Oncogenic and tumor suppressor pathways in subchronic aflatoxicosis in rats: Association with serum and urinary aflatoxin exposure biomarkers.

In this study, the changes in oncogenic and tumor suppressor signaling pathways in liver and their association with serum and urinary biomarkers of aflatoxin exposure were evaluated in Wistar rats fed diets containing aflatoxin B1 (AFB1) for 90 days. Rats were divided into four groups (n = 15 per group) and assigned to dietary treatments containing 0 (control), 50 (AFB50), 100 (AFB100) and 200 µg AFB1 kg-1 diet (AFB200). Multiple preneoplastic foci of hepatocytes marked with glutathione-S-transferase-placental form (GST-P) were identified in AFB100 and AFB200 groups. Hepatocellular damage induced by AFB1 resulted in overexpression of cyclin D1 and ß-catenin. The liver expression of retinoblastoma (Rb) and p27Kip1 decreased in AFB100 and AFB200 groups, confirming the favorable conditions for neoplastic progression to hepatocellular carcinoma. All samples from rats fed AFB1-contaminated diets had quantifiable AFB1-lysine in serum or urinary AFM1 and AFB1-N7-guanine, with mean levels of 20.42-50.34 ng mL-1, 5.31-37.68 and 39.15-126.37 ng mg-1 creatinine, respectively. Positive correlations were found between AFB1-lysine, AFM1 or AFB1-N7-guanine and GST-P+, ß-catenin+ and cyclin D1+ hepatocytes, while Rb + cells negatively correlated with those AFB1 exposure biomarkers. The pathways evaluated are critical molecular mechanisms of AFB1-induced hepatocarcinogenesis in rats.

Determination of aflatoxin M1 and deoxynivalenol biomarkers in infants and children urines from Bangladesh.

The mycotoxins aflatoxin B1 (AFB1) and deoxynivalenol (DON) are found worldwide in crops and dietary staples. The prevalence and levels of these contaminants can vary greatly, and data in Bangladeshi food commodities are scarce. To characterize human exposure, we have conducted biomonitoring, analyzing AFM1 (a metabolite of AFB1) and DON levels in urines of adult cohorts in Bangladesh. Yet, AFM1 and DON occurrence has not been studied in the very young population of this country. Thus, the same methods, HPLC-FD for AFM1 and LC-MS/MS for DON analysis, were now applied to determine these biomarkers in urines of infants (n = 49) and young children (n = 105) in Rajshahi and Dhaka district. Overall, AFM1 and DON detection frequency was 43.5% and 33.4%, with 34.7% and 11.5% in infant and 47.6% and 39.4% in children urines, respectively. The mean AFM1 levels in all infants (9.1 ± 14.3, max 55.6 pg/mL) and children (8.8 ± 12.9, max 75.3 pg/mL) were not significantly different. The AFM1 mean level was slightly higher in Dhaka (9.4 ± 12.4) compared to Rajshahi (8.5 ± 13.9 pg/mL) district. The average DON level was about 2-fold higher in infant (3.8 ± 2.9, max 6.8 ng/mL) than children urines (1.6 ± 1.8, max 8.6 ng/mL), and higher in Rajshahi (2.1 ± 2.3 ng/mL) than Dhaka (1.4 ± 1.6 ng/mL) district. The biomarker-based estimated average daily DON intake (29.6 ± 108.3 ng/kg bw in infants and 36.4 ± 81.8 ng/kg bw in children) or the maximum exposure (560 ng/kg bw) do not exceed the current maximum provisional tolerable daily intake value of 1 µg/kg bw for DON, although DON exposure in infants and children is higher than that of Bangladeshi adults. The AFM1 urine levels in young children are somewhat lower than those found previously in adult cohorts in Bangladesh, but the frequent detection of this biomarker for AFB1 exposure raises further concerns, also for this vulnerable part of the population. Therefore, continuous surveillance for aflatoxins in Bangladeshi food commodities is clearly required, first to identify major sources of intake and then to reduce exposure.

Biomonitoring of Aflatoxin B1 and Deoxynivalenol in a Rural Pakistan Population Using Ultra-Sensitive LC-MS/MS Method.

There are limited data on exposure to mycotoxins in Pakistan. Here, we measured exposure to deoxynivalenol (DON), a common contaminant of wheat, and aflatoxin B1 (AFB1), a known contaminant of rice, using biomarkers of exposure. Wheat (n = 195) and rice (n = 62) samples were analyzed for AFB1 and DON levels, and the corresponding urinary biomarkers were analyzed in urine samples from a rural population (n = 264, aged 4-80 years, male 58%) using ultra-sensitive liquid chromatography-tandem mass spectrometry. AFB1 was detected in 66% of rice (5.04 ± 11.94 µg/kg) and 3% of wheat samples. AFM1 (hydroxylated form of AFB1)was detected in 69% of urine samples, mean 0.023 ± 0.048 ng/mL and DON was detected in 20% of urine samples, mean 0.170 ± 0.129 ng/mL. The maximum probable daily intake for DON derived from the urinary biomarker was 59.8 ng/kg b.w./day, which is below the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives' tolerable daily intake (1000 ng/kg b.w./day). However, for aflatoxin, the derived margin of exposure (MoE) of (13.2) was well below the safe MoE (10,000) suggested by the European Food Safety Authority. The calculated aflatoxin-associated cancer risk of 0.514/105 individuals/year suggests that measures should be taken to reduce the AFB1 contamination in food, particularly rice, in Pakistan.

Screening of the aflatoxin M1 metabolite in urine samples of residents in Terengganu, Malaysia.

A study was conducted to screen the occurrence and level of aflatoxin M1 (AFM1) in urine samples of 206 urban and rural residents in Terengganu, Malaysia. The level of AFM1 was quantified by competitive enzyme-linked immune-absorbent assay (ELISA). Of the 206 samples, 84 were positive for AFM1 (40.8%) in a range of 0.07-5.53 ng/ml (mean = 0.589 ng/ml). Residents of Terengganu are moderately exposed to AFM1. Age, ethnicity, marital status and employment status were associated with urinary level of AFM1. Subjects aged 30 years and above, non-Malays, married, and those unemployed had significantly higher levels of urinary AFM1 (p < 0.05). Since aflatoxin is recognised as a potent-carcinogen for liver cancer and a continuous exposure to this toxin can be fatal, the present findings could provide a baseline for future studies where larger samples and more advanced techniques might be used to find the possible effects of the exposure of this toxin on the community's health.

Association between Urinary Levels of Aflatoxin and Consumption of Food Linked to Maize or Cow Milk or Dairy Products.

The aim of this analysis was to assess the association between consumption of maize and dairy products and urine and serum levels of aflatoxin FM1 (AFM1) in a sample of 59 males occupationally exposed (29) and non-exposed (30) to aflatoxins. Two urine samples were collected for each person; each sample was accompanied by a questionnaire on food consumption in the preceding 96 h. Given the similar levels of contamination found in exposed and non-exposed workers, the association between food consumption and AFM1 levels was analyzed by pooling samples from exposed and non-exposed workers. No serum sample was found to be positive for AFM1, whereas 74% of the urine samples were positive; the average concentration of positive samples was 0.042 ng/mL (range < limit of detection (LoD) (0.002)-0.399 ng/mL). Of the 21 samples from maize consumers, 13 were positive for AFM1 (62%), with a mean concentration of 0.026 ng/mL (range 0.006-0.088 ng/mL), while 76% (74/94) of the samples from maize non-consumers were positive (mean 0.045, range < LoD (0.002)-0.399 ng/mL). No association was found with milk or dairy products. The high urine level of aflatoxins found in both exposed and non-exposed workers was not associated with the consumption of maize or cow milk products.

Optimization and validation of a LC-HRMS method for aflatoxins determination in urine samples.

Mycotoxins' exposure by inhalation and/or dermal contact can occur in different branches of industry especially where heavily dusty settings are present and the handling of dusty commodities is performed. This study aims to explore the possible contribution of the occupational exposure to aflatoxins by analysing urine samples for the presence of aflatoxins B1 and M1 and aflatoxin B1-N7-guanine adduct. The study was conducted in 2017 on two groups of volunteers, the workers group, composed by personnel employed in an Italian feed plant (n = 32), and a control group (n = 29), composed by the administrative employees of the same feed plant; a total of 120 urine samples were collected and analysed. A screening method and a quantitative method with high-resolution mass spectrometry determination were developed and fully validated. Limits of detections were 0.8 and 1.5 pg/mLurine for aflatoxin B1 and M1, respectively. No quantitative determination was possible for the adduct aflatoxin B1-N7-guanine. Aflatoxin B1 and its adduct were not detected in the analysed samples, and aflatoxin M1, instead, was found in 14 samples (12%) within the range 1.9-10.5 pg/mLurine. Only one sample showed a value above the limit of quantification (10.5 pg/mLurine). The absence of a statistical difference between the mean values for workers and the control group which were compared suggests that in this specific setting, no professional exposure occurs. Furthermore, considering the very low level of aflatoxin M1 in the collected urine samples, the contribution from the diet to the overall exposure is to be considered negligible.

Frequency and levels of aflatoxin M1 in urine of children in Bogota, Colombia.

A study was conducted to investigate the frequency and levels of AFM1 and AFM2 in urine from children who attended the emergency service of a pediatric referral hospital in Bogota, Colombia. A survey on the consumption of foods likely to be a source of aflatoxins and on sociodemographic variables was conducted as well. The frequency of AFM1 in urine was found to be 41.7% with an average concentration in positive samples of 16 pg mL-1 ± 10.7 pg mL-1 (range > LOD-48.5 pg mL-1). The presence of AFM1 in the urine was related to the consumption of cereals likely to be contaminated with AFB1, especially corn and rice. No detectable levels of AFM2 were found in any sample. The results show that children's exposure to aflatoxins in Colombia is indeed a problem and should be one of the priorities of the health authorities. Continuous monitoring of aflatoxins in foods should be carried out, in compliance with Colombian regulations, using analytical methods that allow determination and quantification of aflatoxins in different biological and non-biological matrices at trace levels.

Study of aflatoxicosis reduction: effect of Alchornea cordifolia on biomarkers in an aflatoxin B1 exposed rats.

The toxicity of aflatoxins results in cancer and liver disease. Several natural substances such as plants exhibited their ability to inhibit the initiation of aflatoxin carcinogenesis. The aim of this study was to evaluate the effect of Alchornea cordifolia on biomarkers in an aflatoxin B1 (AFB1) exposed rats. The contents of polyphenols, flavonoids and the antioxidant activity of A. cordifolia ethanolic leaf extract (EELac) were assessed. Groups of rats were treated orally with a daily dose of a mixture of AFB1 at a dose of 150 µg/kg body weight and EELac (50, 100 and 300 mg/kg body weight) for 21 days. Biomarkers of AFB1, such as the AFB1-lysine adduct and aflatoxin M1 were assayed in blood and urine, respectively, using an HPLC system with a fluorescence detector. The contents of polyphenols and flavonoids were 6783.23 ± 272.76 µg EAG/g and 10.54 ± 3.15% of dry matter, respectively. EELac showed a good antioxidant activity (IC50 = 12.65 ± 0.13 µg/mL). The administration of the mixture (AFB1 + EELac) at different doses significantly reduced the level of AFB1-lysine adduct from 14.04 ± 2.1 to 4.13 ± 0.9 ng/mg albumin and that of Aflatoxin M1 (AFM1) from 456 ± 16 to 220 ± 24 ng/mL (p <0.05). The rate of reduction was 70.58% for AFB1-lysine adduct and 51.75% for AFM1. A. cordifolia could be used in the prevention of toxicity induced by AFB1 on account of its high content in phenolic compounds.

Urinary aflatoxin exposure monitoring in rural and semi-urban populations in Ogun state, Nigeria.

Aflatoxins are a major class of fungal toxins that have food safety importance due to their economic and health impacts. This pilot aflatoxin exposure biomonitoring study on 84 individuals was conducted in a rural (Ilumafon) and a semi-urban community (Ilishan Remo) of Ogun state, Nigeria, to compare aflatoxin exposures among the two population cohorts. First morning urine samples were obtained from the participants, and the urinary aflatoxin M1 (AFM1) levels were measured by a quantitative Helica Biosystems Inc. ELISA kit assay. About 99% (83 out of 84) of the urine samples had detectable AFM1 levels in the range of 0.06 to 0.51 ng mL-1 (median: 0.27 ng mL-1). The mean urinary AFM1 levels were significantly (p = 0.001) higher in the semi-urban population (0.31 ± 0.09 ng mL-1) compared to the rural population (0.24 ± 0.07 ng mL-1). There were, however, no significant differences in mean urinary AFM1 levels of males and females, and among children, adolescents and adults. This study indicates high aflatoxin exposure to the extent of public health concerns in the studied populations. Thus, more efforts are required for aflatoxin exposure monitoring and control in high-risk regions.

Association between Urinary Aflatoxin (AFM1) and Dietary Intake among Adults in Hulu Langat District, Selangor, Malaysia.

Aflatoxin is a food contaminant and its exposure through the diet is frequent and ubiquitous. A long-term dietary aflatoxin exposure has been linked to the development of liver cancer in populations with high prevalence of aflatoxin contamination in foods. Therefore, this study was conducted to identify the association between urinary aflatoxin M1 (AFM1), a biomarker of aflatoxin exposure, with the dietary intake among adults in Hulu Langat district, Selangor, Malaysia. Certain food products have higher potential for aflatoxin contamination and these were listed in a Food Frequency Questionnaire, which was given to all study participants. This allowed us to record consumption rates for each food product listed. Concomitantly, urine samples were collected, from adults in selected areas in Hulu Langat district, for the measurement of AFM1 levels using an ELISA kit. Of the 444 urine samples collected and tested, 199 were positive for AFM1, with 37 of them exceeding the limit of detection (LOD) of 0.64 ng/mL. Cereal products showed the highest consumption level among all food groups, with an average intake of 512.54 g per day. Chi-square analysis showed that consumption of eggs (X² = 4.77, p = 0.03) and dairy products (X² = 19.36, p < 0.01) had significant associations with urinary AFM1 but both food groups were having a phi and Cramer's V value that less than 0.3, which indicated that the association between these food groups' consumption and AFM1 level in urine was weak.

Comparison of urinary aflatoxin M1 and aflatoxin albumin adducts as biomarkers for assessing aflatoxin exposure in Tanzanian children.

PURPOSE: To determine levels of urinary aflatoxin M1 (AFM1) in children and correlate the concentrations with previously reported aflatoxin albumin adduct (AF-alb) levels in these children. MATERIALS AND METHODS: Matched urine and blood samples were collected from 84 Tanzanian children aged 6-14 months old. From 31 children in one village (Kigwa), samples were collected at three time points six months apart. Samples were collected from 31 and 22 children from two different regions at the second time point only. Urinary AFM1 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit with a modified protocol to improve sensitivity. AF-alb was measured using an established ELISA method. RESULTS: The relative ranking of the three villages for exposure to aflatoxin based on either AFM1 or AF-alb biomarker measurements was the same. In Kigwa village, both AFM1 and AF-alb levels were higher at six months post-harvest compared to baseline. However, at the next visit, the AFM1 levels dropped from a GM (interquartile range) of 71.0 (44.7, 112.6) at visit two to 49.3 (31.5, 77.3) pg/ml urine, whereas AF-alb levels increased from 47.3 (29.7, 75.2) to 52.7 (35.4, 78.3) pg/mg albumin between these two visits, reflecting the fact that AFM1 measures short-term exposure, whereas AF-alb measures longer term exposure. There was a correlation between AFB1 intake and AFM1 excretion (r= 0.442, p ≤ 0.001). CONCLUSIONS: Urinary AFM1 is a good biomarker for AFB1 exposure in Tanzanian children, reflecting geographical and temporal variations in exposure to this foodborne toxin.

An investigation into aflatoxin M 1 in slaughtered fattening pigs and awareness of aflatoxins in Vietnam.

BACKGROUND: Aflatoxin M1 (AFM1) is a hydroxylated metabolite formed after aflatoxin B1 (AFB1) is consumed by humans and animals; it can be detected in urine, milk and blood. It is well recognized that AFB1 is toxic to humans and other animals. The International Agency for Research on Cancer (IARC) classifies aflatoxins as group 1 carcinogens and AFM1 as group 2B carcinogen. The main objective of this study was to evaluate the exposure of pigs to aflatoxins as well as to assess the public awareness of aflatoxins among people in five provinces in Vietnam. RESULTS: A total of 1920 urine samples were collected from slaughterhouses located in five provinces. Overall, the positive rate of AFM1 was 53.90% (95% confidence interval 51.64-56.15) using a cut-off of 0.15 µg/kg (range: limit of detection to 13.66 µg/kg, median: 0.2 µg/kg and mean: 0.63 µg/kg). A total of 252 people from the general population were interviewed from 5 provinces, and overall 67.86% reported being aware of aflatoxins. We also found that men and more highly educated had significantly increased awareness of aflatoxins compared to the females and primary/secondary school group. The respective odds ratios (ORs) were as follows: "male" group (OR: 2.64), "high school educated" group (OR: 3.40) and "college/university or more educated" group (OR: 10.20). CONCLUSIONS: We can conclude that pigs in Vietnam are exposed to aflatoxins to varying degrees, and there may be a risk that pork products could contain AFM1. Further investigation is needed into the possible health impacts as well as to aid in establishing regulations for animal feed to reduce the health impacts in humans and animals.

Intra-laboratory Development and Evaluation of a Quantitative Method for Measurement of Aflatoxins B1, M1 and Q1 in Animal Urine by High Performance Liquid Chromatography with Fluorescence Detection.

Mycotoxins negatively impact animal health. Aflatoxins (AFs) are the most common mycotoxins affecting both large and small animals and are a common cause of toxin-related pet food recalls. Definitive diagnosis of aflatoxicosis is constrained by a lack of validated ante-mortem analytical methods for detection and quantitation of AFs and their metabolites in biological specimens. Herein, we developed and evaluated a urine-based quantitative method for measurement of aflatoxin B1 (AFB1) and its metabolites aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1) in animal urine. (Some of the results have been presented at 59th AAVLD conference, Greensboro, North Carolina, October 13-19th, 2016.) This method uses an immuno-affinity column for clean-up and pre-column derivatization followed by high performance liquid chromatography analysis with fluorescence detection. The method has high selectivity, recovery (>81%) and sensitivity with an instrument limit of detection of 0.20-1.02 pg; instrument limit of quantitation of 0.77-4.46 pg; and a method lower limit of quantitation of 0.30-2.5 ng/mL. The method has high accuracy, repeatability, and is rugged against minor changes. However, because of poor sensitivity of AFQ1 at low concentrations we recommend this method for quantitative determination of AFB1 and AFM1, and for qualitative measurement of AFQ1 in animal urine for diagnosis of aflatoxicosis.

Determinants of recent aflatoxin exposure among pregnant women in rural Zimbabwe.

SCOPE: Aflatoxins (AFs) are toxic secondary metabolites of Aspergillus species that contaminate staple foods such as maize and groundnuts. AF exposure during pregnancy has been associated with adverse birth outcomes in limited-scale surveys in sub-Saharan Africa. The objective of this study was to describe the determinants of AF exposure, using urinary aflatoxin M1 (AFM1) biomarkers and data generated by the Sanitation Hygiene Infant Nutrition Efficacy (SHINE) trial for rural Zimbabwean women in early pregnancy. Sanitation Hygiene Infant Nutrition Efficacy is a large, cluster-randomized community-based trial in Zimbabwe designed to investigate the independent and combined effects of nutrition and hygiene interventions on early child growth. METHODS AND RESULTS: Urine samples collected from 1580 pregnant women in rural Zimbabwe at median gestational age of 13.9 wk were measured for AFM1. AFM1 was detected in 30% of samples (median of exposed, 162 pg AFM1/mg creatinine; range 30-6046 pg AFM1/mg). In multivariable ordinal logistic models, geographical location (p<0.001), seasonality (p < 0.001) and dietary practices (p = 0.011) were significant predictors of urinary AFM1. CONCLUSION: This is the largest AF biomarker survey conducted in Zimbabwe, and demonstrated frequent exposure in pregnant women with clear temporal and spatial variability in AF biomarker levels.

Detection of trace aflatoxin M1 in human urine using a commercial ELISA followed by HPLC.

Aflatoxin is a liver carcinogen, and rapid, inexpensive methods to detect its urinary biomarkers are needed. We used a commercial enzyme-linked immuno-sorbent assay (ELISA) for aflatoxin M1 in urine (Helica Biosystems) to test 52 Haitian samples. Using this ELISA, we detected traces above the limit of detection (0.2 ng/ml urine) but below the limit of quantitation (0.4 ng/ml) in 14 samples. Liquid chromatography of all 52 Haitian urine samples revealed that only 11 had quantifiable AFM1 (mean: 29.5 pg/ml, standard error: 10.8, range: 2.94-96.5 pg/ml). The Helica ELISA may have detected forms of aflatoxin other than AFM1 in the Haitian samples, or matrix enhancement may have affected results at low AFM1 concentrations. This ELISA may serve as an initial, qualitative indicator of aflatoxin exposure for epidemiological purposes. But this method's utility as a precise and specific indicator of AFM1 concentrations will require additional refinement and validation.

Determination of aflatoxin M1 in urine samples indicates frequent dietary exposure to aflatoxin B1 in the Bangladeshi population.

Aflatoxin B1 (AFB1), a hepatocarcinogen and highly toxic mycotoxin, is a contaminant of food commodities, especially in hot and humid climates that favour the growth of aflatoxin-producing fungi. As data on AFB1 contamination of food and feed in Bangladesh are scarce, we conducted an initial screening by ELISA on the occurrence of the metabolite and biomarker aflatoxin M1 (AFM1) in urines from Bangladesh which indicated frequent exposure. This finding led us to conduct a follow-up study where we applied a more sensitive method (IAC clean-up and HPLC-FD analysis) to determine AFM1 concentrations in a larger set of urine samples. To account for possible seasonal and regional differences in mycotoxin exposure, in total 218 urines were collected in two districts of Bangladesh: 164 urines (n=69 in summer, n=95 in winter) from residents of a rural and an urban area in Rajshahi district, among them 62 participants enrolled in both sampling periods, and 54 urine samples obtained from pregnant women in Dhaka district. AFM1 was detected in>40% of all Rajshahi urine samples at a range of 1.7-104pg/mL in summer and at a range of 1.8-190pg/mL in winter season. The mean level of urinary AFM1 was higher in winter (27.7±42.6pg/mL) than in summer (13.6±21.2pg/mL) season, and differences were observed at the mean AFM1 level between the rural and the urban Rajshahi cohort. AFM1 was found less frequently in the Dhaka pregnant women (31% above LOD, mean 13.9±33.3pg/mL), but in a similar concentration range (1.7-141pg/mL) as in the Rajshahi cohort. Urinary AFM1 levels did not show significant associations with the participants food consumption pattern. In conclusion, when compared to biomarker data from other countries, detection frequency and urinary AFM1 levels in our Bangladeshi cohorts raise concerns regarding their exposure to potent carcinogenic aflatoxins.

Urinary aflatoxin M1 in Port-au-Prince and a rural community in north-east Haiti.

Aflatoxins (AFs) are hepatocarcinogenic mycotoxins that can contaminate grains and oil seeds in tropical and sub-tropical areas and have been detected in maize and peanut products of Haiti. The first objective was to assess human exposure to AFs among Haitians at an urban hospital (GHESKIO) and a rural health centre (HCBH). The second objective was to test the association between AF exposure and reported dietary intake of potentially contaminated foods, such as maize, peanut products and milk. Measurement of urinary AFM1 by HPLC revealed that among 367 participants 14% and 22% at GHESKIO and HCBH, respectively, had detectable AFM1. The maximum and median AFM1 concentrations for all detected samples were 700 pg AFM1 ml(-1) and 11.7 pg ml(-1), respectively. Detection of AFM1 was significantly associated with peanut consumption (p < 0.05). Controlling for diet and age group in a logit model, patients who reported peanut consumption the day of the survey and patients from HCBH had greater log odds of excreting detectable AFM1 (p < 0.001 and 0.002, respectively); females had lower log odds (p = 0.020). Recalled frequency of consuming non-dairy animal-sourced foods, an indicator of diet quality, approached significance (p = 0.056) as an inverse predictor of urinary AFM1 detection. The findings augur the need for interventions that will improve food safety in Haiti and limit exposure to AFs, particularly among rural communities.

Association between Aflatoxin M1 and Liver Disease in HBV/HCV Infected Persons in Ghana.

Aflatoxins are produced by the fungi Aspergillus flavus and Aspergillus parasiticus and are common food contaminants in tropical developing countries. Extensive aflatoxin consumption has been shown to be highly associated with liver disease. A case-control study was conducted to determine the association between aflatoxin and liver disease in Kumasi, Ghana. A questionnaire was administered to examine socio-demographic characteristics and food storage and consumption practices, and urine samples were collected to measure levels of the aflatoxin metabolite (AFM1). Two hundred and seventy-six people participated in the study; 38 had liver disease (cases), 136 had neither hepatitis B/C nor liver disease (negative controls), and 102 were hepatitis B/C positive without liver cancer (positive controls). A much higher percent of participants in each group was male (76% of cases, 88% of negative controls and 65% of positive controls). Multivariate analysis showed that age was a significant predictor for being a case when cases were compared to negative controls. The odds of being a case was 70% less for participants aged 25-34 years (odds ratios (OR) 0.30; 95% confidence interval (CI) 0.10-0.88) compared to those ≥45 years. For cases; Akans were seven times more likely to have AFM1 levels below the median when compared to other ethnic groups (OR 7; CI 1.41-34.68). When cases were compared to positive controls, they were 2.29 times more likely to report awareness of aflatoxin contamination of groundnuts (95% CI 1.06-4.91). Cases were also two times more likely to report awareness of aflatoxin contamination of maize than all controls combined (95% CI 1.02-4.11). However, most cases reported that aflatoxin contamination does not cause sickness in humans. This shows that there is awareness of aflatoxin contamination without proper understanding of the serious potential adverse health impacts among these study participants. These findings indicate that educational interventions that stress the harmful health effects of aflatoxin in food, with an emphasis on the higher risk for males, are urgently needed. The reasons for lower aflatoxin levels among Akans need to be determined, and the findings used to design interventions that benefit other ethnic groups in the society.

Assessment of aflatoxin exposure using serum and urinary biomarkers in São Paulo, Brazil: A pilot study.

The aim of this study was to evaluate the human exposure of individuals from Pirassununga, Brazil, to dietary aflatoxins B1 (AFB1) and M1 (AFM1) by determination of serum AFB1-lysine and urinary aflatoxin biomarkers (AFM1 and AFB1-N(7)-guanine). The participants were recruited among employees from a Campus of the University of São Paulo, which provided food samples from their homes, as well as serum and urine samples four times every three months, from June 2011 until March 2012. The probable daily intake (PDI) of aflatoxin was estimated by using the results from analysis of food products collected by the time of samples collection, and data from a 24-hour dietary recall questionnaire. Analyses of AFB1 and AFM1 in food samples were conducted by high-performance liquid chromatography with fluorescence detection. Biomarkers in serum and urine were determined by tandem mass spectrometry. AFB1 and AFM1 were detected in 38 samples of cereals (28%, N=136) and 31 milk products (36%, N=86), respectively. AFB1-lysine and AFB1-N(7)-guanine and were not detected in serum or urine samples, respectively. However, AFM1 was found in 74 urine samples (65%), at mean levels in the 4 sampling times ranging from 0.37±0.23 to 1.70±2.88pg/mg creatinine. The mean PDI varied among different sampling times, ranging from 0.09±0.09 to 1.35±5.98ng/kg body weight/day. A modest though significant correlation (r=0.45; p=0.03; N=23) was found for the first time in Brazil between the AFM1 concentration in urine and the PDI for total aflatoxins (AFB1+AFM1) in sampling 1 (June 2011). Urinary AFM1 was confirmed as very sensitive for monitoring the human exposure to dietary aflatoxin. Further studies using serum and urinary biomarkers are needed to estimate the aflatoxin exposure of populations in higher risk areas in Brazil.

Occurrence of aflatoxin M1 in urines from rural and urban adult cohorts in Bangladesh.

Aflatoxins are important mycotoxins produced by Aspergillus flavus and A. parasiticus, moulds which contaminate mainly grains and nuts, especially in hot and humid climate. Presence of aflatoxin B1 (AFB1), the most toxic one and a potent hepatocarcinogen, has been reported in food and feed in Bangladesh and raised concerns about mycotoxin exposure in the population. Biomonitoring provides the best approach to assess human exposure from various sources and by all routes. Part of the ingested AFB1 is converted in the body to aflatoxin M1 (AFM1), a metabolite that has served as biomarker of AFB1 exposure, as it is excreted in urine, and thus enables non-invasive sampling, a relevant aspect in field studies. This investigation measured the AFM1 concentration in urines collected from adult residents of a rural (n = 52) and an urban (n = 43) area in the Rajshahi district of Bangladesh. The urinary levels of AFM1 were determined by enzyme-linked immunosorbent assay. AFM1 was detected in 46 % of all urine samples at a range of 31-348 pg/mL. The median and mean concentration of AFM1 in urine was 61 and 80 ± 60 pg/mL, respectively. A significant difference (p < 0.05) was found at the mean level of AFM1 between the rural (99 ± 71 pg/mL) and urban (54 ± 15 pg/mL) cohort. Urinary AFM1 levels did not show significant correlations with food frequency data or age, gender and body mass index of the participants. Among them, the highest mean AFM1 level (101 ± 71 pg/mL) was observed in the 50-60 years age group. In conclusion, detection frequency and urinary AFM1 levels in the Bangladeshi adults support concerns regarding their dietary exposure to AFB1. These first data warrant further biomarker-based studies in children and in cohorts of other parts of the country.