Search

One of several challenges in design of clinical chemoprevention trials is the selection of the dose, formulation, and dose schedule of the intervention agent. Therefore, a cross-over clinical trial was undertaken to compare the bioavailability and tolerability of sulforaphane from two of broccoli sprout–derived beverages: one glucoraphanin-rich (GRR) and the other sulforaphane-rich (SFR). Sulforaphane was generated from glucoraphanin contained in GRR by gut microflora or formed by treatment of GRR with myrosinase from daikon (Raphanus sativus) sprouts to provide SFR. Fifty healthy, eligible participants were requested to refrain from crucifer consumption and randomized into two treatment arms. The study design was as follows: 5-day run-in period, 7-day administration of beverages, 5-day washout period, and 7-day administration of the opposite intervention. Isotope dilution mass spectrometry was used to measure levels of glucoraphanin, sulforaphane, and sulforaphane thiol conjugates in urine samples collected daily throughout the study. Bioavailability, as measured by urinary excretion of sulforaphane and its metabolites (in approximately 12-hour collections after dosing), was substantially greater with the SFR (mean = 70%) than with GRR (mean = 5%) beverages. Interindividual variability in excretion was considerably lower with SFR than with GRR beverage. Elimination rates were considerably slower with GRR, allowing for achievement of steady-state dosing as opposed to bolus dosing with SFR. Optimal dosing formulations in future studies should consider blends of sulforaphane and glucoraphanin as SFR and GRR mixtures to achieve peak concentrations for activation of some targets and prolonged inhibition of others implicated in the protective actions of sulforaphane. Cancer Prev Res; 4(3); 384–95. ©2011 AACR.

Supplementary Materials and Methods.

Supplementary Table 1 This Table presence of SF metabolites in patient urine specimens.

Supplementary Tables from Bioavailability of Sulforaphane from Two Broccoli Sprout Beverages: Results of a Short-term, Cross-over Clinical Trial in Qidong, China

Supplementary Figures 1, 2, 3, 4, 5 Supplementary Fig 1 shows SF induction of NRF2 in HPV-negative and HPV-positive cell lines. Supplementary Fig 2 shows induction of NRF2 by proteasome inhibitors. Supplementary Fig 3 shows SF induction of the NRF2 target gene NQO1 in HPV-negative and HPV-positive cell lines. Supplementary Fig 4 shows SF induction of the NRF2 target gene GCLC in HPV-negative and HPV-positive cell lines. Supplementary Fig 5 shows dose-response (cell death) curves for SF in HPV-negative and HPV-positive cell lines.

Supplementary Figure from Bioavailability of Sulforaphane from Two Broccoli Sprout Beverages: Results of a Short-term, Cross-over Clinical Trial in Qidong, China

Supplementary Figure Legends and Table Legends.

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.

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.

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.

P450 activity is required to metabolically activate many chemical carcinogens, rendering them highly genotoxic. CYP3A4 is the most abundantly expressed P450 enzyme in the liver, accounting for most drug metabolism and constituting 50% of all hepatic P450 activity. CYP3A4 is also expressed in extrahepatic tissues, including the intestine. However, the role of CYP3A4 in activating chemical carcinogens into potent genotoxins is unclear. To facilitate efforts to determine whether CYP3A4, per se, can activate carcinogens into potent genotoxins, we expressed human CYP3A4 in the DNA-repair mutant (rad4 rad51) strain of budding yeast Saccharomyces cerevisiae and tested the novel, recombinant yeast strain for ability to report CYP3A4-mediated genotoxicity of a well-known genotoxin, aflatoxin B1 (AFB1 ). Yeast microsomes containing human CYP3A4, but not those that do not contain CYP3A4, were active in hydroxylation of diclofenac, a known CYP3A4 substrate drug, a result confirming CYP3A4 activity in the recombinant yeast strain. In cells exposed to AFB1 , the expression of CYP3A4 supported DNA adduct formation, chromosome rearrangements, cell death, and expression of the large subunit of ribonucleotide reductase, Rnr3, a marker of DNA damage. Expression of CYP3A4 also conferred sensitivity in rad4 rad51 mutants exposed to colon carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). These data confirm the ability of human CYP3A4 to mediate the genotoxicity of AFB1 , and illustrate the usefulness of the CYP3A4-expressing, DNA-repair mutant yeast strain for screening other chemical compounds that are CYP3A4 substrates, for potential genotoxicity. Environ. Mol. Mutagen. 58:217-227, 2017. © 2017 Wiley Periodicals, Inc.

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.

Objective:Chronic aflatoxin exposure has been associated with childhood stunting (length-for-age/height-for-age < –2 sd), while data lacks for Bangladesh, a country with substantial burden of childhood stunting. This paper examined the association between aflatoxin exposure and childhood stunting in a slum setting of Dhaka city.Design:In this MAL-ED aflatoxin birth cohort study, plasma samples were assayed for aflatoxin B1-lysine adduct (AFB1-lys) by MS at 7, 15, 24 and 36 months of age for 208, 196, 173 and 167 children to assess chronic aflatoxin exposure. Relationship between aflatoxin exposure and anthropometric measures was examined by mixed-effects logistic regression models.Setting and participants:The study was conducted in Mirpur, Dhaka, where children were followed from birth to 36 months.Results:Prevalence of stunting increased from 21 % at 7 months to 49 % at 36 months of age. Mean AFB1-lys concentrations at 7, 15, 24 and 36 months were 1·30 (range 0·09–5·79), 1·52 (range 0·06–6·35), 3·43 (range 0·15–65·60) and 3·70 (range 0·09–126·54) pg/mg albumin, respectively, and the percentage of children with detectable AFB1-lys was 10, 21, 18 and 62 %, respectively. No association was observed between aflatoxin exposure and stunting in multivariable analyses. Factors associated with childhood stunting were age, low birth weight, maternal height, stool myeloperoxidase and number of people sleeping in one room.Conclusions:A relatively lower exposure to aflatoxin may not influence the linear growth of children. This finding indicates a threshold level of exposure for linear growth deficit and further investigation in other areas where higher concentrations of aflatoxin exposure exist.

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.

Glucosinolates (GS) are metabolized to isothiocyanates that may enhance human healthspan by protecting against a variety of chronic diseases. Moringa oleifera, the drumstick tree, produces unique GS but little is known about GS variation within M. oleifera, and even less in the 12 other Moringa species, some of which are very rare. We assess leaf, seed, stem, and leaf gland exudate GS content of 12 of the 13 known Moringa species. We describe 2 previously unidentified GS as major components of 6 species, reporting on the presence of simple alkyl GS in 4 species, which are dominant in M. longituba. We document potent chemoprotective potential in 11 of 12 species, and measure the cytoprotective activity of 6 purified GS in several cell lines. Some of the unique GS rank with the most powerful known inducers of the phase 2 cytoprotective response. Although extracts of most species induced a robust phase 2 cytoprotective response in cultured cells, one was very low (M. longituba), and by far the highest was M. arborea, a very rare and poorly known species. Our results underscore the importance of Moringa as a chemoprotective resource and the need to survey and conserve its interspecific diversity.

Dietary exposure to aflatoxin is implicated in growth faltering of children. Despite the high burden of childhood stunting in urban Bangladesh, there are no data on long-term exposure to aflatoxin. This study aimed to explore aflatoxin exposure levels in a group of children followed longitudinally. The current study used data and biospecimens collected during 2010–2014 as part of the MAL-ED birth cohort study in an urban slum of Mirpur, Dhaka where children were followed from birth to 36 months. AFB1-lysine adduct concentrations were determined by isotope dilution mass spectrometry from plasma samples collected at 7, 15, 24, and 36 months of age. The limit of detection was 0.5 pg of AFB1-lys/mg albumin. In 744 plasma samples, the geometric mean of AFB1-lysine/mg albumin was 1.07 pg (range 0.04–123.5 pg/mg albumin). The proportion of children with detectable aflatoxin exposure was 10.1, 20.9, 17.9, and 61.7% for 7, 15, 24, and 36 months, respectively. Reduction in breastfeeding prevalence (80% at 24 months vs. 38% in 36 months) corresponded with the high-level detection of AFB1-lysine at the age of 36 months. AFB1-lysine concentrations were the highest at the end of monsoon. This study reveals that 62% of children in slum settlement were exposed to aflatoxin by the end of the third year of life. High aflatoxin exposure was detected at the end of rainy season and with the introduction of family food. These findings suggest interventions to ameliorate the problem of chronic aflatoxin exposure including childhood stunting.

Pregnancy is a complex physiological state, in which the metabolism of endogenous as well as exogenous agents is ostensibly altered. One exogenous agent of concern is the hepatocarcinogen aflatoxin B1(AFB1), a foodborne fungal toxin, that requires phase I metabolic oxidation for conversion to its toxic and carcinogenic form, the AFB1-8,9-exo-epoxide. The epoxide interacts with cellular targets causing toxicity and cell death; these targets include the covalent modification of DNA leading to mutations that can initiate malignant transformation. The main detoxification pathway of the AFB1- epoxide involves phase II metabolic enzymes including the glutathione-S-transferase (GST) family. Pregnancy can modulate both phase I and II metabolism and alter the biological potency of AFB1. The present work investigated the impact of pregnancy on AFB1exposure in mice. A single IP dose of 6 mg/kg AFB1was administered to pregnant C57BL/6 J mice at gestation day 14 and matched non-pregnant controls. Pregnant mice accumulated 2-fold higher AFB1-N7-guanine DNA adducts in the liver when compared with nonpregnant controls 6h post-exposure. Enhanced DNA adduct formation in pregnant animals paralleled elevated hepatic protein expression of mouse CYP1A2 and mouse homologs of human CYP3A4, phase I enzymes capable of bioactivating AFB1. Although phase II enzymes GSTA1/2 showed decreased protein expression, GSTA3, the primary enzymatic protection against the AFB1-epoxide, was unaffected at the protein level. Taken together, our results reveal that pregnancy may constitute a critical window of susceptibility for maternal health, and provide insight into the biochemical factors that could explain the underlying risks., National Institutes of Health (U.S.) (Grant T32-ES007020), National Institutes of Health (U.S.) (Grant P30-ES002109), National Institutes of Health (U.S.) (Grant R01-ES016313), National Institutes of Health (U.S.) (Grant R01-CA080024), National Institutes of Health (U.S.) (Grant P30-CA006973)

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.

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.

It has become axiomatic that critical windows of susceptibility to genotoxins exist and that genetic damage in utero may be a trigger for later life cancers. Data supporting this critical window hypothesis are remarkably few. This study provides a quantitative bridge between DNA damage by the liver carcinogen aflatoxin B1 (AFB1) during prenatal development and the risk of later life genetic disease. AFB1 was given to pregnant C57BL/6J mice, carrying F1 gestation day 14 (GD14) embryos of the B6C3F1 genotype. Ultra-high performance liquid chromatography and mass spectrometry (UPLC-MS) using aflatoxin-15N5-guanine adduct standards afforded measurement of the AFB1-N7-Gua and AFB1-FAPY adducts six hours post dosing in liver DNA of mothers and embryos. A parallel cohort gave birth and the livers of the F1 were analyzed for mutations in the gpt gene at three and ten weeks of age. The data revealed mutational spectra dominated by G:C to T:A mutations in both the mother and offspring that are characteristic of AFB1 and distinct from background. It was shown that adducts in GD14 embryos were 20-fold more potent inducers of mutagenesis than adducts in parallel-dosed adults. This sensitivity enhancement correlated with Ki67 staining of the liver, reflecting the proliferative potential of the tissue. Taken together, these data provide insight into the relative genetic risks of prenatal and adult exposures to AFB1. Early life exposure, especially during the embryonic period, is strikingly more mutagenic than treatment later in life. Moreover the data provide a baseline against which risk prevention strategies can be evaluated.

Broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can generate the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases (GST) and other cytoprotective enzymes. A broccoli sprout–derived beverage providing daily doses of 600 μmol glucoraphanin and 40 μmol sulforaphane was evaluated for magnitude and duration of pharmacodynamic action in a 12-week randomized clinical trial. Two hundred and ninety-one study participants were recruited from the rural He-He Township, Qidong, in the Yangtze River delta region of China, an area characterized by exposures to substantial levels of airborne pollutants. Exposure to air pollution has been associated with lung cancer and cardiopulmonary diseases. Urinary excretion of the mercapturic acids of the pollutants, benzene, acrolein, and crotonaldehyde, were measured before and during the intervention using liquid chromatography tandem mass spectrometry. Rapid and sustained, statistically significant (P ≤ 0.01) increases in the levels of excretion of the glutathione-derived conjugates of benzene (61%), acrolein (23%), but not crotonaldehyde, were found in those receiving broccoli sprout beverage compared with placebo. Excretion of the benzene-derived mercapturic acid was higher in participants who were GSTT1-positive than in the null genotype, irrespective of study arm assignment. Measures of sulforaphane metabolites in urine indicated that bioavailability did not decline over the 12-week daily dosing period. Thus, intervention with broccoli sprouts enhances the detoxication of some airborne pollutants and may provide a frugal means to attenuate their associated long-term health risks. Cancer Prev Res; 7(8); 813–23. ©2014 AACR.

Mice are resistant to aflatoxin hepatotoxicity, primarily due to high expression of glutathione S-transferases (GSTs), and in particular the GSTA3 subunit. Nuclear factor erythroid 2 related factor 2 (Nrf2) signaling, which controls a broad-based cytoprotective response, was activated either genetically or pharmacologically in an attempt to rescue GSTA3 knockout mice from aflatoxin genotoxicity. Genetic activation of Nrf2 signaling was attained in a GSTA3: hepatocyte-specific Keap1 double knockout (DKO) mouse whereas pharmacologic activation of Nrf2 was achieved through pretreatment of mice with the triterpenoid 1-[2-cyano-3-,12-dioxoleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) prior to aflatoxin B1 exposure. Following oral treatment with aflatoxin, urine was collected from mice for 24 h and hepatic and urinary aflatoxin metabolites then quantified using isotope dilution-mass spectrometry. Although Nrf2 was successfully activated genetically and pharmacologically, neither means affected the response of GSTA3 knockout mice to chemical insult with aflatoxin. Hepatic aflatoxin B1-N(7)-guanine levels were elevated 120-fold in GSTA3 knockout mice compared with wild-type and levels were not attenuated by the interventions. This lack of effect was mirrored in the urinary excretion of aflatoxin B1-N(7)-guanine. By contrast, urinary excretion of aflatoxin B1-N-acetylcysteine was200-fold higher in wild-type mice compared with the single GSTA3 knockout or DKO mouse. The inability to rescue GSTA3 knockout mice from aflatoxin genotoxicity through the Nrf2 transcriptional program indicates that Gsta3 is unilaterally responsible for the detoxication of aflatoxin in mice.

Human susceptibility to environmental carcinogens is highly variable and depends on multiple genetic factors, including polymorphisms in cytochrome P450 genes. Although epidemiological studies have identified individual polymorphisms in cytochrome P450 genes that may alter cancer risk, there is often conflicting data about whether such polymorphisms alter the genotoxicity of environmental carcinogens. This is particularly true of the CYP1A2 polymorphisms that confer differential activation of multiple human carcinogens. To determine whether a single cytochrome P450 polymorphism confers higher levels of carcinogen-associated genotoxicity, we chose an organism that lack enzymes to metabolically activate aflatoxins and expressed individual human P450 genes in budding yeast. We measured the frequencies of recombination, Rad51 foci formation, 7-methoxyresorufin O-demethylase activities, and the concentrations of carcinogen-associated DNA adducts in DNA repair proficient yeast expressing P450 polymorphisms after exposure to aflatoxin B1 (AFB1).We measured growth of rad4 rad51 cells expressing CYP1A2 polymorphisms while exposed to AFB1. We observed that there was significantly less AFB1-associated genotoxicity in yeast expressing CYP1A2 I386F, while yeast expressing CYP1A2 C406Y exhibited intermediate levels of genotoxicity compared to yeast expressing CYP1A2 D348N or wild type. We conclude that differences in carcinogen genotoxicity can be observed in yeast expressing different CYP1A2 alleles. This is the first report that carcinogen-associated P450 polymorphisms can be studied in yeast.

Exposure to aflatoxin, a mycotoxin common in many foods, has been associated with child growth impairment in sub-Saharan Africa. To improve our understanding of growth impairment in relation to aflatoxin and other risk factors, we assessed biospecimens collected in Nepalese children at 15, 24, and 36 months of age for aflatoxin exposure. Children (N = 85) enrolled in the Bhaktapur, Nepal MAL-ED study encompassed the cohort analysed in this study. Exposure was assessed through a plasma biomarker of aflatoxin exposure: the AFB1-lysine adduct. The aflatoxin exposures in the study participants were compared to anthropometrics at each time period (length-for-age [LAZ], weight-for-age [WAZ], and weight-for-length [WLZ] z-scores), growth trajectories over time, age, and breastfeeding status. Results demonstrated chronic aflatoxin exposure in this cohort of children, with a geometric mean of 3.62 pg AFB1-lysine/mg albumin. However, the chronic aflatoxin exposure in this cohort was not significantly associated with anthropometric z-scores, growth trajectories, age, or feeding status, based on the available time points to assess aflatoxin exposure. Low mean levels of aflatoxin exposure and infrequent occurrence of stunting, wasting, or underweight z-score values in this cohort are possible contributing factors to a lack of evidence for an association. Further research is needed to examine whether a threshold dose of aflatoxin exists that could induce child growth impairment.

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

The transcription factor Nrf2 (NF-E2-related-factor 2) regulates a battery of antioxidative stress-response genes and detoxication genes, and Nrf2 knockout lines of mice have been contributing critically to the clarification of roles that Nrf2 plays for cell protection. However, there are apparent limitations in use of the mouse models. For instance, rats exhibit more suitable features for toxicological or physiological examinations than mice. In this study, we generated 2 lines of Nrf2 knockout rats by using a genome editing technology; 1 line harbors a 7-bp deletion (Δ7) and the other line harbors a 1-bp insertion (+1) in the Nrf2 gene. In the livers of rats homozygously deleting the Nrf2 gene, an activator of Nrf2 signaling, CDDO-Im, could not induce expression of representative Nrf2 target genes. To examine altered toxicological response, we treated the Nrf2 knockout rats with aflatoxin B1 (AFB1), a carcinogenic mycotoxin that elicits gene mutations through binding of its metabolites to DNA and for which the rat has been proposed as a reasonable surrogate for human toxicity. Indeed, in the Nrf2 knockout rat livers the enzymes of the AFB1 detoxication pathway were significantly downregulated. Single dose administration of AFB1 increased hepatotoxicity and binding of AFB1-N7-guanine to hepatic DNA in Nrf2 knockout rats compared with wild-type. Nrf2 knockout rats repeatedly treated with AFB1 were prone to lethality and CDDO-Im was no longer protective. These results demonstrate that Nrf2 knockout rats are quite sensitive to AFB1 toxicities and this rat genotype emerges as a new model animal in toxicology.

Epidemiological evidence has suggested that consumption of a diet rich in cruciferous vegetables reduces the risk of several types of cancers and chronic degenerative diseases. In particular, broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can release the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases. Two broccoli sprout-derived beverages, one sulforaphane-rich (SFR) and the other glucoraphanin-rich (GRR), were evaluated for pharmacodynamic action in a crossover clinical trial design. Study participants were recruited from the farming community of He Zuo Township, Qidong, China, previously documented to have a high incidence of hepatocellular carcinoma with concomitant exposures to aflatoxin and more recently characterized with exposures to substantive levels of airborne pollutants. Fifty healthy participants were randomized into two treatment arms. The study protocol was as follows: a 5 days run-in period, a 7 days administration of beverage, a 5 days washout period and a 7 days administration of the opposite beverage. Urinary excretion of the mercapturic acids of acrolein, crotonaldehyde, ethylene oxide and benzene were measured both pre- and postinterventions using liquid chromatography tandem mass spectrometry. Statistically significant increases of 20–50% in the levels of excretion of glutathione-derived conjugates of acrolein, crotonaldehyde and benzene were seen in individuals receiving SFR, GRR or both compared with their preintervention baseline values. No significant differences were seen between the effects of SFR versus GRR. Intervention with broccoli sprouts may enhance detoxication of airborne pollutants and attenuate their associated health risks.

One of several challenges in design of clinical chemoprevention trials is the selection of the dose, formulation, and dose schedule of the intervention agent. Therefore, a cross-over clinical trial was undertaken to compare the bioavailability and tolerability of sulforaphane from two of broccoli sprout–derived beverages: one glucoraphanin-rich (GRR) and the other sulforaphane-rich (SFR). Sulforaphane was generated from glucoraphanin contained in GRR by gut microflora or formed by treatment of GRR with myrosinase from daikon (Raphanus sativus) sprouts to provide SFR. Fifty healthy, eligible participants were requested to refrain from crucifer consumption and randomized into two treatment arms. The study design was as follows: 5-day run-in period, 7-day administration of beverages, 5-day washout period, and 7-day administration of the opposite intervention. Isotope dilution mass spectrometry was used to measure levels of glucoraphanin, sulforaphane, and sulforaphane thiol conjugates in urine samples collected daily throughout the study. Bioavailability, as measured by urinary excretion of sulforaphane and its metabolites (in approximately 12-hour collections after dosing), was substantially greater with the SFR (mean = 70%) than with GRR (mean = 5%) beverages. Interindividual variability in excretion was considerably lower with SFR than with GRR beverage. Elimination rates were considerably slower with GRR, allowing for achievement of steady-state dosing as opposed to bolus dosing with SFR. Optimal dosing formulations in future studies should consider blends of sulforaphane and glucoraphanin as SFR and GRR mixtures to achieve peak concentrations for activation of some targets and prolonged inhibition of others implicated in the protective actions of sulforaphane. Cancer Prev Res; 4(3); 384–95. ©2011 AACR.

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)

Aflatoxin B1 (AFB1) is a major risk factor for hepatocellular carcinoma (HCC) in humans. However, mice, a major animal model for the study of AFB1 carcinogenesis, are resistant, due to high constitutive expression, in the mouse liver, of glutathione S-transferase A3 subunit (mGSTA3) that is lacking in humans. Our objective was to establish that a mouse model for AFB1 toxicity could be used to study mechanisms of toxicity that are relevant for human disease, i.e., an mGSTA3 knockout (KO) mouse that responds to toxicants such as AFB1 in a manner similar to humans. Exons 3-6 of the mGSTA3 were replaced with a neomycin cassette by homologous recombination. Southern blotting, RT-PCR, Western blotting, and measurement of AFB1-N(7)-DNA adduct formation were used to evaluate the mGSTA3 KO mice. The KO mice have deletion of exons 3-6 of the mGSTA3 gene, as expected, as well as a lack of mGSTA3 expression at the mRNA and protein levels. Three hours after injection of 5 mg/kg AFB1, mGSTA3 KO mice have more than 100-fold more AFB1-N(7)-DNA adducts in their livers than do similarly treated wild-type (WT) mice. In addition, the mGSTA3 KO mice die of massive hepatic necrosis, at AFB1 doses that have minimal toxic effects in WT mice. We conclude that mGSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of AFB1, confirming the crucial role of GSTA3 subunit in protection of normal mice against AFB1 toxicity. We propose the mGSTA3 KO mouse as a useful model with which to study the interplay of risk factors leading to HCC development in humans, as well as for testing of additional possible functions of mGSTA3.

We report validation of the first high-pressure liquid chromatography isotope-dilution mass spectrometry method to measure sulforaphane (SFN) and its glutathione-derived conjugates in human urine. As epidemiological evidence continues to mount that the consumption of a diet rich in cruciferous vegetables may reduce the risk of certain cancers, the development of analytical methodologies to accurately measure isothiocyanates (ITCs) and their subsequent metabolic products becomes paramount. SFN, the principal ITC produced by broccoli, is an effective chemopreventive agent with multiple modes of action. SFN and SFN conjugates have often been measured collectively utilizing a cyclocondensation assay with 1,2-benzenedithiol. More recently, some of the major SFN conjugates have been determined using mass spectrometry. Here, triple-quadrupole mass spectrometry has been coupled with the use of stable isotope-labeled internal standards of D8-SFN and all four D8-SFN mercapturic acid pathway conjugates to provide an accurate, precise, sensitive, and specific method for analysis of these compounds. Using urine samples collected during an earlier intervention with broccoli sprouts, the concentrations of SFN, SFN-cysteine, and the mercapturic acid SFN- N-acetylcysteine were sufficiently high such that as little as 50 nL of urine was required for analysis. Although each study participant received an equivalent dose of broccoli sprout preparation, the interindividual conversion of the precursor glucosinolate to SFN varied over 100-fold. These 98 urines provided an ideal sample set for examining the robustness of the assay. The mean urinary concentrations +/- standard deviations in overnight voids following ingestion of the first dose were 4.7 +/- 5.1, 0.03 +/- 0.05, 0.06 +/- 0.06, 18 +/- 15, and 42 +/- 23 nmol/mg creatinine for SFN, SFN-glutathione, SFN-cysteine-glycine, SFN-cysteine, and SFN- N-acetylcysteine, respectively. This method determines SFN and all four SFN glutathione-derived metabolites with minimal sample preparation and will be extremely useful in understanding the role of SFN-rich foods in preventing cancer and other chronic diseases.

A novel acetylenic tricyclic bis-(cyano enone), TBE-31, is a lead compound in a series of tricyclic compounds with enone functionalities in rings A and C. Nanomolar concentrations of this potent multifunctional molecule suppress the induction of the inflammatory protein, inducible nitric oxide synthase, activate phase 2 cytoprotective enzymes in vitro and in vivo, block cell proliferation, and induce differentiation and apoptosis of leukemia cells. Oral administration of TBE-31 also significantly reduces formation of aflatoxin-DNA adducts and decreases size and number of aflatoxin-induced preneoplastic hepatic lesions in rats by >90%. Because of the two cyano enones in rings A and C, TBE-31 may directly interact with DTT and protein targets such as Keap1 that contain reactive cysteine residues. The above findings suggest that TBE-31 should also be tested for chemoprevention and chemotherapy in relevant models of cancer and against other chronic, degenerative diseases in which inflammation and oxidative stress contribute to disease pathogenesis. [Cancer Res 2008;68(16):6727–33]

The hepatocarcinogen aflatoxin B(1) (AFB(1)) is a potent recombinagen but weak mutagen in the yeast Saccharomyces cerevisiae. AFB(1) exposure induces DNA damage-inducible genes, such as RAD51 and those encoding ribonucleotide reductase (RNR), through a MEC1 (ATR homolog)-dependent pathway. Previous studies have indicated that MEC1 is required for both AFB(1)-associated recombination and mutation, and suggested that AFB(1)-DNA adducts are common substrates for recombination and mutagenesis. However, little is known about the downstream effectors of MEC1 required for genotoxic events associated with AFB(1) exposure. Here we show that AFB(1) exposure increases frequencies of RAD51-dependent unequal sister chromatid exchange (SCE) and activates Rad53 (CHK2). We found that MEC1, RAD53, and DUN1 are required for both AFB(1)-associated mutation and SCE. Deletion of SML1, which encodes an inhibitor of RNR, did not suppress the DUN1-dependent requirement for AFB(1)-associated genetic events, indicating that higher dNTP levels could not suppress the dun1 phenotype. We identified AFB(1)-DNA adducts and show that approximately the same number of adducts are obtained in both wild type and rad53 mutants. Since DUN1 is not required for UV-associated mutation and recombination, these studies define a distinct role for DUN1 in AFB(1)-associated mutagenesis and recombination. We speculate that AFB(1)-associated DNA adducts stall DNA replication, a consequence of which can either be mutation or recombination.

The reduction of the aflatoxin B 1 (AFB 1) dialdehyde metabolite to its corresponding mono and dialcohols, catalyzed by aflatoxin B 1-aldehyde reductase (AFAR, rat AKR7A1, and human AKR7A3), is greatly increased in livers of rats treated with numerous chemoprotective agents. Recombinant human AKR7A3 has been shown to reduce the AFB 1-dialdehyde at rates greater than those of the rat AKR7A1. The activity of AKR7A1 or AKR7A3 may detoxify the AFB 1-dialdehyde, which reacts with proteins, and thereby inhibits AFB 1-induced toxicity; however, direct experimental evidence of this hypothesis was lacking. Two human B lymphoblastoid cell lines, designated pMF6/1A2/AKR7A1 and pMF6/1A2, were genetically engineered to stably express AKR7A1 and/or cytochrome P4501A2 (1A2). The pMF6/1A2/AKR7A1 cells were refractory to the cytotoxic effects of 3 ng/mL AFB 1, in comparison to pM6/1A2 cells, which were more sensitive. Diminished protection occurred at higher concentrations of AFB 1 in pMF6/1A2/AKR7A1 cells, suggesting that additional factors were influencing cell survival. COS-7 cells were transfected with either vector control, rat AKR7A1, or human AKR7A3, and the cells were treated with AFB 1-dialdehyde. There was a 6-fold increase in the dialdehyde LC 50, from 66 microM in vector-transfected cells to 400 microM in AKR7A1-transfected cells, and an 8.5-fold increase from 35 microM in vector-transfected cells to 300 microM in AKR7A3-transfected cells. In both cases, this protective effect of the AFAR enzyme was accompanied by a marked decrease in protein adducts. Fractionation of the cellular protein showed that the mitochondria/nuclei and microsomal fractions contained the highest concentration of protein adducts. The levels of human AKR7A3 and AKR7A2 were measured in 12 human liver samples. The expression of AKR7A3 was detectable in all livers and lower than those of AKR7A2 in 11 of the 12 samples. Overall, these results provide the first direct evidence of a role for rat AKR7A1 and human AKR7A3 in protection against AFB 1-induced cytotoxicity and protein adduct formation.

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

Exposure to aflatoxin, a mycotoxin common in maize and groundnuts, has been associated with childhood stunting in sub-Saharan Africa. In an effort to further our understanding of growth impairment in relation to mycotoxins and other risk factors, biospecimens from a cohort of children enrolled in the Bhaktapur, Nepal MAL-ED study were assessed for aflatoxin exposure at 15, 24, and 36 months of age. Exposure was assessed through a well-established serum biomarker, the AFB1-lysine adduct. In this manuscript, the levels of aflatoxin exposure in the Nepal cohort were compared with those observed in aflatoxin studies, with child growth parameters as a health outcome. Results from this preliminary analysis demonstrated chronic aflatoxin exposure in children residing in Bhaktapur with a geometric mean of 3.62 pg AFB1-lysine/mg albumin. The range of exposure in this population is similar to those in African populations where associations with aflatoxin biomarkers and poor child growth have been observed. Future work will analyze the relationships between aflatoxin levels, growth, and other risk factors collected by the MAL-ED study.

We report validation of the first isotope dilution mass spectrometry method for determination of aflatoxin B(1)-N(7)-guanine (AFB(1)-N(7)-Gua), a major human aflatoxin-DNA adduct that is excreted in the urine. Measurement of urinary AFB(1)-N(7)-Gua, a biomarker of the biologically effective dose following dietary aflatoxin B(1) (AFB(1)) exposure, has helped identify AFB(1) as a risk factor in the development of hepatocellular carcinoma, a common cancer worldwide. Triple-quadrupole mass spectrometry, coupled with the use of a stable isotope-labeled internal standard (AFB(1)-N(7)-(15)N(5)-Gua) and better solid phase extraction and immunoaffinity column chromatography, have enabled us to greatly improve accuracy, precision, specificity, and sensitivity over previously published determinations. The limit of quantitation for AFB(1)-N(7)-Gua was 0.8 pg/20 mL urine (0.07 pg/mg creatinine). The method was validated for accuracy and precision over the range of 0.8-25 pg/20 mL urine, with between-day and within-day reproducibility for analysis of six aliquots of a human urine sample containing 6.0 pg/20 mL measured at

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.

Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part due to consumption of aflatoxin-contaminated foods, and are exposed to high levels of phenanthrene, a sentinel of hydrocarbon air toxics. Cruciferous vegetables, such as broccoli, contain anticarcinogens. Glucoraphanin, the principal glucosinolate in broccoli sprouts, can be hydrolyzed by gut microflora to sulforaphane, a potent inducer of carcinogen detoxication enzymes. In a randomized, placebo-controlled chemoprevention trial, we tested whether drinking hot water infusions of 3-day-old broccoli sprouts, containing defined concentrations of glucosinolates, could alter the disposition of aflatoxin and phenanthrene. Two hundred healthy adults drank infusions containing either 400 or

Plant chlorophylls and carotenoids are highly colored, conjugated polyenes that play central roles in photosynthesis. Other porphyrins (tetrapyrroles), such as cytochromes, which are structurally related to chlorophyll, participate in redox reactions in many living systems. An unexpected new property of tetrapyrroles, including tetramethyl coproporphyrin III, tetrabenzoporphine, copper chlorin e4 ethyl ester, and of carotenoids including zeaxanthin and alpha-cryptoxanthin is their ability to induce mammalian phase 2 proteins that protect cells against oxidants and electrophiles. The capacity of these compounds to induce the phase 2 response depends upon their ability or that of their metabolites to react with thiol groups, a property shared with all other classes of phase 2 inducers, which show few other structural similarities. Pseudo second-order rate constants of these inducers are correlated with their potency in inducing the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in murine hepatoma cells. One of the most potent inducers was isolated from chlorophyllin, a semisynthetic water-soluble chlorophyll derivative. Although chlorophyll itself is low in inducer potency, it may nevertheless account for some of the disease-protective effects attributed to diets rich in green vegetables because it occurs in much higher concentrations in those plants than the widely studied 'phytochemicals'.

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.

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