rates from this primary hepatobiliary cancer, particularly over the past decade, has coincided with a rapidly growing interest among clinicians, investigators, and patient advocates to seek greater mechanistic insights and more effective biomarker-driven targeted approaches for managing and/or preventing this challenging liver cancer. In addition to discussing challenges posed by this aggressive cancer, this review will emphasize recent epidemiological, basic, and translational research findings for iCCA. In particular, we will highlight emerging demographic changes and evolving risk factors, the critical role of the tumor microenvironment, extracellular vesicle biomarkers and therapeutics, intertumoral and intratumoral heterogeneity, and current and emerging targeted therapies regarding iCCA. Specifically, recent evidence linking non-bile duct medical conditions, such as nonalcoholic fatty liver disease and nonspecific cirrhosis, to intrahepatic cholangiocarcinogenesis together with geographic and ethnic variation will be assessed. Recent developments concerning the roles played by transforming growth factor-β and platelet-derived growth factor D in driving the recruitment and expansion of cancer-associated myofibroblasts within cholangiocarcinoma (CCA) stroma as well as their therapeutic implications will also be discussed. In addition, the potential significance of extracellular vesicles as bile and serum biomarkers and therapeutic delivery systems for iCCA will be described. An integrated systems approach to classifying heterogeneous iCCA subtypes will be further highlighted, and recent clinical trials and emerging targeted therapies will be reviewed, along with recommendations for future translational research opportunities. Established international CCA networks are now facilitating collaborations aimed at advancing iCCA translational and clinical research.

Background Despite a strong statistical correlation between dietary aflatoxin B1 (AFB1)-exposure and childhood stunting, the causal mechanism remains speculative. This issue is important because of emerging interest in reduction of human aflatoxin exposure to diminish the prevalence and complications of stunting. Pediatric liver diseases cause growth impairment, and AFB1 is hepatotoxic. Thus, liver injury might mediate AFB1-associated growth impairment. We have developed a rat model of dietary AFB1-induced stunting to investigate these questions. Methods Newly-weaned rats were given AFB1-supplemented- or control-diets from age 3-9 weeks, and then euthanized for serum- and tissue-collection. Food intake and weight were serially assessed, with tibial-length determined at the experimental endpoint. Serum AFB1-adducts, hepatic gene and protein expression, and liver injury markers were quantified using established methodologies. Results AFB1-albumin adducts correlated with dietary toxin contamination, but such contamination did not affect food consumption. AFB1-exposed animals exhibited dose-dependent wasting and stunting, liver pathology, and suppression of hepatic targets of growth hormone (GH) signaling, but did not display increased mortality. Conclusion These data establish toxin-dependent liver injury and hepatic GH-resistance as candidate mechanisms by which AFB1-exposure causes growth impairment in this mammalian model. Interrogation of modifiers of stunting using this model could guide interventions in at-risk and affected children.

Aflatoxin B1 is a potent carcinogen, occurring from mold growth that contaminates staple grains in hot, humid environments. In this investigation, aflatoxin B1-lysine albumin biomarkers were measured by mass spectrometry in rural South Asian women, during the first and third trimester of pregnancy, and their children at birth and at two years of age. These subjects participated in randomized community trials of antenatal micronutrient supplementation in Sarlahi District, southern Nepal and Gaibandha District in northwestern Bangladesh. Findings from the Nepal samples demonstrated exposure to aflatoxin, with 94% detectable samples ranging from 0.45 to 2939.30 pg aflatoxin B1-lysine/mg albumin during pregnancy. In the Bangladesh samples the range was 1.56 to 63.22 pg aflatoxin B1-lysine/mg albumin in the first trimester, 3.37 to 72.8 pg aflatoxin B1-lysine/mg albumin in the third trimester, 4.62 to 76.69 pg aflatoxin B1-lysine/mg albumin at birth and 3.88 to 81.44 pg aflatoxin B1-lysine/mg albumin at age two years. Aflatoxin B1-lysine adducts in cord blood samples demonstrated that the fetus had the capacity to convert aflatoxin into toxicologically active compounds and the detection in the same 2-year-old children illustrates exposure over the first 1000 days of life.

Background & Aims Aflatoxin, which causes hepatocellular carcinoma, may also cause gallbladder cancer. We investigated whether patients with gallbladder cancer have higher exposure to aflatoxin than patients with gallstones. Methods We measured aflatoxin B 1 (AFB 1 )–lysine adducts in plasma samples from the Shanghai Biliary Tract Cancer case-control study, conducted from 1997 through 2001. We calculated age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) and the population-attributable fraction for 209 patients with gallbladder cancer and gallstones vs 250 patients with gallstones without cancer (controls). In 54 patients with gallbladder cancer, tumor tissue was examined for the R249S mutation in TP53 , associated with aflatoxin exposure, through targeted sequencing. Results The AFB 1 –lysine adduct was detected in 67 (32%) of 209 patients with gallbladder cancer and 37 (15%) of the 250 controls (χ 2 P 1 –lysine, the median level of AFB 1 –lysine was 5.4 pg/mg in those with gallbladder cancer, compared with 1.2 pg/mg in controls. For patients in the fourth quartile of AFB 1 –lysine level vs the first quartile, the OR for gallbladder cancer was 7.61 (95% CI, 2.01–28.84). None of the 54 gallbladder tumors sequenced were found to have the R249S mutation in TP53 . The population-attributable fraction for cancer related to aflatoxin was 20% (95% CI, 15%–25%). Conclusions In a case-control study of patients with gallbladder cancer and gallstones vs patients with gallstones without cancer, we associated exposure to aflatoxin (based on plasma level of AFB 1 –lysine) with gallbladder cancer. Gallbladder cancer does not appear associate with the R249S mutation in TP53 . If aflatoxin is a cause of gallbladder cancer, it may have accounted for up to 20% of the gallbladder cancers in Shanghai, China, during the study period, and could account for an even higher proportion in high-risk areas. If our findings are verified, reducing aflatoxin exposure might reduce the incidence of gallbladder cancer.

Liver cancer is an emerging global health issue, with rising incidence in both the United States and the economically developing world. Although Guatemala experiences the highest rates of this disease in the Western hemisphere and a unique 1:1 distribution in men and women, few studies have focused on this population. Thus, we determined the prevalence and correlates of aflatoxin B1 (AFB1) exposure and hepatitis virus infection in Guatemalan adults. Healthy men and women aged ≥40 years (n = 461), residing in five departments of Guatemala, were enrolled in a cross-sectional study from May-October of 2016. Serum AFB1-albumin adducts were quantified using isotope dilution mass spectrometry. Multivariate linear regression was used to assess relationships between AFB1-albumin adduct levels and demographic factors. Biomarkers of hepatitis B virus and hepatitis C virus infection were assessed by immunoassay and analyzed by Fisher's exact test. AFB1-albumin adducts were detected in 100% of participants, with a median of 8.4 pg/mg albumin (range, 0.2-814.8). Exposure was significantly higher (p

In the original version of [this article][1] ([1][2]), authors Thomas Karasic and Jeffrey Meyer are mistakenly omitted from the author list. This error has been corrected in the latest online HTML and PDF versions of the article and the author contributions and disclosures have been updated as

5530 Background: Cervical cancer is the leading cause of cancer-related deaths among women living in Africa. Only a small proportion of HPV-infected women develop cervical cancer and other cofactors may increase a woman’s risk of developing cervical cancer. Aflatoxin, a potent carcinogen and immunosuppressive agent, is produced by fungi that contaminate corn and other staple foods in sub-Saharan Africa. Women who ingest aflatoxin may be more likely to have persistent infections with oncogenic HPV type. Methods: Demographics, behavioral data, plasma, and cervical swabs were collected from HIV-uninfected women 18 and 45 years of age who presented for cervical cancer screening at Moi Referral and Teaching Hospital (Eldoret, Kenya) and had normal VIA examination. HPV testing was performed on cervical swabs using the Roche Linear Array Assay. Aflatoxin-albumin adduct (AFB1-lys) was detected and quantified in plasma. The association of plasma AFB1-lys detection and concentration and the detection of HPV was examined. Results: Sufficient plasma was available from 88 HIV-uninfected women and was transported to the U.S. for aflatoxin testing. Valid HPV testing results were available for 86 of these women (mean age 34.0 years); 49 women (57.0%) had detectable AFB1-lys and 37 (43.0%) had no detection. Substantial variation existed in plasma AFB1-lys concentrations among the 49 women (range 0.02 to 0.21 pg/µL). Detection of AFB1-lys was not associated with age, and other behavioral factors such as number of lifetime partners, marital status and age at first sex. AFB1-lys detection was associated with detection of A9 HPV types (HPV 16, 31, 33, 35, 52, and 58) as a group in cervical swabs (p = 0.029) as well as A9 types excluding HPV 16 (p = 0.020), but not with individual A9 types, A7 HPV types (such as HPV 18), or low-risk HPV types. A concentration dependent association of AFB1-lys was seen with detection of A9 HPV types as a group (p = 0.009), non-HPV 16 A9 types (p = 0.005), and HPV 52 (p = 0.042), but not with the A7 HPV types. Conclusions: AFB1-lys was detected in 57% of HIV-uninfected Kenyan women without cervical dysplasia. AFB1-lys-positive women were more likely than AFB1-lys-negative women to have oncogenic HPV A9 types detected. Higher plasma AFB1-lys concentrations were associated with increased likelihood of oncogenic HPV A9 type detection. Further studies are needed to determine if chronic exposure to aflatoxin interacts with HPV infection (and possibly HIV co-infection) to modulate the risk of cervical cancer in women in Kenya and other developing countries.

Abstract. Selenium deficiency or excess may have public health consequences, yet selenium status is infrequently characterized in populations, perhaps due to challenges in methodology. We are seeking to identify plasma proteins, using proteomics discovery and validation approaches, to serve as proxies for micronutrient status, including selenium, which may in the future be more readily assessed by robust, affordable field methods. In a sample of rural Nepalese children 6 - 8 years old (n = 500), the prevalence of selenium deficiency was 13.6 and 60.9 % at plasma selenium concentrations < 0.60 and < 0.89 µmol/L, respectively, assessed by atomic absorption spectroscopy. Relative abundance of selenoprotein P isoform 1 (SEPP1), glutathione reductase-3, and apolipoprotein A2 from discovery-based experiments was correlated with plasma selenium with a false discovery rate < 10 % (i. e., q < 0.10), all with p < 0.001. In linear mixed effects regression models to predict plasma selenium, only SEPP1 was significant (R2 = 0.63), estimating 8.2 % (95 % CI: 3.9 - 12.6) and 65.5(61.4 - 69.7)% of the in-sample population as deficient at each respective cut-off. Targeted quantification of SEPP1 in a preliminary series of specimens (n = 19) as a validation of the discovery approach revealed a high correlation with plasma selenium (r = 0.757, p = 0.0002). Plasma proteomics can identify valid plasma protein indicators of micronutrient status, as shown with selenium, comprising a step toward making population assessment of selenium status in vulnerable groups more accessible.

Vitamin K (VK) is a fat-soluble vitamin whose deficiency disrupts coagulation and may disturb bone and cardiovascular health. However, the scale and systems affected by VK deficiency in pediatric populations remains unclear. We conducted a study of the plasma proteome of 500 Nepalese children 6–8 years of age (male/female ratio = 0.99) to identify proteins associated with VK status. We measured the concentrations of plasma lipids and protein induced by VK absence-II (PIVKA-II) and correlated relative abundance of proteins quantified by mass spectrometry with PIVKA-II. VK deficiency (PIVKA-II >2 μg/L) was associated with a higher abundance of low-density lipoproteins, total cholesterol, and triglyceride concentrations (p 10% of the children, five proteins were associated with PIVKA-II and seven proteins were differentially abundant between VK deficient versus sufficient children, including coagulation factor-II, hemoglobin, and vascular endothelial cadherin, passing a false discovery rate (FDR) threshold of 10% (q 0.7). Untargeted proteomics offers a novel systems approach to elucidating biological processes of coagulation, vascularization, and erythrocyte oxidative stress related to VK status. The results may help elucidate subclinical metabolic disturbances related to VK deficiency in populations.

Aflatoxins are highly toxic and carcinogenic naturally occurring products of spoilage molds that are widely encountered as contaminants of commonly consumed food staples, including cereal grains and nuts. This human exposure scenario is especially prominent in hot, humid environments found in low-income countries. Aflatoxin B 1 , the most abundant and potent member of a group of related compounds, has been designated as a major risk factor for human liver cancer and an important contributor to nearly 700 000 cancer deaths per year. Importantly, in experimental systems, it also induces pleiotropic toxic effects such as growth retardation and immune suppression, but the potential for aflatoxins to induce maternal and early-life toxicities in children has received only limited study. However, this possibility is gaining attention since nutritional interventions alone have had only modest effects in reducing the prevalence of health problems such as stunting and wasting. Thus, mitigation of aflatoxin exposures through primary and secondary prevention strategies holds promise for broadly improving health status in developing countries.

Characterization of normal changes in the serum proteome during pregnancy may enhance understanding of maternal physiology and lead to the development of new gestational biomarkers of health. In 23 Nepalese pregnant women who delivered at term two-dimensional difference in-gel electrophoresis (DIGE) was used to assess changes in relative protein abundance between paired serum samples collected in the first and third trimesters. One-hundred and forty-five of over 700 protein spots in DIGE gels (pI 4.2–6.8) exhibited nominally significant (p

In regions with high prevalence of chronic hepatitis B virus (HBV) infection and dietary aflatoxin B(1) (AFB(1)) exposure, hepatocellular carcinomas (HCCs) often contain TP53 mutation at codon 249 (R249S). Furthermore, a C-terminal truncated HBx protein expressed from hepatocyte integrated HBV is associated with HCC development. This study evaluates the association between R249S and HBX status in relation to HCC in West African population. HBX (complete or 3'-truncated) and HBS genes were assessed by PCR in cell-free DNA (CFDNA) from plasma of subjects recruited in a hospital-based case-control study (325 controls, 78 cirrhotic patients and 198 HCC cases) conducted in The Gambia. These samples had been previously analyzed for R249S and HBV serological status. Complete HBX sequence was frequently detected in CFDNA of HCC-R249S positive (77%, 43/56) compared with HCC-R249S-negative cases (44%, 22/50). Conversely, the proportion of 3'-truncated HBX gene was significantly higher in HCC-R249S negative than positive cases (34%, 17/50, compared with 12%, 7/56) (χ(2) = 12.12; P = 0.002; distribution of R249S negative and positive according to HBX status). Occult HBV infection (detected by PCR) was present in 24% of HCC previously considered as negative by HBV serology. Moreover, HBV mutation analysis revealed that double mutation at nucleotides 1762(T)/1764(A) was associated with diagnosis of cirrhosis or HCC {cirrhosis: odds ratio (OR): 9.50 [95% confidence interval (CI) 1.50-60.11]; HCC: OR: 11.29 [95% CI 2.07-61.47]}. These findings suggest that in HCC from The Gambia, complete HBX sequences are often associated with the presence of TP53 R249S mutation.

Abstract 13 Background: The proportion of liver cancer (LC) that is linked to metabolic risk factors has been increasing in many countries. Guatemala has the highest reported incidence of LC in the Americas, 1 but the prevalence of metabolic risk factors is not clear. We undertook this work to examine the prevalence of metabolic risk factors for LC in Guatemala by sex and residence. Methods: We conducted a cross-sectional study of 461 adults older than 40 years who resided in rural and urban areas. Risk factors were defined by using physical exam and laboratory data, including anthropometry, blood pressure, fasting plasma glucose, serum liver enzymes, and lipids. Fatty liver disease (FLD) was defined as a fatty liver index score of > 60 and liver fibrosis (LF) as defined by a FIB-4 score of > 2.67. 2 , 3 Results: Among participants, 66% resided in rural areas and 57% were women. Mean ages of men and women were 58.0 ± 11.3 and 53.4 ± 9.8 years, respectively. Compared with men, women had higher prevalence of obesity (15% v 41%; P ≤ .001), metabolic syndrome (46% v 74%; P < .001), and FLD (67% v 52%; P < .001), but not LF (6% v 4%; P = .238). Compared with men and women from rural areas, those from urban areas had higher prevalence of diabetes (10% v 27%; P = .002; and 14% v 32%; P < .001) FLD (42% v 67%; P < .001; and 59% v 79%; P < .001), and metabolic syndrome (37% v 58%; P = .005; and 69% v 81%; P = .032), respectively. There was no difference in prevalence of LF by area among either men (6% v 6%; P = .86) or women (3% v 4%; P = .75). Conclusion: This study highlights a high prevalence of metabolic risk factors for LC in Guatemala, especially among women in urban areas. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Alvaro Rivera-Andrade No relationship to disclose Maria Fernanda Kroker-Lobos No relationship to disclose Mariana Lazo No relationship to disclose Neal Freedman No relationship to disclose John Groopman No relationship to disclose Eliseo Guallar No relationship to disclose Carlos Mendoza-Montano No relationship to disclose Katherine McGlynn No relationship to disclose Josh Smith Research Funding: Abbott Nutrition Manuel Ramirez-Zea No relationship to disclose

Background: Chronic hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) exposure are etiological factors for hepatocellular carcinoma (HCC) in countries with hot, humid climates. HCC often harbors a TP53 (tumor protein p53) mutation at codon 249 (R249S). In chronic carriers, 1762T/1764A mutations in the HBV X gene are associated with increased HCC risk. Both mutations have been detected in circulating cell-free DNA (CFDNA) from asymptomatic HBV carriers. Objective: We evaluated seasonal variation in R249S and HBV in relation to AFB1 exposure. Methods: R249S was quantitated by mass spectrometry in CFDNA in a cross-sectional survey of 473 asymptomatic subjects (237 HBV carriers and 236 noncarriers) recruited in three villages in the Gambia over a 10-month period. 1762T/1764A HBV mutations were detected by quantitative polymerase chain reaction. In addition, the HBV S gene was sequenced in 99 subjects positive for HBV surface antigen (HBsAg). Results: We observed a seasonal variation of serum R249S levels. Positivity for R249S and average concentration were significantly higher in HBsAg-positive subjects surveyed during April–July (61%; 5,690 ± 11,300 R249S copies/mL serum) than in those surveyed October–March [32% and 480 ± 1,030 copies/mL serum (odds ratio = 3.59; 95% confidence interval: 2.05, 6.30; p < 0.001)]. Positivity for HBV e antigen (HBeAg) (a marker of HBV replication) and viral DNA load also varied seasonally, with 15–30% of subjects surveyed between April and June HBeAg positive, compared with < 10% surveyed during other months. We detected 1762T/1764A mutations in 8% of carriers, half of whom were positive for R249S. We found HBV genotype E in 95 of 99 HBsAg-positive subjects. Conclusion: R249S is detectable in CFDNA of asymptomatic subjects. Evidence of temporal and quantitative variations suggests an interaction among AFB1 exposure, HBV positivity, and replication on TP53 mutation formation or persistence.

Aflatoxin B1 (AFB1) is a DNA-binding toxin that contributes to the burden of liver cancer in tropical areas. AFB1-DNA adducts are powerful biomarkers that discern individual and population risk from exposure to this carcinogen. The discovery of concordance between the metabolic pathways of the male Fischer rat and humans allowed data from rats to guide the development of chemoprevention strategies employed in clinical trials in high-risk regions. In this study, the variables of strain and sex are studied in the rat model, as a step toward understanding how ethnic differences and sex influence DNA adduct formation and the induction of enzymes by chemoprotective agents. Sulforaphane (SF), which induces phase II enzymes including glutathione S-transferases (GSTs), was evaluated for its ability to induce GST activity and reduce the AFB1-DNA adducts in livers of both sexes of two rat strains that differ in susceptibility to AFB1 hepatocarcinogenesis. A dose-dependent relationship was found for SF for both induction of GST and reduction in of AFB1-N7-guanine in both Fischer (sensitive to AFB1) and Sprague-Dawley rats (relatively resistant). Sprague-Dawley rats exhibited the greatest increase in GST levels and the largest reduction in AFB1-N7-guanine in liver DNA. Males and females of each strain were also compared to determine if the ability of SF to induce GST and reduce AFB1-N7-guanine correlated with gender differences in sensitivity to AFB1 carcinogenesis. No gender-specific responses to SF were observed. These results support the view that SF induction of liver GST activity may play a role in its chemoprotective activity., National Institutes of Health (U.S.) (Grant R01 ES016313), National Institutes of Health (U.S.) (Grant P30 ES002109), National Institutes of Health (U.S.) (Grant P01 ES006052), National Institutes of Health (U.S.) (Grant P30 ES003819)

Hepatocellular carcinoma (HCC) continues to be a major cause of cancer death globally, with aflatoxin B1 (AFB1) as a prevalent risk factor in low- and middle-income countries. MicroRNAs have been shown to be differentially expressed in HCC and may serve as predictive biomarkers. In this study, we analyzed tumor and non-tumor tissue from rats dosed with AFB1 (200 µg/kg BW) for 28 consecutive days that received vehicle only or AFB1 plus the chemopreventive agent CDDO-Im (30 µmol), and control animals (Johnson et al., CaPR, 2014). Total RNA was isolated from tumor or non-tumor tissue at the time of sacrifice and sequenced. MicroRNA transcriptomic analysis revealed 17 miRNAs significantly upregulated (> 5 fold) in tumors compared to non-tumor tissue. The top ten dysregulated miRNAs determined by fold change and biological significance were selected for further investigation: rno-miR- 205, 200b-3p, 182, 429, 31a-5p, 10b-5p, 141-3p, 132-3p, 802-5p. Validation of sequencing results by quantitative PCR (qPCR) confirmed the upregulation of the majority of candidate miRNAs in tumors and rno-miR-224-5p as the most dysregulated miRNA (over 400 fold). We also examined the levels of these candidates in terminal sera of the same animals by qPCR. Circulating miRs-224-5p, 182, and 122-5p were increased (> 1.5 fold) in animals diagnosed with HCC compared to untreated animals and those previously dosed with AFB1 plus CDDO-Im. Analysis of tracking of serum miR-182 by generalized estimating equations (GEE) revealed significantly increased levels (5 fold; CI: 2.17- 2.50) in animals that developed HCC. This sustained increase in serum miR-182 is seen months before any tumors or other symptoms are present, and highlights this miRNA as a potential predictive biomarker in AFB1-induced HCC. Supported by T32ES007141-31A1 and CA197222. Citation Format: Merricka C. Livingstone, Natalie M. Johnson, Bill D. Roebuck, Thomas W. Kensler, John D. Groopman. Dysregulated microRNAs in aflatoxin-induced hepatocellular carcinoma: Serum miR-182 as a potential predictive biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5401.

Liver cancer is the leading cause of cancer death in many regions of the world. With the goal to discover biomarkers that reflect subsets of high-risk individuals and their prognosis, we nested our study in a male cohort of 5,581 hepatitis B surface antigen carriers in Qidong, People's Republic of China, who were recruited starting in 1989. By December 2003, 667 liver cancer cases were diagnosed in this group and plasma samples collected at the initial screening at enrollment were available in 515 cases who had succumbed to liver cancer. Hepatitis B virus (HBV) DNA could be isolated in 355 (69%) of these samples. In 14%, 15%, 19%, 31%, and 22%, screening took place at ≤1.5, 1.51 to 3, 3.01 to 5, 5.01 to 9, and >9 years before death, respectively; and 39% died at age below 45 years. The relation between mutations in HBV and time to death were determined by logistic regression for the odds of mutation and by survival analyses methods with age as the time scale. In 279 (79%) of these individuals, the samples contained a two-nucleotide 1762T/1764A HBV mutation. Sixteen samples lacking the 1762T/1764A mutation had novel mutations elsewhere in the 1761 to 1767 region of the HBV genome. There was a statistically significant difference (P = 0.012) for the high prevalence of the HBV mutations in the men who died from hepatocellular carcinoma under the age of 45 years relative to those who died after 55 years of age and HBV mutations accelerated death (relative hazard, 1.40; 95% confidence interval, 1.06-1.85) and that the effect was attenuated by age from 2.04 for age 35 years to 1.0 for age 65 years with the 90% confidence band being above 1 for ages

Background Hepatitis B (HB) infection is common in Mali. However, there is little information on molecular and biochemical characteristics of HB carriers. Methods A group of 1466 adult volunteers was recruited in the district of Bamako. Confirmed HB carriers were tested for HB viral load by quantitative PCR and HBV was genotyped by sequencing of HBS. Fibrosis and hepatitis activity were measured using the Fibrotest-Actitest. A mutation of TP53 at codon 249 (R249S), specific for exposure to aflatoxin, was detected in cell-free DNA extracted from plasma. Results Overall, 276 subjects were HBsAg-positive (18.8%). Among 152 subjects tested for HBV load, 49 (32.2%) had over 104 copies/mL and 16 (10.5%) had levels below the limit of detection. The E genotype was found in 91.1% of carriers. Fibrotest scores ≥ F2 were observed in 52 subjects (35.4%). Actitest scores ≥ A2 were detected in 15 subjects (10.2%) and were correlated with Fibrotest scores (p = 0.0006). Among 105 subjects tested, 60% had detectable levels of R249S copies (>40 copies/mL plasma). Conclusion Chronic HB carriage in adults in Bamako district is well over epidemic threshold. About 1/3 of carriers have moderate to severe liver fibrosis and 60% have detectable aflatoxin-related TP53 R249S mutation. These results support introduction of anti-HB therapies to reduce the progression towards severe liver disease.

Background: The term vitamin E describes a family of 8 vitamers, 1 of which is α-tocopherol, that is essential for human health. Vitamin E status remains largely unknown in low-income countries because of the complexity and cost of measurement. Quantitative proteomics may offer an approach for identifying plasma proteins for assessing vitamin E status in these populations. Objective: To improve options for vitamin E status assessment, we sought to detect and quantify a set of plasma proteins associated with α- and γ-tocopherol concentrations in a cohort of 500 rural Nepalese children aged 6–8 y and, based on nutrient-protein associations, to predict the prevalence of vitamin E deficiency (α-tocopherol 10% of all child samples, of which 119 correlated with α-tocopherol (false discovery rate, q < 0.10). Proteins were primarily involved in lipid transport, coagulation, repair, innate host defenses, neural function, and homeostasis. Six proteins [apolipoprotein (apo)C-III; apoB; pyruvate kinase, muscle; forkhead box 04; unc5 homolog C; and regulator of G-protein signaling 8] explained 71% of the variability in plasma α-tocopherol, predicting an in-sample population prevalence of vitamin E deficiency of 51.4% (95% CI: 46.4%, 56.3%) compared with a measured prevalence of 54.8%. Plasma γ-tocopherol was associated with 12 proteins (q < 0.10), 2 of which (apoC-III and Misato 1) explained 20% of its variability. Conclusions: In this undernourished population of children in South Asia, quantitative proteomics identified a large plasma α-tocopherome from which 6 proteins predicted the prevalence of vitamin E deficiency. The findings illustrate that protein biomarkers, once absolutely quantified, can potentially predict micronutrient deficiencies in populations. The maternal micronutrient supplementation trial from which data were derived as a follow-up activity was registered with clinicaltrials.gov as NCT00115271.

Aromatic amines such as 2-naphthylamine and 4-aminobiphenyl are established human bladder carcinogens. Experimental evidence for carcinogenicity of monocylic aromatic amines is limited mostly to other organs, but a recent epidemiologic study of bladder cancer found that 2,6-dimethyl- (2,6-DMA), 3,5-dimethyl- (3,5-DMA), and 3-ethylaniline (3-EA) may play a significant role in the etiology of this disease in man. The present work was undertaken to test whether a genotoxic mechanism can account for the presumptive activity of 2,6-DMA, 3,5-DMA, and 3-EA by quantifying the binding of these compounds to DNA in vivo. Each of these three [(14)C]alkylanilines was administered at approximately 100 microg/kg to C57BL/6 mice, which were subsequently sacrificed 2, 4, 8, 16, and 24 h post-dosing. Bladder, colon, kidney, liver, lung, and pancreas were harvested from each animal, and DNA was isolated from each tissue. Adduct levels were determined by quantifying bound isotope using accelerator mass spectrometry. Adducts were detectable in the bladder and liver DNA samples from every animal at every time point at levels that ranged from 3 per 10(9) to 1.5 per 10(7) nucleotides. Adduct levels were highest in animals given 3,5-DMA and lowest in those given 3-EA. Levels in both bladder and liver declined by severalfold over the course of the experiment. Adducts were detected less frequently in the other four tissues. Taken together, the results strongly suggest that these three alkylanilines are metabolized in vivo to electrophilic intermediates that covalently bind to DNA and that adducts are formed in the DNA of bladder, which is a putative target organ for these alkylanilines.

Cancer research is drawing on the human genome project to develop new molecular-targeted treatments. This is an exciting but insufficient response to the growing, global burden of cancer, particularly as the projected increase in new cases in the coming decades is increasingly falling on developing countries. The world is not able to treat its way out of the cancer problem. However, the mechanistic insights from basic science can be harnessed to better understand cancer causes and prevention, thus underpinning a complementary public health approach to cancer control. This manuscript focuses on how new knowledge about the molecular and cellular basis of cancer, and the associated high-throughput laboratory technologies for studying those pathways, can be applied to population-based epidemiological studies, particularly in the context of large prospective cohorts with associated biobanks to provide an evidence base for cancer prevention. This integrated approach should allow a more rapid and informed translation of the research into educational and policy interventions aimed at risk reduction across a population.

The use of biomarkers in molecular epidemiology studies for identifying stages in the progression of development of the health effects of environmental agents has the potential for providing important information for critical regulatory, clinical and public health problems. Investigations of aflatoxins probably represent one of the most extensive data sets in the field and this work may serve as a template for future studies of other environmental agents. The aflatoxins are naturally occurring mycotoxins found on foods such as corn, peanuts, various other nuts and cottonseed and they have been demonstrated to be carcinogenic in many experimental models. As a result of nearly 30 years of study, experimental data and epidemiological studies in human populations, aflatoxin B(1) was classified as carcinogenic to humans by the International Agency for Research on Cancer. The long-term goal of the research described herein is the application of biomarkers to the development of preventative interventions for use in human populations at high-risk for cancer. Several of the aflatoxin-specific biomarkers have been validated in epidemiological studies and are now being used as intermediate biomarkers in prevention studies. The development of these aflatoxin biomarkers has been based upon the knowledge of the biochemistry and toxicology of aflatoxins gleaned from both experimental and human studies. These biomarkers have subsequently been utilized in experimental models to provide data on the modulation of these markers under different situations of disease risk. This systematic approach provides encouragement for preventive interventions and should serve as a template for the development, validation and application of other chemical-specific biomarkers to cancer or other chronic diseases.

Hepatocellular carcinoma is one of the leading causes of cancer death worldwide. The etiology of liver cancer is multifactorial, and infection with hepatitis B virus (HBV), whose pathogenesis is exacerbated by the acquisition of mutations that accelerate carcinogenesis, or hepatitis C virus (HCV) and dietary exposure to aflatoxin B1 all contribute to elevating one's risk for this disease. In this study, we sought to determine the contributions of these agents by measuring the occurrence of an HBV 1762T/1764A double mutation, an aflatoxin-specific 249G→T mutation of the p53 gene, and HCV in plasma of 34 HCC cases and 68 age- and gender-matched controls, and in 25 liver tumors from northern Thailand. In total, 14 cases, 5 controls, and 19 tumors had detectable levels of HBV DNA. All 14 cases, 2 controls (2.9%), and 17 tumors (89.5%) were positive for the HBV double mutation. Nine cases (26.5%), 10 controls (14.7%), and 6 tumors (24%) were positive for the p53 mutation. Five cases (14.7%), no controls, and 4 tumors (16%) had both mutations. The median age of HCC diagnosis in these 5 cases was 34 years versus 51 years for other cases. Five cases (14.7%) and 1 control (1.5%) were HCV enzyme immunoassay positive. Thus, specific HBV, HCV, and aflatoxin biomarkers reveal the complexity of risks contributing to HCC in northern Thailand and suggest further application of these biomarkers as intermediate end points in prevention, intervention trials, and etiologic investigations.

A major risk factor for hepatocellular carcinoma (HCC) is hepatitis B virus (HBV), whose pathogenesis is exacerbated by the acquisition of mutations that accelerate carcinogenesis. We examined, with mass spectrometry, the temporality of an HBV 1762 T /1764 A double mutation in plasma and tumors. Initial studies found that 52 of 70 (74.3%) tumors from patients residing in Qidong, People's Republic of China, contained this HBV mutation. Paired plasma samples were available for six of the tumor specimens; four tumors had the HBV 1762 T /1764 A mutation, whereas three of the paired plasma samples were also positive. The potential predictive value of this biomarker was explored by using stored plasma samples from a study of 120 residents of Qidong who had been monitored for aflatoxin exposure and HBV infection. After 10 years of passive follow-up, there were six cases of major liver disease including HCC (four cases), hepatitis (one case), and cirrhosis (one case). All six cases had detectable levels of the HBV 1762 T /1764 A mutation up to 8 years before diagnosis. Finally, 15 liver cancers were selected from a prospective cohort of 1,638 high-risk individuals in Qidong on the basis of available plasma samples spanning the years before and after diagnosis. The HBV 1762 T /1764 A mutation was detected in 8 of the 15 cases (53.3%) before cancer. The persistence of detection of this mutation was statistically significant ( P = 0.022, two-tailed). We therefore found that a prediagnosis biomarker of specific HBV mutations can be measured in plasma and suggest this marker for use as an intermediate endpoint in prevention and intervention trials.

Epidemiological studies have shown that exposure to aflatoxin B(1) (AFB(1)) and concurrent infection with hepatitis B lead to a multiplicative risk of developing liver cancer. This chemical-viral interaction can be recapitulated in the tree shrew (Tupia belangeri chinensis). As an initial characterization of this model, the metabolism of AFB(1) in tree shrews has been examined and compared to a sensitive bioassay species, the rat. Utilizing LC/MS/MS, an unreported product, aflatoxin M(1)-N(7)-guanine (AFM(1)-N(7)-guanine), was detected in urine and hepatic DNA samples 24 h after administration of 400 microg/kg AFB(1). In hepatic DNA isolated from tree shrews, AFM(1)-N(7)-guanine was the predominant adduct, 0.74 +/- 0.14 pmol/mg DNA, as compared to 0.37 +/- 0.07 pmol/mg DNA of AFB(1)-N(7)-guanine. Conversely, in rat liver, 6.56 +/- 2.41 pmol/mg DNA of AFB(1)-N(7)-guanine and 0.42 +/- 0.13 pmol/mg DNA of AFM(1)-N(7)-guanine were detected. Rats excreted 1.00 +/- 0.21 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.29 +/- 0.10 pmol AFM(1)-N(7)-guanine/mg creatinine as compared to 0.60 +/- 0.12 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.69 +/- 0.16 pmol AFM(1)-N(7)-guanine/mg creatinine excreted by the tree shrew. Furthermore, tree shrew urine contained 40 times more of the hydroxylated metabolite, AFM(1), than was excreted by rats. In vitro experiments confirmed this difference in oxidative metabolism. Hepatic microsomes isolated from tree shrews failed to produce aflatoxin Q(1) or aflatoxin P(1) but formed a significantly greater amount of AFM(1) than rat microsomes. Bioassays indicated that the tree shrew was considerably more resistant than the rat to AFB(1) hepatocarcinogenesis, which may reflect the significant differences in metabolic profiles of the two species.

A specific missense mutation in the p53 tumor gene at codon 249 has been reported in over 50% of hepatocellular carcinoma (HCC) tumors and in paired blood samples from areas of high dietary exposure to aflatoxin B1, including Qidong, People's Republic of China. Using a combination of pre-digestion with HaeIII, PCR and mass spectrometry, the temporality of this mutation in plasma before and after the clinical diagnosis of HCC was examined. Sixteen liver cancer cases, diagnosed between 1997 and 2001, were selected from a prospective cohort of 1638 high-risk individuals in Qidong on the basis of available annual plasma samples spanning the years before and after diagnosis. The codon 249 mutation was detected in plasma samples obtained after diagnosis in seven of the 15 cases (46.7%) with PCR amplifiable DNA, which is in accord with the reported prevalence of this mutation in HCC. The persistent detection of this mutation in plasma collected annually following diagnosis was statistically significant (P = 0.024, two-tailed) in repetitive samples following diagnosis. Moreover, the mutation was detected in the plasma of four of eight cases positive at the time of diagnosis at least 1 year and in one case 5 years prior to diagnosis. Tracking of the marker in pre-diagnostic samples was borderline statistically significant (P = 0.066). None of the 18 healthy US control plasma samples had any detectable mutations. We have therefore found that pre-diagnosis biomarkers of specific p53 mutations can be measured in plasma and this suggests a paradigm for developing these markers for use in prevention and intervention trials.

Cancer contributes to a large proportion of the mortality and morbidity in the United States and worldwide. Despite advances in diagnosis and treatment of various cancers, early detection and treatment of cancer remain a challenge. Diagnosis of cancer often occurs once the disease has progressed to a point where currently available intervention options provide limited success. Therefore, techniques that enable early detection followed by targeted interventions would influence stage at diagnosis and, in turn, mortality associated with cancer. Identification of molecular biomarkers, especially those that are associated with cancer initiation and progression, shows promise as an effective strategy in this regard. One potential early detection biomarker is DNA methylation of the promoter region of certain cancer-associated genes, which results in gene inactivation. Examination of serum for circulating tumor DNA with abnormal methylation patterns offers a possible method for early detection of several cancers and serves as a point for early intervention and prevention strategies. Additionally, it is imperative to consider how such a screening mechanism can be implemented in populations at risk, especially in resource-poor settings. Thus, the challenge is to validate DNA methylation as a cancer-specific biomarker, with the ultimate goal of designing a research plan that integrates the current knowledge base regarding cancer detection and diagnosis into specific prevention and intervention strategies that can be applied at a population level.

Background: Aflatoxin, which is a risk factor for developing hepatocellular carcinoma, may also cause gallbladder cancer (GBC). We hypothesized that GBC patients have higher aflatoxin exposure than gallstone patients. Methods: We used samples and data from the Shanghai Biliary Tract Cancer Case-control Study conducted from 1997-2001. Of the 267 GBC and 730 gallstone participants with plasma available, 250 GBC patients and 250 controls with gallstones were randomly selected for measurement of aflatoxin B1 (AFB1)-lysine plasma adducts using isotope dilution mass spectrometry. In 54 GBC patients, tumor tissue was examined for the aflatoxin-related R249S TP53 mutation through targeted sequencing. Results: We calculated age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals (CIs), as well as the population attributable fraction (PAF). AFB1-lysine adduct detection was nearly 3-fold higher in GBC patients (OR, 2.96; 95% CI, 1.90 to 4.61). Among the 85 GBC patients (34%) with detectable AFB1-lysine adduct levels, the median was 4.6 pg/mg, compared to 1.2 pg/mg among the 37 gallstone-patient controls (15%) with detectable levels, and the OR was 9.97(95% CI, 2.73 to 36.44) for quartile 4 versus 1 in GBC compared to gallstone patients. The PAF for cancer related to aflatoxin was 22% (95% CI: 17%-27%). In 54 GBC patients, tumor tissue was examined for the aflatoxin-related R249S TP53 mutation through targeted sequencing, however, all patients were negative for the mutation. Conclusions: Aflatoxin is significantly associated with GBC, although it does not appear to be correlated with the R249S TP53 mutation in the gallbladder. If aflatoxin is shown to cause GBC, it may have accounted for up to 22% of the gallbladder cancers in Shanghai during the study period and may account for an even higher proportion in high-risk areas. If verified, reducing aflatoxin exposure may reduce GBC incidence. Citation Format: Jill E. Koshiol, Yu-Tang Gao, Michael Dean, Patricia Egner, Chirag Nepal, Kristine Jones, Bingsheng Wang , Asif Rashid, Wen Lou, Alison Van Dyke, Catterina Ferreccio, Michael Malasky, Ming-Chang Shen, Bin zhu, Jesper Andersen, Allan Hildesheim, Ann W. Hsing, John Groopman. Association of aflatoxin and gallbladder cancer in Shanghai [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-157. doi:10.1158/1538-7445.AM2017-LB-157

Low-income countries across the world are increasingly burdened with growing rates of cancer incidence and mortality. Opportunities for prevention in these populations may be realized most readily for cancer sites with known etiologies that are amenable to intervention. Liver cancer is the sixth-most commonly occurring cancer worldwide and is the second-most common contributor to global cancer mortality. Aflatoxin B1 (AFB1) is a known and potent human hepatocarcinogen, with exposure occurring via ingestion of grains - especially maize - contaminated with Aspergillus flavus. Previous work in regions with endemic levels of liver cancer has shown that reducing AFB1 exposure attenuated liver cancer mortality - even in the presence of other known etiological exposures, such as hepatitis B virus. Guatemala experiences high rates of liver cancer - which is uniquely distributed equally between men and women - and consumes maize as a dietary staple, making the assessment of AFB1 exposure a high priority for cancer prevention efforts in this country. Therefore, we measured circulating levels of the AFB1-albumin adduct, an established biomarker of AFB1 exposure, in adults residing in five geographically and ecologically dispersed departments of Guatemala (Guatemala, Suchitepéquez, Sololá, Escuintla, and Quiché). Healthy men and women aged 40 and older were recruited for the study. Pregnant women or individuals with implanted cardiac devices were excluded from participation. Subjects (n=436) gave informed consent and provided demographic information and a venipuncture blood sample during a clinic visit. Serum was separated from whole blood, digested with protease, subjected to solid-phase extraction, and analyzed with isotope dilution ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometry. Serum albumin was measured by ELISA. AFB1-albumin adducts (pg AFB1-lysine adduct/mg albumin) were detectable in 100% of samples, with a mean ± SEM of 21.6 ± 2.5 and range of 0.2 - 814.8. These levels are similar to those observed in regions of China in the 1980s, prior to reductions in AFB1 exposure and liver cancer mortality. Stratification by demographic factors revealed significant differences in aflatoxin levels in urban (9.6 ± 1.3) versus rural (28.8 ± 3.9) subjects (p ≤ 0.001). Accordingly, AFB1-albumin adduct levels were lowest in the urban municipality of Escuintla (5.8 ± 1.1) and highest in rural Suchitepéquez (36.4 ± 4.8). Exposure was significantly different by sex, with men having higher levels compared to women (30.0 ± 5.4 vs. 15.3 ± 1.3, p = 0.01). These data show that AFB1 exposure in Guatemalan adults is similar to levels previously shown to increase liver cancer risk. Larger cohort and prospective studies are warranted to assess the relationship between liver cancer incidence and AFB1 exposure in this population. Citation Format: Joshua W. Smith, Patricia A. Egner, Mariana Lazo, Alvaro Rivera, Fernanda Kroker, Neal D. Freedman, Katherine A. McGlynn, Manuel Ramirez-Zea, Eliseo Guallar, John D. Groopman. Quantitative measurement of aflatoxin-serum albumin adducts reveals substantial exposure in Guatemala, a country with high rates of liver cancer mortality in men and women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4247. doi:10.1158/1538-7445.AM2017-4247

In experimental animals and humans, aflatoxin B1 (AFB1) is a potent hepatic toxin and carcinogen. The synthetic oleanane triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a powerful activator of Keap1-Nrf2 signaling, protects against AFB1-induced toxicity and preneoplastic lesion formation (GST-P–positive foci). This study assessed and mechanistically characterized the chemoprotective efficacy of CDDO-Im against AFB1-induced hepatocellular carcinoma (HCC). A lifetime cancer bioassay was undertaken in F344 rats dosed with AFB1 (200 μg/kg rat/day) for four weeks and receiving either vehicle or CDDO-Im (three times weekly), one week before and throughout the exposure period. Weekly, 24-hour urine samples were collected for analysis of AFB1 metabolites. In a subset of rats, livers were analyzed for GST-P foci. The comparative response of a toxicogenomic RNA expression signature for AFB1 was examined. CDDO-Im completely protected (0/20) against AFB1-induced liver cancer compared with a 96% incidence (22/23) observed in the AFB1 group. With CDDO-Im treatment, integrated level of urinary AFB1-N7-guanine was significantly reduced (66%) and aflatoxin-N-acetylcysteine, a detoxication product, was consistently elevated (300%) after the first AFB1 dose. In AFB1-treated rats, the hepatic burden of GST-P–positive foci increased substantially (0%–13.8%) over the four weeks, but was largely absent with CDDO-Im intervention. The toxicogenomic RNA expression signature characteristic of AFB1 was absent in the AFB1 + CDDO-Im–treated rats. The remarkable efficacy of CDDO-Im as an anticarcinogen is established even in the face of a significant aflatoxin adduct burden. Consequently, the absence of cancer requires a concept of a threshold for DNA damage for cancer development. Cancer Prev Res; 7(7); 658–65. ©2014 AACR.