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Background: Aflatoxin B1causes damage to the DNA by the alkylation of bases and P53 mutation. Exposure to this mycotoxin is associated with the development of liver cancer. Measures to reduce grain and cereal contamination have been a focus however, the effects of these measures are still lagging behind and exposure continues to occur even in populations at risk of developing liver cancer., Objective: To quantify aflatoxin B1 exposure in a population of HIV infected patients with and without HCC., Method: This was a cross-sectional study among 196 patients with HIV and or HCC. We evaluated the exposure to aflatoxin B1 using the Aflatoxin M1 metabolite by ELISA on urine samples., Results: A total of 196 participants consisting of 163 (83.2%) HIV positive and 28 (14.3%) HCC. Mean age is 46.64±10.8 years. The median aflatoxin (IQR) aflatoxin M1level is 177.3(112.5-272) pg/ml. Only 8(4.1%) of the participant had no exposure to aflatoxin B1. The median (IQR) aflatoxin for fibrosis score ≥ 13kpa (178.7(112.9-286.8) pg/ml) VS < 13kpa (173.5(107.9-250.4)), p = 0.046., Conclusion: There is high prevalence of aflatoxin B1 exposure in this population. Concerted efforts must be put in place to mitigate exposure because of the potential effects of short- and long-term exposure to aflatoxin., (© 2023 Davwar P et al.)

Analysis of body fluids and tissues of aflatoxin exposed individuals for the presence of aflatoxins and aflatoxin metabolites has emerged as a reliable indicator of exposure and metabolism of aflatoxins. However, current aflatoxin biomarkers are not appropriate for investigating the long-term effects of aflatoxin exposure. In this explorative study, we investigated the analysis of hair as a complementary or alternative matrix for the assessment of biomarkers of long-term aflatoxin exposure. Three groups of guinea pigs were orally dosed with 5 ugkg -1 bw -1 , 50 ugkg -1 bw -1 , and 100 ugkg -1 bw -1 of AFB1. Urine and hair samples were collected on days 0, 1, 2, 3, 7, 30, 60, and 90 and analysed for AFB1 and AFM1 using UHPLC-MS/MS. AFB1 and AFM1 were detected in 75% and 13.6%, respectively, of the day 1 to day 7 urine samples. AFB1 was detected in hair samples collected from day 3 up to day 60. This is the first report to confirm the deposition of AFB1 in the hair of experimental animals. These findings indicate that hair analysis has the potential to provide an accurate long-term historical record of aflatoxin exposure with potentially important implications for the field of aflatoxin biomarkers.

This case-control study adds to the growing body of knowledge on the medical, nutritional, and environmental factors associated with Nodding Syndrome (NS), a seizure disorder of children and adolescents in northern Uganda. Past research described a significant association between NS and prior history of measles infection, dependence on emergency food and, at head nodding onset, subsistence on moldy maize, which has the potential to harbor mycotoxins. We used LC-MS/MS to screen for current mycotoxin loads by evaluating nine analytes in urine samples from age-and-gender matched NS cases (n = 50) and Community Controls (CC, n = 50). The presence of the three mycotoxins identified in the screening was not significantly different between the two groups, so samples were combined to generate an overall view of exposure in this community during the study. Compared against subsequently run standards, α-zearalenol (43 ± 103 µg/L in 15 samples > limit of quantitation (LOQ); 0 (0/359) µg/L), T-2 toxin (39 ± 81 µg/L in 72 samples > LOQ; 0 (0/425) µg/L) and aflatoxin M1 (4 ± 10 µg/L in 15 samples > LOQ; 0 (0/45) µg/L) were detected and calculated as the average concentration ± SD; median (min/max). Ninety-five percent of the samples had at least one urinary mycotoxin; 87% were positive for two of the three compounds detected. While mycotoxin loads at NS onset years ago are and will remain unknown, this study showed that children with and without NS currently harbor foodborne mycotoxins, including those associated with maize.

Human biomonitoring is an important tool to assess human exposure to chemicals, contributing to describe trends of exposure over time and to identify population groups that could be under risk. Aflatoxins are genotoxic and carcinogenic food contaminants causing hepatocellular carcinoma, the third leading cause of cancer deaths worldwide. In Portugal, scarce data are available regarding exposure to aflatoxins and no previous study used human biomonitoring data to comprehensively characterize the associated burden of disease. 24 h urine and first-morning urine paired samples were collected by 94 participants and were analyzed by liquid chromatography-tandem mass spectrometry for the quantitative determination of aflatoxins (B 1 , B 2 , G 1 , G 2 and M 1 ). Deterministic and probabilistic models were developed to assess the Portuguese exposure to aflatoxins and to estimate the health impact of this exposure, estimating the attributed Disability-Adjusted Life Years (DALYs). Aflatoxins were detected in a maximum of 13% (AFB 1 ), 16% (AFB 2 ), 1% (AFG 1 ), 2% (AFG 2 ) and 19% (AFM 1 ) of the urine samples. Data obtained through the probabilistic approach revealed an estimated mean probable daily intake of 13.43 ng/kg body weight per day resulting in 0.13 extra cases of hepatocellular carcinoma, corresponding to mean annual DALYs of 172.8 for the Portuguese population (10291027 inhabitants). The present study generated for the first time and within a human biomonitoring study, reliable and crucial data to characterize the burden associated to the exposure to aflatoxins of the Portuguese population. The obtained results constitute an imperative support to risk managers in the establishment of preventive policy measures that contribute to ensure public health protection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The study aimed to quantify the immediate effects of dietary diversification, food safety, and hygiene interventions on child undernutrition in four rural villages in Kongwa district of central Tanzania. One hundred mothers with their children of less than 24 months old were recruited for this study. The difference-in-difference (DID) method was used to assess the effects of intensive intervention through a learning-by-doing process on the topic of aflatoxin free diversified food utilization and improved hygiene practices. Periodic anthropometric measurements were conducted on the 0th, 7th, 14th, and 21st days, and DID estimator showed the significant and positive average marginal effects of the intervention on Z-Scores being 0.459, 0.252, and 0.493 for wasting, stunting, and underweight, respectively. Notably, at the end of the study, the mean aflatoxin M 1 level in urine samples decreased by 64% in the intervention group, while it decreased by 11% in the control group. The study provides quantitative evidence on intensive 21-day training for mothers incorporating integrated technologies yielded positive impacts on their children's nutritional outcomes.

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 B 1 and M 1 and aflatoxin B 1 -N 7 -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/mL urine for aflatoxin B 1 and M 1 , respectively. No quantitative determination was possible for the adduct aflatoxin B 1 -N 7 -guanine. Aflatoxin B 1 and its adduct were not detected in the analysed samples, and aflatoxin M 1 , instead, was found in 14 samples (12%) within the range 1.9-10.5 pg/mL urine . Only one sample showed a value above the limit of quantification (10.5 pg/mL urine ). 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 M 1 in the collected urine samples, the contribution from the diet to the overall exposure is to be considered negligible.

Cereal foods are commonly contaminated with multiple mycotoxins resulting in frequent human mycotoxin exposure. Children are at risk of high-level exposure because of their high cereal intake relative to body weight. Hence, this study aims to assess multimycotoxin exposure in UK children using urinary biomarkers. Spot urines ( n = 21) were analyzed for multimycotoxins (deoxynivalenol, DON; nivalenol, NIV; ochratoxin A, OTA; zearalenone, ZEN; α-zearalenol, α-ZEL; β-zearalenol, β-ZEL; T-2 toxin, T-2; HT-2 toxin, HT-2; and aflatoxin B 1 and M 1, AFB 1 , AFM 1 ) using liquid chromatography-coupled tandem mass spectrometry. Urine samples frequently contained DON (13.10 ± 12.69 ng/mL), NIV (0.36 ± 0.16 ng/mL), OTA (0.05 ± 0.02 ng/mL), and ZEN (0.09 ± 0.07 ng/mL). Some samples (1-3) contained T-2, HT-2, α-ZEL, and β-ZEL but not aflatoxins. Dietary mycotoxin estimation showed that children were frequently exposed to levels exceeding the tolerable daily intake (52 and 95% of cases for DON and OTA). This demonstrates that UK children are exposed to multiple mycotoxins through their habitual diet.

A study was conducted to investigate the frequency and levels of AFM 1 and AFM 2 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 AFM 1 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 AFM 1 in the urine was related to the consumption of cereals likely to be contaminated with AFB 1 , especially corn and rice. No detectable levels of AFM 2 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.

Objective: The present study examined the impacts of training on nutrition, hygiene and food safety designed by the Nutrition Working Group, Child Survival Collaborations and Resources Group (CORE)., Design: Adapted from the 21d Positive Deviance/Hearth model, mothers were trained on the subjects of appropriate complementary feeding, water, sanitation and hygiene (WASH) practices, and aflatoxin contamination in food. To assess the impacts on child undernutrition, a randomised controlled trial was implemented on a sample of 179 mothers and their children (<2 years old) in two districts of Malawi, namely Mzimba and Balaka. Settings A 21d intensive learning-by-doing process using the positive deviance approach., Subjects: Malawian children and mothers., Results: Difference-in-difference panel regression analysis revealed that the impacts of the comprehensive training were positive and statistically significant on the Z-scores for wasting and underweight, where the effects increased constantly over time within the 21d time frame. As for stunting, the coefficients were not statistically significant during the 21d programme, although the level of significance started increasing in 2 weeks, indicating that stunting should also be alleviated in a slightly longer time horizon., Conclusions: The study clearly suggests that comprehensive training immediately guides mothers into improved dietary and hygiene practices, and that improved practices take immediate and progressive effects in ameliorating children's undernutrition.

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 AFM 1 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.

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., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The direct measurement of biomarkers of exposure in biological fluids such as urine has become important for assessing aflatoxin exposure in humans as it is the only tool that integrates exposures from various routes. For this reason, a study was conducted to assess aflatoxin exposure among young children in Ethiopia using urinary biomarkers. A cross-sectional study was conducted in ten Woredas (Districts) from Amhara and Tigray regional states of Ethiopia including 200 children (aged 1-4 years). A total of 200 urine samples were collected from 200 children and assessed for the levels of aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), aflatoxin G 2 (AFG 2 ) and aflatoxin M 1 (AFM 1 ) using a validated LC-MS/MS method. Aflatoxins were detected in 34/200 (17%) of the urine samples whereby four out of five analysed aflatoxins were detected. AFM 1 was detected in 14/200 (7%) of the urine samples in a range of 0.06-0.07 ng/mL. AFB 2 , AFG 2 and AFG 1 were detected in respectively 9/200 (4.5%), 6/200 (3%) and 5/200 (2.5%) of the urine samples whereas AFB 1 was not detected in any of the samples. In this study, there was no association between the different malnutrition categories (stunted, wasting and underweight) and aflatoxin exposure. However, the biomarker analysis showed a clear exposure of young children to aflatoxins. Therefore, awareness to the public is important to prevent potential health consequences of aflatoxins.

Feed mill workers may handle or process maize contaminated with aflatoxins (AFs). This condition may lead to an unacceptable intake of toxins deriving from occupational exposure. This study assessed the serological and urinary levels of AFs in workers exposed to potentially contaminated dusts in two mills. From March to April 2014, blood and urine samples were collected, on Monday and Friday morning of the same working week from 29 exposed workers and 30 non-exposed controls. AFs (M₁, G₂, G₁, B₁, B₂) and aflatoxicol (AFOH) A were analyzed. Each subject filled in a questionnaire to evaluate potential food-borne exposures to mycotoxins. AFs contamination in environmental dust was measured in both plants. No serum sample was found to be positive. Seventy four percent of urine samples (73.7%) revealed AFM₁ presence. AFM₁ mean concentration was 0.035 and 0.027 ng/mL in exposed and non-exposed workers, respectively ( p = 0.432); the concentration was slightly higher in Friday's than in Monday's samples, in exposed workers, 0.040 versus (vs.) 0.031 and non-exposed controls (0.030 vs. 0.024, p = 0.437). Environmental AFs contamination ranged from 7.2 to 125.4 µg/kg. The findings of this study reveal the presence of higher AFs concentration in exposed workers than in non-exposed controls, although these differences are to be considered consistent with random fluctuations.

Background: Aflatoxin and fumonisin are toxic food contaminants. Knowledge about effects of their exposure and coexposure on child growth is inadequate., Objective: We investigated the association between child growth and aflatoxin and fumonisin exposure in Tanzania., Methods: A total of 166 children were recruited at 6-14 months of age and studied at recruitment, and at the 6th and 12th month following recruitment. Blood and urine samples were collected and analyzed for plasma aflatoxin-albumin adducts (AF-alb) using ELISA, and urinary fumonisin B1 (UFB1) using liquid chromatography-mass spectrometry, respectively. Anthropometric measurements were taken, and growth index z-scores were computed., Results: AF-alb geometric mean concentrations (95% CIs) were 4.7 (3.9, 5.6), 12.9 (9.9, 16.7), and 23.5 (19.9, 27.7) pg/mg albumin at recruitment, 6 months, and 12 months from recruitment, respectively. At these respective sampling times, geometric mean UFB1 concentrations (95% CI) were 313.9 (257.4, 382.9), 167.3 (135.4, 206.7), and 569.5 (464.5, 698.2) pg/mL urine, and the prevalence of stunted children was 44%, 55%, and 56%, respectively. UFB1 concentrations at recruitment were negatively associated with length-for-age z-scores (LAZ) at 6 months (p = 0.016) and at 12 months from recruitment (p = 0.014). The mean UFB1 of the three sampling times (at recruitment and at 6 and 12 months from recruitment) in each child was negatively associated with LAZ (p < 0.001) and length velocity (p = 0.004) at 12 months from recruitment. The negative association between AF-alb and child growth did not reach statistical significance., Conclusions: Exposure to fumonisin alone or coexposure with aflatoxins may contribute to child growth impairment.

Scope: In this study, the exposure of a German population (n = 101) to mycotoxins was assessed using an LC-MS/MS urinary multibiomarker approach. Food consumption of the participants was documented with a food frequency questionnaire to correlate mycotoxin exposure with individual nutritional habits., Methods and Results: The presence of 23 urinary biomarkers including trichothecenes (deoxynivalenol (DON), DON-3-glucuronide (DON-3-GlcA), T-2 toxin, HT-2 toxin (HT-2, HT-2-toxin-4-glucuronide (HT-2-GlcA), fumonisins (fumonisin B1, fumonisin B2), aflatoxins (aflatoxin B1, aflatoxin G2, aflatoxin B2, aflatoxin M1), zearalenone and derivatives (zearalanone, α-zearalenol, β-zearalenol, zearalenone-14-O-glucuronide, zearalanone-14-O-glucuronide, α-zearalenol-14-O-glucuronide/β-zearalenol-14-O-glucuronide), ochratoxin A, ochratoxin alpha, enniatin B and dihydrocitrinone was evaluated using a validated, sensitive "dilute and shoot"-LC-MS/MS method applying Scheduled MRM(TM) technology. Six mycotoxins and urinary metabolites were detected (DON, DON-3-GlcA, zearalenone-14-O-glucuronide, T-2 toxin, enniatin B, and dihydrocitrinone) in 87% of the samples in single- or co-occurence. Only DON and DON-3-GlcA were detectable in quantifiable amounts. A provisional mean daily intake of 0.52 μg DON/kg body weight was calculated. No statistical evidence for the correlation of staple food intake and urinary biomarker concentration could be determined., Conclusion: The results of this study suggest a low everyday exposure of the investigated German population to mycotoxins, but reveal peak exposures above the widely accepted tolerable daily intake to DON in parts of the population., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recently, an association between childhood growth stunting and aflatoxin (AF) exposure has been identified. In Ghana, homemade nutritional supplements often consist of AF-prone commodities. In this study, children were enrolled in a clinical intervention trial to determine the safety and efficacy of Uniform Particle Size NovaSil (UPSN), a refined calcium montmorillonite known to be safe in adults. Participants ingested 0.75 or 1.5 g UPSN or 1.5 g calcium carbonate placebo per day for 14 days. Hematological and serum biochemistry parameters in the UPSN groups were not significantly different from the placebo-controlled group. Importantly, there were no adverse events attributable to UPSN treatment. A significant reduction in urinary metabolite (AFM1) was observed in the high-dose group compared with placebo. Results indicate that UPSN is safe for children at doses up to 1.5 g/day for a period of 2 weeks and can reduce exposure to AFs, resulting in increased quality and efficacy of contaminated foods., (© The American Society of Tropical Medicine and Hygiene.)

In the present study, a longitudinal assessment was carried out to evaluate the short-term human exposure to aflatoxins in Pirassununga region, São Paulo, Brazil, by determination of urinary aflatoxins by a liquid chromatography coupled to mass sprectrometry (UPLC-MS/MS) method. Sixteen volunteers with ages ranging from 14 to 55 years old were instructed to collect the early morning first urine four times every three months, from June 2011 to March 2012, totaling 64 samples. Aflatoxin M1 (AFM1) was found in 39 samples (61%) at levels ranging from 0.19 to 12.7 pg·mg-1 creatinine (mean: 1.2 ± 2.0 pg·mg-1 creatinine). Residues of aflatoxins B1, B2, G1, G2 and aflatoxicol were not identified in any urine sample. No significant difference was found among the AFM1 mean levels in urine samples collected in the four sampling periods. The levels of AFM1 found in urine samples indicate a low short-term exposure of the population studied to aflatoxins through the diet, although further investigations are needed to assess other long-term biomarkers of exposure to AFB1.

Over the past 20 years, exposure to mycotoxin producing mold has been recognized as a significant health risk. Scientific literature has demonstrated mycotoxins as possible causes of human disease in water-damaged buildings (WDB). This study was conducted to determine if selected mycotoxins could be identified in human urine from patients suffering from chronic fatigue syndrome (CFS). Patients (n = 112) with a prior diagnosis of CFS were evaluated for mold exposure and the presence of mycotoxins in their urine. Urine was tested for aflatoxins (AT), ochratoxin A (OTA) and macrocyclic trichothecenes (MT) using Enzyme Linked Immunosorbent Assays (ELISA). Urine specimens from 104 of 112 patients (93%) were positive for at least one mycotoxin (one in the equivocal range). Almost 30% of the cases had more than one mycotoxin present. OTA was the most prevalent mycotoxin detected (83%) with MT as the next most common (44%). Exposure histories indicated current and/or past exposure to WDB in over 90% of cases. Environmental testing was performed in the WDB from a subset of these patients. This testing revealed the presence of potentially mycotoxin producing mold species and mycotoxins in the environment of the WDB. Prior testing in a healthy control population with no history of exposure to a WDB or moldy environment (n = 55) by the same laboratory, utilizing the same methods, revealed no positive cases at the limits of detection.

Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M(1) (AFM(1)), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alpha-zearalenol (α-ZOL)/beta-zearalenol (β-ZOL), and fumonisin B(1) (FB(1)) can be used as urinary biomarkers. A liquid chromatographic-tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed-phase chromatography using a multi-step linear methanol-water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC-ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03-12 ng mL(-1), using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3-20%. Enzymatic digestion with β-glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time.

This was a screening study that aimed to determine the presence of nephrotoxic mycotoxins in urine samples from patients with chronic kidney disease of uncertain etiology in the North Central Province of Sri Lanka. The percentage detection of aflatoxins, ochratoxins and fumonisins in 31 patients were 61.29%, 93.5% and 19.4%, respectively. Geometric means of urinary aflatoxins and ochratoxins were 30.93 creatinine and 34.62 ng/g creatinine in chronic kidney disease of uncertain etiology stage 1-2 patients and 84.12 ng/g creatinine and 63.52 ng/g creatinine in unaffected relatives of patients. In chronic kidney disease of uncertain etiology stage 3-5 patients, geometric means of urinary aflatoxins and ochratoxins were 10.40 and 17.08 ng/g creatinine, respectively. Non-affected relatives of patients (n = 6) had comparable levels of these mycotoxins, but healthy Japanese individuals (n = 4) had lower levels than in Sri Lanka. The higher detection rate of urinary ochratoxins in Sri Lankans indicates that exposure is common in the region.

We report a single, reliable, non-invasive analytical method for monitoring the fetal level of exposure to different mycotoxins. We assessed by tandem mass spectrometry levels up to 200 nanog/l for ochratoxin A and 500 nanog/l for aflatoxins in sample of serum (n= 71), urine (n= 18) and amniotic fluid (n= 21) of pregnant women. Aflatoxin G1 was present in one sample of serum (3.48 microg/l) and in four samples of urine (ranging from 14.0 to 18.8 microg/l), ochratoxin A was present in one sample of amniotic fluid (4.26 microg/l), whereas aflatoxin B1 (ranging from 0.4-2 microg/l) and B2 (ranging from 0.3-3 microg/l) were contextually present in two samples of urine. The very few contaminated samples did not allow statistical comparison between subjects grouped according to the frequency of consumption of commonly contaminated foods. Data confirm that mycotoxins can occur in fetal-maternal biological fluids. However, the incidence and the level of exposure to the investigated mycotoxins do not appear to pose risk for the mother and the fetus.

Aflatoxins are food contaminants usually associated with hepatitis, immunodepression, impairment of fertility and cancer. The present work was to determine the presence of aflatoxins in eggs, milk, urine, and blood samples that were collected from various sources and periods; and hepatitis B virus antigen in blood samples. Aflatoxin was found in eggs (45.2%), cow raw milk (15.9%), breast milk (4.8%), urine from kwashiorkor and marasmic kwashiorkor children (45.5%), and sera from primary liver cancer patients (63.9%); HbsAg was also detected in 69.4% of the serum samples, but there was no association between both factors. Both AF and hepatitis B virus seem to be risk factors that could increase the incidence and prevalence rates of malnutrition and cancer in Cameroon.

The goal of this study was to determine if selected mycotoxins (trichothecenes, aflatoxins, and ochratoxins) could be extracted and identified in human tissue and body fluids from patients exposed to toxin producing molds in their environment. Human urine and methanol extracted tissues and sputum were examined. Trichothecenes were tested using competitive ELISA techniques. Aflatoxins B1, B2, G1, and G2, and ochratoxin A were tested by using immunoaffinity columns and fluorometry. Test sensitivity and specificity were determined. Levels of detection for the various mycotoxins varied from 0.2 ppb for trichothecenes, 1.0 ppb for aflatoxins, and 2.0 ppb for ochratoxins. Trichothecene levels varied in urine, sputum, and tissue biopsies (lung, liver, brain) from undetectable (<0.2 ppb) to levels up to 18 ppb. Aflatoxin levels from the same types of tissues varied from 1.0 to 5.0 ppb. Ochratoxins isolated in the same type of tissues varied from 2.0 ppb to > 10.0 ppb. Negative control patients had no detectable mycotoxins in their tissues or fluids. These data show that mycotoxins can be detected in body fluids and human tissue from patients exposed to mycotoxin producing molds in the environment, and demonstrate which human tissues or fluids are the most likely to yield positive results.

The efficacy of NovaSil clay (NS) to reduce aflatoxin (AF) biomarkers of exposure was evaluated in 656 blood samples and 624 urine samples collected from study participants during a 3-month phase IIa clinical intervention trial in Ghana. NS was delivered before meals via capsules. Serum AFB (1)-albumin adduct was measured by radioimmunoassay and urinary AFM (1) metabolites were quantified by immunoaffinity-high-performance liquid chromatography (HPLC)-fluorescence methods. Levels of AFB (1) -albumin adduct in serum samples collected at baseline and at 1 month were similar (p = 0.2354 and p = 0.3645, respectively) among the placebo (PL), low dose (LD, 1.5 g NS day (-1)), and high dose (HD, 3.0 g NS day (-1)) groups. However, the levels of AFB (1)-albumin adduct at 3 months were significantly decreased in both the LD group (p < 0.0001) and the HD group (p < 0.0001) compared with levels in the PL group. Levels of AFM(1) in urine samples collected at baseline and at 1 month were not statistically different among the three study groups. However, a significant decrease (up to 58%) in the median level of AFM (1) in samples collected at 3 months was found in the HD group when compared with the median level in the PL group (p < 0.0391). In addition, significant effects were found for dose, time, and dose-time interaction with serum AFB(1)-albumin adduct and dose-time interaction with urinary AFM (1) metabolites. The results suggest that capsules containing NS clay can be used to reduce effectively the bioavailability of dietary AF based on a reduction of AF-specific biomarkers.

This study investigates the occurrence of aflatoxins in Ecuador. Early investigators proved the presence of aflatoxins in human and animal food, but the disturbing data lead to the formation of two research teams at Guayaquil University and the Agrarian University of Ecuador to investigate aflaxotins and other mycotoxins in food and their relationship to human health. Because the concept of mycotoxicosis as a result of the secondary metabolites produced by different species of moulds could cause different clinical patterns, the research team includes Aspergillus metabolites found in the urine of a patient with pulmonary aspergilloma. We considered that the body itself could create secondary metabolites. An ELISA method was used to detect mycotoxins with the specific reactive compounds using a company base assay. This allows the detection quantitative of such metabolites in 24 h collected urine. The patient was treated with itraconazole for nine months, after clinical, radiological and aflatoxins testing. We also investigated three other cases in children with a second level of malnutrition and only with vomitoxins results and in three investigated cases of otomycosis caused by Aspergillus niger only in one case traces of aflatoxins were found.

Aflatoxins are a major risk factor for hepatocellular carcinoma (HCC), and thus understanding the pattern of aflatoxin exposure in different regions is important in order to develop targeted intervention strategies. Given the early onset of HCC in many countries early life exposures may be important. This study investigated aflatoxin exposure in Egyptian children (n=50, aged 1-2.5 years) by assessing urinary aflatoxin metabolite (AFM(1), AFB(1), AFB(2), AFG(1), AFG(2)) levels. Samples from Guinean children (n=50, aged 2-4 years) were analyzed in parallel providing a comparison to a region of established frequent aflatoxin exposure. Aflatoxins were isolated from urine using C18-cartridges followed by immunoaffinity clean-up, and quantified by HPLC with fluorescence detection. Overall aflatoxins were less frequently present in Egyptian (38%) than Guinean urine samples (86%) (p<0.001), which was particularly related to differences in detection rates of AFM(1) (8% compared to 64%, respectively, (p<0.001)). For AFM(1) the geometric mean level in Guinea (16.3 pg/ml; 95% CI: 10.1, 26.6 pg/ml) was 6-fold higher (p<0.001) than in Egypt (2.7 pg/ml; 95% CI: 2.5, 2.8 pg/ml). Urinary aflatoxins from healthy children in these two regions have not previously been reported, and exposure appears modest in Egypt compared to Guinea. These data suggest that measures to reduce aflatoxin exposure in both regions are important, though particularly in Guinea.

To evaluate the efficacy of green tea polyphenols (GTPs) in modulating aflatoxin B(1) (AFB(1)) biomarkers, a total of 352 serum samples and 352 urine samples collected from a 3 month chemoprevention trial with 500 mg GTPs, 1000 mg GTPs and a placebo were measured for AFB(1)-albumin adducts (AFB-AA), aflatoxin M(1) (AFM(1)) and aflatoxin B(1)-mercapturic acid (AFB-NAC). Levels of AFB-AA at baseline were comparable for all three dose groups (P = 0.506). No significant differences were observed in AFB-AA levels in the placebo group over the 3 month period (P = 0.252). However, a significant reduction in AFB-AA levels was observed in the 500 mg group (P = 0.002). A marginally significant reduction in AFB-AA levels was also found in the 1000 mg group over the 3 month intervention period (P = 0.051). An analysis using a mixed-effects model indicated that the reduction in AFB-AA levels over time was dose and time dependent (dose-time interaction P = 0.049). There were no significant differences in median AFM(1) levels among the three study groups at the baseline (P = 0.832), 1 month (P = 0.188) and 3 months (P = 0.132) of the GTP intervention; however, reduction of 42 and 43% in median AFM(1) levels, as compared with the placebo, were found in 500 mg (P = 0.096) and 1000 mg (P = 0.072) groups at 3 months of the intervention. Significant elevations in median AFB-NAC levels and the ratio of AFB-NAC:AFM(1) were found in both 500 and 1000 mg groups compared with the placebo group at both 1 month (P < 0.001) and 3 months (P < 0.001) of GTPs intervention. These results demonstrate that GTPs effectively modulate AFB(1) metabolism and metabolic activation.

Automated immunoaffinity solid-phase extraction followed by liquid chromatography-tandem mass spectrometry and chemical analogue internal standardization is employed to detect and quantify the aflatoxins AFB(1), AFB(2), AFG(1), AFG(2), and the metabolites AFM(1) and AFP(1) in urine. The dynamic range of the method is nearly three orders of magnitude with limits of detection in the low femtogram on column range. The method was validated over a 12-day period by eight analysts. This method is suitable for agricultural, forensic, and public health laboratories during an accidental outbreak or a chemical terrorism event where a rapid and accurate diagnosis of aflatoxicosis is needed.

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 < 3 micromol glucoraphanin nightly for 2 weeks. Adherence to the study protocol was outstanding; no problems with safety or tolerance were noted. Urinary levels of aflatoxin-N(7)-guanine were not different between the two intervention arms (P = 0.68). However, measurement of urinary levels of dithiocarbamates (sulforaphane metabolites) indicated striking interindividual differences in bioavailability. An inverse association was observed for excretion of dithiocarbamates and aflatoxin-DNA adducts (P = 0.002; R = 0.31) in individuals receiving broccoli sprout glucosinolates. Moreover, trans, anti-phenanthrene tetraol, a metabolite of the combustion product phenanthrene, was detected in urine of all participants and showed a robust inverse association with dithiocarbamate levels (P = 0.0001; R = 0.39), although again no overall difference between intervention arms was observed (P = 0.29). Understanding factors influencing glucosinolate hydrolysis and bioavailability will be required for optimal use of broccoli sprouts in human interventions.

Our study was designed to assess the fecal and urinary excretion of 3 aflatoxin B1 (AFB1) metabolites, aflatoxins M1 (AFM1) and Q1 (AFQ1) and aflatoxin B1-N7-guanine (AFB-N7-guanine) that are produced by the predominant forms of cytochrome P450 enzymes responsible for the biotransformation of AFB1. Fecal and urinary AFM1, AFQ1 and urinary AFB-N7-guanine were assessed in 83 young Chinese males selected from a larger population (n = 300) based on detectable urinary AFM1. The concentration of fecal AFQ1 (median 137 ng/g fresh weight, IQR 9.1 to 450) was approximately 60 times higher than that of AFM1 (2.3 ng/g, IQR 0.0 to 7.3). In urine, the median AFQ1 was 10.4 ng/ml (IQR 3.4 to 23.3), and the median AFM1 and AFB-N7-guanine 0.04 ng/ml (IQR 0.01 to 0.33) and 0.38 ng/ml (IQR 0.0 to 2.15), respectively. A subgroup (n = 14) with hepatitis B virus (HBV) infection had significantly higher fecal concentrations of AFQ1 (p = 0.043) and AFM1 (p = 0.001) than those who were hepatitis B-virus antigen (HBsAg) negative, and the respective differences in urinary AFQ1 and AFM1 concentrations approached statistical significance (p = 0.054, p = 0.138). Our study demonstrates that AFQ1 is excreted in urine and feces at higher levels than AFM1, and feces are an important route of excretion of these AFB1 metabolites. AFQ1 should be further assessed for its predictive value as a marker for exposure and risk of dietary aflatoxins., (Copyright 2005 Wiley-Liss, Inc.)

The aim of the present work was to study the presence of aflatoxins in blood and urine of infants with protein-energy malnutrition (PEM). The study was conducted on 60 infants, 30 with kwashiorkor and 30 with marasmus, with 10 age-matched healthy infants studied as a control group. Complete blood count, liver function tests, and determination of the level of aflatoxins (B1, B2, G1, G2, M1, M2, G2a, B3, GM1, P, and aflatoxicol R0) in blood and urine were carried out in all studied infants. Serum aflatoxins were detected in more infants with kwashiorkor (80%) than in those with marasmus (46.7%). The mean serum levels of total aflatoxins, AFB1, AFG1, and AFB2a, were significantly higher in infants with kwashiorkor (p <.001). Aflatoxin B1 (AFB1) was the most commonly detected type. The prevalence of aflatoxin excretion in the urine of infants with kwashiorkor was 80%, a higher value than that in infants with marasmus (46.7%). The mean urinary concentration of total aflatoxins followed the same pattern of distribution (p < .052). There were no significant differences between groups in the mean urinary concentrations of AFB1, AFG1, AFB2a, AFM1, and AFG2a. Aflatoxins were not detected in any of the serum or urine samples of the control group. Aflatoxins are highly prevalent in this study population and show a high degree of correlation with severe PEM.

Hydrated sodium calcium aluminosilicate (HSCAS) is a phyllosilicate clay commonly used as an anticaking agent in animal feeds. HSCAS tightly and selectively adsorbs aflatoxin. In 1998, 55 dogs died in Texas after eating dog food containing aflatoxin (150-300 ppb). The corn in the diets was contaminated with aflatoxin. Six dogs were given a low-level, sub-clinical dose of aflatoxin B(1). On average, 71.5% of aflatoxin M(1) cleared within 6 h after dosing, increasing to 90.4% after 12 h. Aflatoxin M(1) was no longer detectable in urine after 48 h. Aflatoxin P(1) was not found in urine compared to large amounts of M(1) and trace amounts of Q(1). In a crossover study, six dogs randomly fed a commercial dog food (no-clay control) or coated with HSCAS (0.5% by weight) were subsequently administered a sub-clinical dose of aflatoxin B(1). Diets were switched and the process repeated. The HSCAS-coated diet significantly reduced urinary aflatoxin M(1) by 48.4%+/-16.6 SD versus the control diet. In conclusion, HSCAS protects dogs fed diets with even minimal aflatoxin contamination. Despite regular and careful ingredient screening for aflatoxin, low concentrations may reach the final product undetected. Therefore, HSCAS may provide the pet food industry further assurance of canine diet safety.

Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part from consumption of foods contaminated with aflatoxins. Chlorophyllin, a mixture of semisynthetic, water-soluble derivatives of chlorophyll that is used as a food colorant and over-the-counter medicine, has been shown to be an effective inhibitor of aflatoxin hepatocarcinogenesis in animal models by blocking carcinogen bioavailability. In a randomized, double-blind, placebo-controlled chemoprevention trial, we tested whether chlorophyllin could alter the disposition of aflatoxin. One hundred and eighty healthy adults from Qidong were randomly assigned to ingest 100 mg of chlorophyllin or a placebo three times a day for 4 months. The primary endpoint was modulation of levels of aflatoxin-N(7)-guanine adducts in urine samples collected 3 months into the intervention measured by using sequential immunoaffinity chromatography and liquid chromatography-electrospray mass spectrometry. This aflatoxin-DNA adduct excretion product serves as a biomarker of the biologically effective dose of aflatoxin, and elevated levels are associated with increased risk of liver cancer. Adherence to the study protocol was outstanding, and no adverse events were reported. Aflatoxin-N(7)-guanine could be detected in 105 of 169 available samples. Chlorophyllin consumption at each meal led to an overall 55% reduction (P = 0.036) in median urinary levels of this aflatoxin biomarker compared with those taking placebo. Thus, prophylactic interventions with chlorophyllin or supplementation of diets with foods rich in chlorophylls may represent practical means to prevent the development of hepatocellular carcinoma or other environmentally induced cancers.

A liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) method for the measurement of aflatoxin biomarkers in urine has been developed and validated. The two major aflatoxin-DNA adducts formed in rat tissues, aflatoxin N(7)-guanine and its imidazole ring opened derivative, 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-aflatoxin B(1), were detected and quantified in urine by the LC-ESI-MS/MS technique. Other metabolites derived from the conjugation and/or oxidation of aflatoxin B(1) measured in the urine of dosed rats included aflatoxin P(1), aflatoxin P(1)-glucuronide, aflatoxin Q(1), aflatoxin M(1), 8,9-dihydro-8,9-dihydroxy aflatoxin B(1), aflatoxin B(1)-mercapturic acid, the aflatoxin-cysteine glycine adduct derived from the aflatoxin-glutathione conjugate, aflatoxin M(1)P(1) and the aflatoxin B(1)-dialcohol. For in vivo studies to determine the dosimetry of certain aflatoxin metabolites, aflatoxin B(2) was used as an internal standard for recovery since this compound is not naturally produced in rats. In the final method using the internal standard, the coefficient of variation of six replicate analyses of in vivo rat urine samples for aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid, and aflatoxin M(1) was 12.5, 12.8, and 5.8%, respectively. Further, the LC-ESI-MS/MS method to detect aflatoxin N(7)-guanine in in vivo rat urine samples was at least 20-fold more sensitive than prior techniques. Using the LC-ESI-MS/MS technique, the dosimetry, on a weekly basis, of major urinary aflatoxin metabolites was assessed in animals chronically dosed over a 5-week period. Of particular importance was the application of this method to determine the modulation of levels of urinary aflatoxin metabolites by treatment with oltipraz, a chemopreventive agent that can completely ablate aflatoxin hepatocarcinogenesis in the rat. After 1 week, oltipraz administration diminished urinary aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid and aflatoxin M(1) levels by 83, 92, and 82%, respectively. The magnitude of this reduction was persistent at the day 14, 21, 28, and 35-day time points with the average decrease of aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid and aflatoxin M(1) being 73, 92, and 90%, respectively. Importantly, even under circumstances where the oltipraz intervention was most efficient in reducing aflatoxin metabolite levels, the LC-ESI-MS/MS method was still sensitive enough to detect the reduced biomarker content. This outcome has important translational implications for the application and analysis of the efficacy of primary and secondary prevention interventions in human populations where ambient exposure levels are low, but the toxicologic hazards of these exposures remain high.

HPLC and Inhibitory Indirect ELISA (I.I. ELISA) methods for quantitation of aflatoxins (AF) in human urine were compared in terms of specificity, sensitivity, easiness and cost. I.I. ELISA was optimized in kind of antibody in use, type of plastic plate, adduct synthesis technique, peroxidase and antibody dilutions, etc. Both polyclonal (Cuban) and monoclonal (British) anti-AF antibodies were statistically studied and the process was standardized. HPLC and electrophoresis were performed while synthetizing AFB(1)-DNA and AFB(1)-Cl-Ovalbumin (AFB(1)-Cl-Ov) adducts. Costar polystyrene plate had the best adherence. Optimum coating dilution was 10 ng of AFB(1)-Cl-Ov per well. Dilutions of 1:1000 of monoclonal antibody from purified culture or 1:300 from monoclonal antibody from tissue culture and 1:1000 of peroxidase anti-mouse conjugate were the best. Optimum separation with HPLC was obtained isocratically with 60% MeOH and 40% distilled water mobile phase. ELISA had a sensitivity of 1 pg mL(-1) AFB(1) and HPLC sensitivity was 0.1 ng mL(-1) AFB(1) with fluorescence detector and 4.5 ng mL(-1) with UV detector. Monoclonal antibody gave more accurate results for determination of free and adducted AFB(1) in urine analysis., (Copyright 1999 John Wiley & Sons, Ltd.)

A method for the determination of aflatoxicol in urine has been developed. The urine samples were cleaned up by an automated procedure using immunoaffinity columns before analysis by high-performance liquid chromatography and fluorescence detection. Post-column derivatization with bromine allowed the simultaneous determination of aflatoxicol and aflatoxins B1, B2, G1, G2, M1, and Q1. Average recovery of aflatoxicol was 99% in the range 4-40 pg ml-1 of spiked urine samples. The relative standard deviations were all between 1% and 3%. The limit of detection was 1 pg ml-1 urine. Authentic samples from exposed feed-factory workers were analysed, but aflatoxin levels were found to be below the detection limit.

To investigate the carcinogenic effect of environmental aflatoxin exposure, 56 cases of hepatocellular carcinoma (HCC) diagnosed between 1991 and 1995 were identified and individually matched by age, sex, residence and date of recruitment to 220 healthy controls from the same large cohort in Taiwan. Blood samples were analyzed for hepatitis B and C viral markers and for aflatoxin-albumin adducts; urine was tested for aflatoxin metabolites. We obtained information about sociodemographic characteristics, habitual alcohol drinking, cigarette smoking and diet in a structured interview. Hepatitis B virus surface antigen (HBsAg) carriers had a significantly increased risk for HCC. After adjustment for HBsAg serostatus, the matched odds ratio (ORm) was significantly elevated for subjects with high levels of urinary aflatoxin metabolites. When stratified into tertiles, a dose-response relationship with HCC was observed. The ORm for detectable aflatoxin-albumin adducts was not significant after adjustment for HBsAg serostatus. HBsAg-seropositive subjects with high aflatoxin exposure had a higher risk than subjects with high aflatoxin exposure only or HBsAg seropositivity only. In male HBsAg-seropositive subjects, adjusted ORs were 2.8 (95% confidence interval [CI] = 0.9-9.1) for detectable compared with non-detectable aflatoxin-albumin adducts and 5.5 (CI = 1.3-23.4) for high compared with low urinary aflatoxin metabolite levels. Our results suggest that environmental aflatoxin exposure may enhance the hepatic carcinogenic potential of hepatitis B virus. A large-scale study will be needed to evaluate the effect of aflatoxin exposure on HBsAg non-carriers.

A method for the determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B1 103%, B2 106%, G1 98% and G2 96% in the range 6.8-73 pg/ml of urine and M1 103% and Q1 100% in the range 18-97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B1, B2, G1 and G2 and 18 pg/ml for M1 and Q1.

The value of aflatoxins is well known as a carcinogenic, mutagenic and teratogenic, likewise its association with the hepatitis B virus. In addition, it is known the incidence of hepatocellular carcinoma in such viral infection. A study was performed on the albumin adducts-aflatoxins levels in sera determined by ELISA method of children within 3-15 years old at the Service of Pediatric Gastroenterology from the National Institute of Gastroenterology. Samples consisted of 40 patients with chronic active hepatitis (CAH) B, 10 HBsAg+ carriers and 20 controls. The CAH group, showed a 32.5% of positiveness with a maximum levels of 25pg aflatoxin lysine/mg albumin while 20% of HBsAg positive carriers showed levels of un 12.3 pg aflatoxin lysine/mg albumin and 15% of the control group 5pg AF lysine/mg albumin. It can be observed that aflatoxin levels in patients of CAH presented values up to 5 times over the control group. This study suggest the validity of aflatoxin-albumin adducts as a marker of chronic exposure to this carcinogen and its importance in relation with the virus of hepatitis B.

A liquid chromatography/electrospray ionization tandem mass spectrometry method is described for the determination of aflatoxins B1, B2, G1 and G2. Samples of naturally contaminated airborne dust and spiked urine were cleaned up on immunoaffinity columns and analysed by liquid chromatography using either mass spectrometry detection or post-column derivatization with bromine and fluorescence detection. With tandem mass spectrometry, detection limits (S/N = 3) calculated as amount ejected on column were: aflatoxin B1 4 pg, B2 4 pg, G1 5 pg, and G2 10 pg.

A liquid chromatographic system with an automated clean-up procedure for aflatoxin Q1 in human urine is described. The samples were cleaned up by using immunoaffinity columns originally designed for aflatoxin M1. The chromatographic system was a C18 column with an acidic mobile phase of acetonitrile-water containing potassium bromide. Fluorescence detection (365/440 nm) of aflatoxin Q1 was enhanced by addition of bromine, using post-column derivatization, which was studied by factorial designs. Average recovery of aflatoxin Q1 in spiked 10-ml urine samples was 88% (R.S.D. = 6.4%) at a level of 50 pg/ml. The determination limit was 49.5 pg/ml urine.

Children suffering from kwashiorkor could be grouped as those in whom aflatoxin was detectable in both serum and urine, and those in whom this mycotoxin was undetectable. Examination of the clinical records of the aflatoxin-positive and -negative children (58% and 42% of the sample, respectively) showed several other differences between the two groups. Compared with the aflatoxin-negative group, the children scored as aflatoxin-positive showed a significantly lower haemoglobin level (P = 0.02), a longer duration of oedema (P = 0.057), an increased number of infections (P = 0.037), and a longer duration of hospital stay (P = 0.008). The present findings suggest that the consumption of a staple food such as maize that is contaminated with the fungus Aspergillus flavus exposes susceptible kwashiorkor children to the metabolic hazards of aflatoxins, resulting in a greater risk of frequent infections.

A cohort of 18,244 mostly middle-aged (45-64 years) men residing in four small geographically defined areas of Shanghai was accrued between January 1986 and September 1989. In addition to an in-person interview regarding dietary and other past exposures, each subject donated a single void urine sample at recruitment so that the presence of aflatoxins in urine could be assessed. In addition, a 1-year survey of market foods in Shanghai was conducted to quantitatively estimate the extent of aflatoxin exposure in the study population. After close to 70,000 person-years of follow-up, 55 incident cases of hepatocellular carcinoma (HCC) had been identified. Levels of urinary aflatoxin B1 and the oxidative metabolites, including the major aflatoxin nucleic acid adduct, aflatoxin-N7-guanine, were determined for 50 of the 55 identified cases of HCC. Two hundred sixty-seven controls were chosen randomly from the cohort; they were matched to the 50 cases by age (within 1 year), time of specimen collection (within 1 month), and residence. After integrating the high-pressure liquid chromatography chromatograms to measure aflatoxin-N7-guanine, aflatoxin M1, aflatoxin P1, and aflatoxin B1, 49, 67, 53, and 71 of the urine samples had detectable levels of these compounds, respectively. The aflatoxin metabolite detected at the highest concentration was aflatoxin P1; the range was 0.59-16.0 ng/ml. The range of aflatoxin M1 in the urine was 0.17-5.2 ng/ml. The aflatoxin-N7-guanine adduct range was 0.3-1.81 ng/ml in the 49 positive samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Using a urinary immunoassay to measure aflatoxin metabolites, we examined the associations between exposure to aflatoxin, chronic infection with the hepatitis-B virus (HBV) and background rates of hepatocellular carcinoma (HCC) mortality in a cross-sectional survey of 250 residents from 8 areas of Taiwan with a 4-fold variation in age-adjusted HCC mortality. Specimens of fasting blood and overnight urines were used to determine HBV carrier status and excretion of aflatoxin in the subjects surveyed. While the prevalence of hepatitis-B virus carriers showed moderate variability, there was a 500-fold range in urinary aflatoxin levels. Mean log-transformed levels of aflatoxin metabolites were similar in males and females and in HBV carriers and non-carriers. In the 8 townships, HCC mortality correlated positively with both area HBV carrier prevalence and mean aflatoxin levels. The primary analyses, however, were conducted at the individual level. Each subject's aflatoxin level was treated as the response variable in a multiple regression model, and the corresponding sex-specific area HCC rate was included as a predictor along with the individual's carrier status, age and sex; alcohol consumption and cigarette smoking were also considered. In these analyses, a significant association was again observed between the marker of aflatoxin exposure and the background rate of HCC mortality. In females, the slope of the regression line was somewhat steeper in HBV carriers, but this pattern was not seen in males and formal testing yielded no statistically significant evidence of an interaction. Our findings are consistent with the hypothesis that aflatoxin plays an independent role in hepatocellular carcinoma in Taiwan.

An automated extraction and clean-up procedure was developed for the determination of aflatoxins in human urine at the 50 pg/ml level. Aflatoxins B1, B2, G1 and G2 are captured on C2 extraction columns and simultaneously cleaned up with the aid of a robotic system. The processed samples are analysed by reversed-phase high-performance liquid chromatography. Fluorescence detection was enhanced for aflatoxins B1 and G1 using factorial design optimization of the post-column reactor. Silylation of the glass vials used in the robotic system was of the utmost importance. With non-silylated glass vials, up to 75% of the analytes were lost. Average aflatoxin recoveries were B1 95%, B2 90%, G1 93% and G2 89%.

Human epidemiology and experimental animal data have provided the statistical association and biological information necessary to propose that aflatoxins are risk factors for human liver cancer. As liver cancer causes at least 200,000 deaths per year, prevention measures must be developed to ameliorate this nearly always fatal disease. Preventive strategies will be facilitated by the identification of individuals at high risk. It is the goal of the molecular dosimetry field to provide facile and accurate biomarkers to identify people at high risk for carcinogen exposure and consequent adverse health effects. We have developed methods to defect the major aflatoxin DNA adduct, aflatoxin N7-guanine (AFB-N7-guanine), in urine, examined the dose-response characteristics in people living in China and The Gambia, and have found an excellent association of this biomarker with exposure. In addition to exposure studies in people, our laboratories have monitored AFB-N7-guanine excretion in the urine of rats whose risk for developing cancer has been modulated with dietary chemoprotective agents such that independent groups of animals receiving the same dosage of aflatoxin B1 were at either high or low risk for tumorigenesis. The production of DNA damage by aflatoxins is not the exclusive mechanism for liver cancer. Many other factors, including hepatitis B virus, cell proliferation, and nutritional status, can exert strong modification effects in human disease. Thus, molecular epidemiological investigations that examine only one biomarker may greatly underestimate or overestimate the risk for an individual.(ABSTRACT TRUNCATED AT 250 WORDS)

An investigation to examine the relationship between Crohn's disease and aflatoxins, a group of structurally related toxic and carcinogenic metabolites, was carried out on 24 patients. Extracts of serum and urine from the patients were assayed qualitatively by thin layer chromatography and the Aflatest method, and quantitatively by fluorimetry. There was evidence that some patients suffering from Crohn's Disease, together with some having coeliac disease and ulcerative colitis, did have varying amounts of aflatoxins in their serum and urine. The presence of aflatoxins may have been due to exposure to food containing these toxins or inability of the patient to excrete aflatoxins on account of some gastro-intestinal derangement. Only long-term investigation would establish the link between dietary history and the presence of aflatoxins in these patients.