Aflatoxin-albumin Adducts Research Articles

Abstract PL02-03: Changing landscape of environmental carcinogenesis in Qidong, China: Lessons for prevention

Abstract Residents of Qidong, China, located at the mouth of the Yangtze River, are undergoing a rapid fluctuation in cancer incidence rates at many organ sites, reflecting a dynamic interplay of socio-behavioral, economic and environmental factors. As tracked by their cancer registry, there are extraordinary changes in the China age-standardized incidence rates for the two leading cancer killers in the Qidong region: one going down and the other one up, liver and lung cancer respectively. In endemic areas such as Qidong, liver cancer arises from chronic infection with hepatitis B virus (HBV) and ingestion of aflatoxins. Agricultural and economic reforms in the 1980s promoted a switch from maize to rice as the dietary staple, with concomitant >40-fold decreases in aflatoxin exposure over this period as measured by aflatoxin-albumin adducts in archived serum samples. Universal, subsidized vaccination against HBV was initiated in the early 2000s and likely does not account for the 45% drop in liver cancer incidence over the past quarter century, but augurs well for the future. Age standardized incidence rates for lung cancer, by contrast, have tripled in men over this period, reflecting the high prevalence of smoking (~60%) in adult males and increasing numbers of cigarettes smoked. Women in Qidong, on the other hand rarely smoke. Nonetheless, their rates of lung cancer incidence have doubled over the past decade only, perhaps related to changing patterns of exposures to indoor and outdoor air pollution. Approaches to modulate the impact of these exposures through chemopreventive interventions will be presented. Both primary and secondary prevention strategies appear to offer opportunities for risk reduction in this shifting landscape of exposures to environmental carcinogens. Citation Format: Thomas W. Kensler, Jian-Guo Chen, Tao-Yang Chen, Patricia Egner, Derek Ng, Alvaro Munoz, John Groopman. Changing landscape of environmental carcinogenesis in Qidong, China: Lessons for prevention. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr PL02-03.

Dietary exposure to aflatoxin and fumonisin among Tanzanian children as determined using biomarkers of exposure.

The study aims to evaluate the status of dietary exposure to aflatoxin and fumonisin in young Tanzanian children, using previously validated biomarkers of exposure. A total of 148 children aged 12-22 months, were recruited from three geographically distant villages in Tanzania; Nyabula, Kigwa, and Kikelelwa. Plasma aflatoxin-albumin adducts (AF-alb) and urinary fumonisin B1 (UFB1) were measured by ELISA and LC-MS, respectively. AF-alb was detectable in 84% of children, was highest in fully weaned children (p < 0.01) with higher levels being associated with higher maize intake (p < 0.05). AF-alb geometric mean (95% CI) was 43.2 (28.7-65.0), 19.9 (13.5-29.2), and 3.6 (2.8-4.7) pg/mg albumin in children from Kigwa, Nyabula, and Kikelelwa, respectively. UFB1 was detectable in 96% of children and the level was highest in children who had been fully weaned (p < 0.01). The geometric UFB1 mean (95% CI) was 327.2 (217.1-493.0), 211.7 (161.1-278.1), and 82.8 (58.3-117.7) pg/mL in Kigwa, Nyabula, and Kikelelwa, respectively. About 82% of all the children were exposed to both mycotoxins. Young children in Tanzania are chronically exposed to both aflatoxin and fumonisin through contaminated diet, although the level of exposure varies markedly between the three villages studied.

Human aflatoxin exposure in Kenya, 2007: a cross-sectional study

Aflatoxins contaminate approximately 25% of agricultural products worldwide. They can cause liver failure and liver cancer. Kenya has experienced multiple aflatoxicosis outbreaks in recent years, often resulting in fatalities. However, the full extent of aflatoxin exposure in Kenya has been unknown. Our objective was to quantify aflatoxin exposure across Kenya. We analysed aflatoxin levels in serum specimens from the 2007 Kenya AIDS Indicator Survey – a nationally representative, cross-sectional serosurvey. KAIS collected 15,853 blood specimens. Of the 3180 human immunodeficiency virus-negative specimens with ≥1 mL sera, we randomly selected 600 specimens stratified by province and sex. We analysed serum specimens for aflatoxin albumin adducts by using isotope dilution MS/MS to quantify aflatoxin B1-lysine, and normalised with serum albumin. Aflatoxin concentrations were then compared by demographic, socioeconomic and geographic characteristics. We detected serum aflatoxin B1-lysine in 78% of serum specimens (range = <LOD–211 pg/mg albumin, median = 1.78 pg/mg albumin). Aflatoxin exposure did not vary by sex, age group, marital status, religion or socioeconomic characteristics. Aflatoxin exposure varied by province (p < 0.05); it was highest in Eastern (median = 7.87 pg/mg albumin) and Coast (median = 3.70 pg/mg albumin) provinces and lowest in Nyanza (median = <LOD) and Rift Valley (median = 0.70 pg/mg albumin) provinces. Our findings suggest that aflatoxin exposure is a public health problem throughout Kenya, and it could be substantially impacting human health. Wide-scale, evidence-based interventions are urgently needed to decrease exposure and subsequent health effects.

Serum concentrations of an aflatoxin-albumin adduct in the National Health and Nutrition Examination Survey (NHANES) 1999–2000

BackgroundDuring 1998, weather conditions in the United States favored the growth of Aspergillus species leading to widespread contamination of Midwestern and Southern corn with hepatotoxic and hepatocarcinogenic aflatoxins. We designed a study to provide the first national prevalence estimate of aflatoxin exposure using the National Health and Nutrition Examination Survey (NHANES), a representative cross-sectional survey of the noninstitutionalized civilian population of the US. MethodsIsotope dilution liquid chromatography–tandem mass spectrometry was used to quantitate serum concentrations of aflatoxin B1-lysine in a one-third random subset of participants from NHANES 1999–2000. ResultsAbout 1% of the U.S. population had detectable levels (≥0.02μg/l) of aflatoxin B1-lysine. Of those with detectable levels, the geometric mean (95% confidence interval) was 0.038 (0.024–0.060) μg/l (equivalent to 0.842 (0.530–1.34) pg/mg albumin). The highest value was 0.2μg/l (4.43pg/mg albumin). Based on liver function biomarkers, there was no evidence of increased liver dysfunction in these persons. ConclusionsDuring a time when exposure to aflatoxins in food products might have been expected to be increased, we identified few exposed persons. Although none of the subgroup analyses provided reliable estimates due to high relative standard errors, they suggested that additional targeted surveillance may be warranted.

Abstract SY14-04: Global burden of liver cancer: Emerging needs in Asia.

Globally, driven by increased life expectancy, the overall incidence of cancer is expected to nearly double over the next 20 years. Nonetheless, the target organs where these diagnoses will occur will still vary widely from country to country because of divergent exposures and other etiologic factors (1). One cancer that currently illustrates this geographic disparity is primary liver cancer (PLC). Currently, PLC is the third leading cause of cancer mortality worldwide with an estimated 696,000 deaths in 2008. Half of these PLC deaths of those deaths occur in the People9s Republic of China. The major etiological factors associated with PLC in China, as established from prospective cohort studies, are chronic infection with hepatitis B virus (HBV) and extended exposure to high levels of aflatoxin in the diet, especially from corn and groundnuts (2). The largest study, comprising more than 18,000 men residing in Shanghai in the 1980s, examined HBV infection and aflatoxin exposure as independent and interactive risk factors for PLC (3). This nested case control study revealed a statistically significant increase in the relative risk (RR) of 7.3 for men who were chronically infected with HBV (HBsAg) but unexposed to aflatoxin. In men on whom urinary aflatoxin but not HBV biomarkers were detected, the RR was 3 (4). Furthermore, in men exhibiting both urinary aflatoxin biomarkers and positive HBsAg status, the RR was 59.4.3 A subsequent cohort study in Taiwan confirmed these results.4 In light of this synergistic interaction, programs to eliminate either factor, or both, should have substantial impact on the burden of PLC. Chronic infection with HBV has been long regarded as the major cause of PLC and initiatives have been undertaken to implement universal immunization programs. It is estimated that more than 90% of countries now routinely vaccinate newborns against HBV, and approximately 70% are now delivering 3 immunization doses. Newborns are targeted because HBV transmission is often vertical from mother to child at birth or during the perinatal period. In 1986, Taiwan became the first region to vaccinate all newborns against HBV. Since then, the number of HBV carriers in the juvenile population has declined dramatically. As of 2009, incidence of hepatocellular carcinoma was significantly lower among children aged 6-19 years in vaccinated compared with antecedent unvaccinated birth cohorts (5). For many years, we have been working with colleagues in Qidong, China, located at the mouth of the Yangtze River, an endemic area for liver cancer, where early age of onset and high mortality rates from PLC are reported (6). HBV vaccination of newborns in the rural townships was initiated during the 1980s but did not become universal until 2002. Crude rates of PLC per 100,000 residents of Qidong rose slightly from 1972 through 1990 at levels around 50/100,000 before increasing in the 1990s to a stable rate of about 75/100,000. Such data have led to the perception that Qidong remains an irrevocably high-risk region (7). However, the latter PLC mortality rate increase largely reflects a dramatically altered age distribution in the population rising from 17.0% over 50 years of age in 1973 to 22.7% in 1988 and 34.2% in 2003. Prognosis for survival from liver cancer following diagnosis remains poor: currently In order to accurately describe aflatoxin exposures over the past quarter century, frozen serum samples collected since 1989 were measured for aflatoxin-albumin adduct content (9–11). There has been a dramatic decline in levels of exposure from the 1980s to the present. Median levels of aflatoxin-albumin adducts from residents of the villages of Daxin and HeZuo declined from 19.3 pg/mg albumin in 1989, to 3.6 in 1995, to 2.3 in 1999, to 1.4 in 2003 and undetectable (i.e., Acknowledgments This work has been funded by the National Institutes of Health USA (P01 ES 006052) and the National Science and Technology Mega-Projects of China (No. 2008ZX10002-015, No. 2012ZX10002-008. Citation Format: John D. Groopman, Thomas W. Kensler. Global burden of liver cancer: Emerging needs in Asia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY14-04. doi:10.1158/1538-7445.AM2013-SY14-04

Abstract CN05-01: Viral and Dietary Factors in Declining Liver Cancer Mortality in Qidong, China

Abstract Liver cancer is the third leading cause of cancer mortality globally. In endemic areas of sub-Saharan Africa and southeast Asia liver cancer largely arises from both chronic infection with hepatitis B virus (HBV) and the ingestion of aflatoxin-contaminated foods such as corn and groundnuts. While synergistic interactions between these viral and chemical carcinogens have been observed in prospective cohort studies in China, the effects of primary prevention against both of these factors has not been examined. Mortality rates from liver cancer by birth cohort in Qidong, China were compared to timelines in the region for the introduction of HBV vaccination and to mandated, wholesale changes in staple foodstuffs from corn to rice. Levels of aflatoxin-albumin adducts in archived serum samples collected in Qidong from 1989 to 2012 were quantified by isotope dilution mass spectrometry as measures of carcinogen exposure. Halving of mortality rates from liver cancer are seen between birth cohorts from the 1960s and 1980s for residents 34 years old and younger. However, prevalence of HBsAg positivity was unchanged in these cohorts as all were born prior to implementation of universal HBV vaccination of newborns. Changing agricultural policies from a planned to a market-oriented economy in the early 1980s led from corn to rice based diets and dramatic decreases in aflatoxin exposures in Qidong. Substantial declines in liver cancer morality in birth cohorts occurring prior to the introduction of a universal HBV vaccination program in Qidong highlight the importance of altered food policies as a means for rapid risk reduction, especially amongst the ∼350 million HBV carriers, many of whom reside in endemic areas. Supported by NIEHS ES 006052 and National Science and Technology of China 2008ZX10002-015. Citation Format: Thomas W. Kensler. Viral and dietary factors in declining liver cancer mortality in Qidong, China. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr CN05-01.

Aflatoxin exposure may contribute to chronic hepatomegaly in Kenyan school children.

Background: Presentation with a firm type of chronic hepatomegaly of multifactorial etiology is common among school-age children in sub-Saharan Africa.Objective: Aflatoxin is a liver toxin and carcinogen contaminating staple maize food. In this study we examined its role in chronic hepatomegaly.Methods: Plasma samples collected in 2002 and again in 2004 from 218 children attending two schools in neighboring villages were assayed for aflatoxin exposure using the aflatoxin-albumin adduct (AF-alb) biomarker. Data were previously examined for associations among hepatomegaly, malaria, and schistosomiasis.Results: AF-alb levels were high in children from both schools, but the geometric mean (95% confidence interval) in year 2002 was significantly higher in Matangini [206.5 (175.5, 243.0) pg/mg albumin] than in Yumbuni [73.2 (61.6, 87.0) pg/mg; p < 0.001]. AF-alb levels also were higher in children with firm hepatomegaly [176.6 (129.6, 240.7) pg/mg] than in normal children [79.9 (49.6, 128.7) pg/mg; p = 0.029]. After adjusting for Schistosoma mansoni and Plasmodium infection, we estimated a significant 43% increase in the prevalence of hepatomegaly/hepatosplenomegaly for every natural-log-unit increase in AF-alb. In 2004, AF-alb levels were markedly higher than in 2002 [539.7 (463.3, 628.7) vs. 114.5 (99.7, 131.4) pg/mg; p < 0.001] but with no significant difference between the villages or between hepatomegaly and normal groups [539.7 (436.7, 666.9) vs. 512.6 (297.3, 883.8) pg/mg], possibly because acute exposures during an aflatoxicosis outbreak in 2004 may have masked any potential underlying relationship.Conclusions: Exposure to aflatoxin was associated with childhood chronic hepatomegaly in 2002. These preliminary data suggest an additional health risk that may be related to aflatoxin exposure in children, a hypothesis that merits further testing.

TP53 R249S Mutations, Exposure to Aflatoxin, and Occurrence of Hepatocellular Carcinoma in a Cohort of Chronic Hepatitis B Virus Carriers from Qidong, China

Hepatocellular carcinoma (HCC) has a high mortality in East Asia and Sub-Saharan Africa, two regions where the main etiologic factors are chronic infections with hepatitis B virus and dietary exposure to aflatoxin. A single base substitution at the third nucleotide of codon 249 of TP53 (R249S) is common in HCC in these regions and has been associated with aflatoxin-DNA adducts. To determine whether R249S may be detected in plasma DNA before HCC diagnosis, we conducted a case-control study nested in a cohort of adult chronic hepatitis B virus carriers from Qidong County, People's Republic of China. Of the 234 plasma specimens that yielded adequate DNA, only 2 (0.9%) were positive for R249S by restriction fragment length polymorphisms, and both of them were controls. Of the 249 subjects tested for aflatoxin-albumin adducts, 168 (67%) were positive, with equal distribution between cases and controls. Aflatoxin-albumin adduct levels were low in the study, suggesting an overall low ongoing exposure to aflatoxin in this cohort. The R249S mutation was detected in 11 of 18 (61%) available tumor tissues. To assess whether low levels of mutant DNA were detectable in pre-diagnosis plasma, 14 plasma specimens from these patients were analyzed by short oligonucleotide mass analysis. Nine of them (64%) were found to be positive. Overall, these results suggest that HCC containing R249S can occur in the absence of significant recent exposure to aflatoxins. The use of short oligonucleotide mass analysis in the context of low ongoing aflatoxin exposure may allow the detection of R249S in plasma several months ahead of clinical diagnosis.

Aflatoxin–albumin adducts and correlation with decreased serum levels of vitamins A and E in an adult Ghanaian population

A study of aflatoxin (AF) exposure and the levels of vitamins A and E was carried out with a group of 507 Ghanaian participants. AFB1–albumin adducts (AFB-AA) were measured by radioimmunoassay and vitamins A and E were measured by high-performance liquid chromatography (HPLC). The average level of serum AFB-AA was 0.94 ± 0.64 (range = 0.1–4.44) pmol mg−1 albumin. Mean levels of vitamins A and E were 1.32 ± 0.48 (range = 0.41–4.85) µmol l−1 and 15.68 ± 4.12 (range = 6.35–30.40) µmol l−1, respectively. A significantly negative correlation was found between serum AFB-AA and vitamin A levels (r = −0.110, p = 0.013). An even stronger, significant negative, correlation was found between serum AFB-AA and vitamin E levels (r = −0.149, p < 0.001). Serum AFB-AA levels were statistically higher (median = 0.985 pmol mg−1 albumin) in subjects who had low levels of both vitamins A and E as compared with the levels (median = 0.741 pmol mg−1 albumin) subjects who had high vitamins A and E levels (p trend = 0.001). To verify these findings, blood samples were again collected from 165 of the 507 people 3 months after the initial collection. Significantly negative correlations were confirmed between levels of serum AFB-AA and both vitamins A (r = −0.232, p = 0.003) and E (r = −0.178, p = 0.023). Again, high serum AFB-AA concentrations (median = 1.578 pmol mg−1 albumin) were found in subjects with low levels of vitamins A and E compared with the concentrations (median = 1.381 pmol mg−1 albumin) in subjects with high levels of vitamins A and E (p trend = 0.002). These data show that AF exposure was associated with decreased levels of serum vitamins A and E in high-risk human populations, which may significantly influence the incidence of AF-related adverse health effects.

Abstract PL02-02: Foodborne toxins in developing countries

Abstract PL02-02 Mycotoxins are fungal metabolites which commonly contaminate the human food supply, particularly cereals (e.g. maize (corn), wheat, barley and oats), oilseeds (e.g. groundnuts (peanuts)) and tree nuts. Estimates are that around 25% of food worldwide contains these toxins. The mycotoxins of most importance from a human health perspective are aflatoxins, fumonisins, ochratoxin A, tricothecenes and zearalenone. Exposure to aflatoxins and fumonisins, both of which are carcinogenic, occurs predominantly in developing countries. This is due to a combination of factors, including the climatic conditions which promote crop stress, fungal growth and toxin production during cultivation and storage; the reliance on a few dietary staple foods; inadequate food sufficiency, translating to consumption of contaminated food; and the absence of regulation, or implementation of regulations, to control mycotoxin levels in the food supply. At the same time, understanding the human health effects of mycotoxins has been hampered, at least in part, by the absence of accurate exposure measurements. In response, biomarkers offer to improve exposure assessment at the individual level. These same biomarkers can be used to evaluate potential approaches to reduce mycotoxin exposure. Aflatoxins have been associated with increased liver cancer risk in interaction with chronic hepatitis B virus (HBV) infection, but the underlying molecular mechanisms remain ill-defined. Genetic polymorphisms in genes coding for aflatoxin metabolising enzymes and DNA repair enzymes were associated with an altered cancer risk1,2 and earlier data suggested that HBV infection could alter expression of some of these metabolising enzymes3. In West Africa there is a high prevalence and level of dietary exposure to aflatoxins, with &amp;gt;90% of the individuals having detectable blood aflatoxin-albumin adduct4. Exposure is life-long, beginning in utero, reducing during breastfeeding, but increasing markedly at weaning. This exposure early in life in West Africa has been associated with a striking impairment of growth in young children5-8. Exposure was also associated with a decrease in salivary IgA levels9. Growth and immune impairment could therefore be critical in pre-disposing children to the infections, including HBV, that result in the high morbidity and mortality in these populations. In parallel, to the studies of disease aetiology, we evaluated a simple post-harvest intervention in Guinea to reduce aflatoxin contamination of the groundnut crop. This involved sun-drying the crop on mats prior to storage; the use of natural fibre bags for storage; the use of wooden pallets to keep the bags off the moist earth in the storage facilities and the application of insecticide on the floor of the facility. This intervention resulted in a &amp;gt;50% mean reduction in aflatoxin-albumin adducts in subjects in the intervention villages five months post-harvest10. In addition, around 20% of subjects in the intervention villages had no detectable aflatoxin-albumin biomarker in their serum compared to less than 3% in the control villages. This demonstrates that relatively simple prevention approaches can make a significant impact on aflatoxin exposure when implemented at the small subsistence farm level. This primary prevention compares well with the alternative approach of chemoprevention using oltipraz, chlorophyllin, green tea or broccoli tea as demonstrated in studies in aflatoxin-exposed individuals in the People’s Republic of China11. More recently the use of absorbent clays in the diet in Ghana has been shown to reduce aflatoxin biomarker levels12. Overall the prevention of hepatocellular carcinoma in developing countries depends on reducing chronic infection with hepatitis B or C viruses as well as a reduction in aflatoxin exposure. It is also notable that the health effects of aflatoxin exposure may extend well-beyond liver cancer if the associations with impaired growth and immunity are causal. This would significantly change the priority assigned to tackling this environmental toxin. Fumonisins are predominantly found in maize. These toxins have been associated with squamous cell carcinoma of the oesophagus in parts of southern Africa and People’s Republic of China and, more recently, with a high incidence of neural tube defects on the Texas-Mexico border. We developed a urinary biomarker for fumonisin exposure and in a study in Mexico showed that tortilla intake, the major source of maize, was correlated with the biomarker13. In the former Transkei region of South Africa a high level of fumonisin exposure was also demonstrated using the urinary biomarker. The effectiveness in reducing fumonisin contamination by hand-washing and sorting maize kernels prior to cooking was evaluated using the biomarker in a study of subsistence farming families. In summary, mycotoxins are common in much of the food supply of the developing world. However, the adverse health effects of these chronic exposures are not fully understood. The ability to measure exposure permits both aetiologic and intervention studies to be conducted in the future. Finally, in assessing the benefits of interventions, the complex range of adverse health effects resulting from mycotoxin exposure needs to be taken into account. Citation Information: Cancer Prev Res 2008;1(7 Suppl):PL02-02.

Human Aflatoxin Albumin Adducts Quantitatively Compared by ELISA, HPLC with Fluorescence Detection, and HPLC with Isotope Dilution Mass Spectrometry

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B(1). In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

Pilot survey of aflatoxin–albumin adducts in sera from Egypt

Aflatoxins are potent liver carcinogens that frequently contaminate cereals in developing countries. Aflatoxin exposure has been predicted in Egypt but, to date, no studies have measured the level of aflatoxin–albumin (AF–alb) adducts as a validated biomarker to assess exposure. In this pilot survey, a limited number of sera samples, available from a hepatocellular carcinoma (HCC) case-control study in Egypt, were analysed. AF–alb was detected in 24/24 samples from HCC-negative individuals (geometric mean 9.0 pg mg−1; range 3.5–25.8pg mg−1), while 7/22 samples from HCC-positive cases had detectable AF–alb (geometric mean 2.6 pg mg−1; range: non-detectable–32.8 pg mg−1). These AF–alb data do not represent a case-control comparison due to inherent difficulties in comparing markers of dietary intake between controls and patients with disease. Although these data are limited, the potential health consequences of aflatoxin exposure in this region merit further investigation.

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 μg pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose–response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-α, IL-1β, IL-6, IFN-γ) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-γ and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1.

Aflatoxin exposure in utero causes growth faltering in Gambian infants

Growth faltering in West African children has previously been associated with dietary exposure to aflatoxins, particularly upon weaning. However, in animal studies in utero exposure to low levels of aflatoxin also results in growth faltering. This study investigated the effect of in utero aflatoxin exposure on infant growth in the first year of life in The Gambia. Height and weight were measured for 138 infants at birth and at regular monthly intervals for one year. Aflatoxin-albumin (AF-alb) adduct level was measured in maternal blood during pregnancy, in cord blood and in infants at age 16 weeks. The geometric mean AF-alb levels were 40.4 pg/mg (range 4.8-260.8 pg/mg), 10.1 pg/mg (range 5.0-189.6 pg/mg) and 8.7 pg/mg (range 5.0-30.2 pg/mg) in maternal, cord and infant blood, respectively. AF-alb in maternal blood was a strong predictor of both weight (P = 0.012) and height (P = 0.044) gain, with lower gain in those with higher exposure. A reduction of maternal AF-alb from 110 pg/mg to 10 pg/mg would lead to a 0.8 kg increase in weight and 2 cm increase in height within the first year of life. This study shows a strong effect of maternal aflatoxin exposure during pregnancy on growth in the first year of life and thus extends earlier observations of an association between aflatoxin exposure during infancy and growth faltering. The findings imply value in targeting intervention strategies at early life exposures.

Polycyclic aromatic hydrocarbon- and aflatoxin-albumin adducts, hepatitis B virus infection and hepatocellular carcinoma in Taiwan

To determine the association between polycyclic aromatic hydrocarbon (PAH) exposure and risk of hepatocellular carcinoma, a case-control study nested within a community-based cohort was conducted in Taiwan. Baseline blood samples, collected from a total of 174 HCC cases and 776 matched controls, were used to determine the level of PAH-albumin adducts by competitive enzyme-linked immunosorbent assay. Conditional logistic regression analysis was used to calculate odds ratios (OR) and 95% confidence intervals (CI) to assess the effect of PAH-albumin adducts on risk of HCC. When compared to subjects in the lowest quantile, there was an increase in risk of HCC, with adjusted ORs (95% CI) of 1.0 (0.5–2.0), 1.2 (0.6–2.4) and 2.0 (1.0–4.2: P trend = 0.08) for subjects in the 2nd, 3rd and 4th quantile, respectively. The corresponding adjusted ORs (95% CI) were 1.9 (0.6–6.1), 1.7 (0.6–4.9) and 2.1 (0.5–8.2; P trend = 0.22), respectively, among subjects with high AFB 1-albumin adducts; and 0.8 (0.3–2.7), 1.5 (0.6–3.5) and 2.9 (1.0–8.6; P trend = 0.06), respectively, for those who were chronically infected with hepatitis B virus (HBV). The combination of PAH- and AFB 1-albumin adducts above the mean and chronic HBV infection resulted in an OR of 8.2 (95% CI, 3.6–19.0; p < 0.0001), compared to those with low adducts and no viral infection. These results demonstrate that PAH-albumin adducts are associated with an increased risk of HCC, especially among those with high aflatoxin exposure and chronic HBV infection. Environmental PAH exposure seems to enhance the hepatocarcinogenicity of chronic HBV infection.

Determinants of the Variability of Aflatoxin–Albumin Adduct Levels in Ghanaians

Hepatocellular carcinoma (HCC) is a multifactorial disease with various host and environmental factors involved in its etiology. Of these, aflatoxin exposure has been established as an important risk factor in the development of HCC; the presence of aflatoxin–albumin (AA) adducts in the blood serves as a valuable biomarker of human exposure. In this study, the relationship between a variety of different HCC host factors and the incidence of AA adduct levels was examined in a Ghanaian population at high risk for HCC. These factors included age, gender, hepatitis virus B (HVB) and hepatitis C virus (HCV) status, and genetic polymorphisms in both microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GSTs). Blood samples were analyzed for AA adducts and HBV and HCV status. GSTM1 and GSTT1 deletion polymorphisms and mEH exon 3 and exon 4 single-nucleotide polymorphisms (SNPs) were determined from urine samples. In univariate analysis, age, HBV and HVC status, and GSTT1 and mEH exon 3 genotypes were not associated with AA adduct levels. However, mean adduct levels were significantly higher in both females and individuals typed heterozygous for mEH exon 4 (vs. wild types). Stratification analysis also showed that gender along with mEH exon 4 genotype and HBV status had a significant effect on adduct levels. Both females typed HBsAg+ and males with mEH exon 4 heterozygote genotypes showed significantly higher adduct levels as compared to the HBsAg– and wild types, respectively. Understanding the relationships between these host factors and the variability in aflatoxin-adduct levels may help in identifying susceptible populations in developing countries and for targeting specific public health interventions for the prevention of aflatoxicoses in populations with HCC and chronic liver diseases.

Quantitative Analysis and Chronic Dosimetry of the Aflatoxin B1 Plasma Albumin Adduct Lys-AFB1 in Rats by Isotope Dilution Mass Spectrometry

Aflatoxin B1 (AFB1) is a major risk factor in the pathogenesis of liver cancer in Asia and sub-Saharan Africa. Biomarkers reflecting exposure will facilitate disease risk assessment and the efficacy of protective interventions in these populations. The Lys-AFB1 adduct in plasma albumin is a candidate biomarker for this role. Although aflatoxin albumin adducts are most frequently measured in epidemiological studies using an enzyme-linked immunosorbent assay, a more specific and 10-fold more sensitive isotopic dilution mass spectrometric assay for Lys-AFB1 has recently become available. Here, the dosimetry of chronically administered AFB1 at lower doses than have been previously studied was explored using this assay. AFB1 was administered to rats for nine consecutive days at eight dose levels ranging from 50 pg to 55 microg/kg body wt. Plasma samples were enzymatically digested and processed by solid phase extraction. Lys-AFB1 was isolated by HPLC and detected via selected reaction monitoring. The dose-response relationship was linear-quadratic exhibiting upward curvature at higher doses. The adduct yield [(pg Lys-AFB1/mg albumin)/(microg AFB1/kg body wt)] increased nonlinearly with the dose by 6-fold between the 0.05 and 55 microg AFB1/kg body wt groups and exhibited the onset of saturation in the highest dose group where the adduct yield was approximately 2%. Incomplete knowledge of the timing of exposure and the complexity of the underlying biology confound the precise determination of prior AFB1 exposures in humans; however, the dosimetry of AFB1 observed in chronically dosed rats conceptually suggests that measurements in humans may underestimate exposure if a constant fraction of the AFB1 dose, approximately 2%, is assumed to be converted to Lys-AFB1 without regard to the dose.

Phase IIa chemoprevention trial of green tea polyphenols in high-risk individuals of liver cancer: modulation of urinary excretion of green tea polyphenols and 8-hydroxydeoxyguanosine

Modulation of urinary excretion of green tea polyphenols (GTPs) and oxidative DNA damage biomarker, 8-hydroxydeoxyguanosine (8-OHdG), were assessed in urine samples collected from a randomized, double-blinded and placebo-controlled phase IIa chemoprevention trial with GTP in 124 individuals. These individuals were sero-positive for both HBsAg and aflatoxin-albumin adducts, and took GTP capsules daily at doses of 500 mg, 1000 mg or a placebo for 3 months. Twenty-four hour urine samples were collected before the intervention and at the first and third month of the study. Urinary excretion of 8-OHdG and GTP components was measured by HPLC-CoulArray electrochemical detection. The baseline levels of 8-OHdG and GTP components among the three groups showed homogeneity (P > 0.70), and a non-significant fluctuation was observed in the placebo group over the 3 months (P > 0.30). In GTP-treated groups, epigallocatechin (EGC) and epicatechin (EC) levels displayed significant and dose-dependent increases in both the 500 mg group and 1000 mg group (P < 0.05). The 8-OHdG levels did not differ between the three groups at the 1 month collection, with medians of 1.83, 2.08 and 1.86 ng/mg-creatinine for placebo, 500 and 1000 mg group, respectively (P = 0.999). At the end of the 3 months' intervention, 8-OHdG levels decreased significantly in both GTP-treated groups, with medians of 2.02, 1.03 and 1.15 ng/mg-creatinine for placebo, 500 mg and 1000 mg group, respectively (P = 0.007). These results suggest that urinary excretions of EGC and EC can serve as practical biomarkers for green tea consumption in human populations. The results also suggest that chemoprevention with GTP is effective in diminishing oxidative DNA damage.

Absence of TP53 Codon 249 Mutations in Young Guinean Children with High Aflatoxin Exposure

Infection with hepatitis viruses and chronic exposure to high levels of dietary aflatoxins are the major etiologic agents for hepatocellular carcinoma in west Africa. A challenge for the prevention of hepatocellular carcinoma in this region is that both hepatitis B virus and aflatoxin exposures start early in life; indeed, aflatoxin exposures can start in utero and continue unabated throughout childhood. A mutation in the TP53 tumor suppressor gene at codon 249 (TP53 Ser249 mutation) has been reported previously for hepatocellular carcinoma tumors and matched plasma DNA samples in individuals from areas with high aflatoxin exposure. We examined whether the TP53 Ser249 mutation could be observed in DNA found in plasma of young children (ages 2-5 years) from Guinea, west Africa, a region of high aflatoxin exposure. Plasma aflatoxin-albumin adducts were present in 119 of 124 (96%) of the children, geometric mean of positives 9.9 pg/mg albumin (95% confidence interval, 8.8-11.0 pg/mg). This is the level and prevalence of exposure observed previously in adults. Following PCR amplification of plasma-derived DNA and detection using mass spectrometry, none of the samples were found to contain the TP53 Ser249 mutation. Because approximately 50% of the hepatocellular carcinomas in adults in west Africa have this specific TP53 Ser249 mutation, a lack of detection in samples from children ages <5 years may indicate that a window of opportunity for intervention exists that could be exploited to lower hepatocellular carcinoma risk.

Dietary exposure to aflatoxin from maize and groundnut in young children from Benin and Togo, West Africa

Aflatoxins are a family of fungal toxins that are carcinogenic to man and cause immunosuppression, cancer and growth reduction in animals. We conducted a cross-sectional study among 480 children (age 9 months to 5 years) across 4 agro-ecological zones (SS, NGS, SGS and CS) in Benin and Togo to identify the effect of aflatoxin exposure on child growth and assess the pattern of exposure. Prior reports on this study [ Gong, Y.Y., Cardwell, K., Hounsa, A., Egal, S., Turner, Hall, A.J., Wild, C.P., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. British Medical Journal 325, 20–21, Gong, Y.Y., Egal, S., Hounsa, A., Turner, P.C., Hall, A.J., Cardwell, K., Wild, C.P., 2003. Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning and weaning foods. International Journal of Epidemiology, 32, 556–562] showed that aflatoxin exposure among these children is widespread (99%) and that growth faltering is associated with high blood aflatoxin-albumin adducts (AF-alb adducts), a measure of recent past exposure. The present report demonstrates that consumption of maize is an important source of aflatoxin exposure for the survey population. Higher AF-alb adducts were correlated with higher A. flavus (CFU) infestation of maize ( p = 0.006), higher aflatoxin contamination (ppb) of maize ( p < 0.0001) and higher consumption frequencies of maize ( p = 0.053). The likelihood of aflatoxin exposure from maize was particularly high in agro-ecological zones where the frequency of maize consumption (SGS and CS), the presence of aflatoxin in maize (SGS) or the presence of A. flavus on maize (NGS and SGS) was relatively high. Socio-economic background did not affect the presence of A. flavus and aflatoxin in maize, but better maternal education was associated with lower frequencies of maize consumption among children from the northernmost agro-ecological zone (SS) ( p = 0.001). The impact of groundnut consumption on aflatoxin exposure was limited in this population. High AF-alb adduct levels were correlated with high prevalence of A. flavus and aflatoxin in groundnut, but significance was weak after adjustment for weaning status, agro-ecological zone and maternal socio-economic status (resp. p = 0.091 and p = 0.083). Ingestion of A. flavus and aflatoxin was high in certain agro-ecological zones (SS and SGS) and among the higher socio-economic strata due to higher frequencies of groundnut consumption. Contamination of groundnuts was similar across socio-economic and agro-ecological boundaries. In conclusion, dietary exposure to aflatoxin from groundnut was less than from maize in young children from Benin and Togo. Intervention strategies that aim to reduce dietary exposure in this population need to focus on maize consumption in particular, but they should not ignore consumption of groundnuts.