Elevated Urinary Levels of Fungal and Environmental Toxins in Patients with Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Risk factors for pancreatic ductal adenocarcinoma (PDAC) include tobacco/alcohol abuse, genetic predisposition, insulin resistance, and pancreatic cysts. Despite these well-established risk factors and the screening of high-risk individuals, some people still develop PDAC. This study aims to explore a potential risk factor for PDAC by investigating the association between fungal toxins (FT) and environmental toxins (ET) and the disease. We predicted that individuals with PDAC would have higher levels of these toxins compared to healthy controls. The rationale behind this hypothesis is that exposure to FT and ET might contribute to the development of PDAC by elevating cancer risk. METHODS: A pilot retrospective cohort study was conducted at Moffitt Cancer Center from 2022 to 2023. This study compared FT and ET levels, demographic data, and PDAC features between subjects with PDAC and healthy controls. RESULTS: Forty subjects were enrolled in the study, comprising 20 with pancreatic ductal adenocarcinoma (PDAC) and 20 healthy controls. Baseline demographics were similar between the two groups. Among the PDAC subjects, the most common tumor location was the head of the pancreas (55%); 30% had locally advanced disease, 45% were borderline resectable, and 10% had metastatic disease. Compared to the controls, subjects with PDAC had significantly higher levels of fungal toxins (FTs) including ochratoxin, gliotoxin, and citrinin (p < 0.05). Additionally, PDAC patients had significantly elevated levels of environmental toxins (ETs) such as methyl tert-butyl ether (MTBE), xylene, styrene, acrylonitrile, perchlorate, diphenyl phosphate, bromopropane, organophosphates, acrolein, tiglylglycine, and diethylphosphate (p < 0.05). CONCLUSION: Our study demonstrates that subjects with PDAC, without other risk factors, have higher FT and ET levels than controls. Further studies are needed to evaluate whether ET and FT exposure can be clinically utilized as a risk factor for PDAC development.

Validation of a UPLC-MS/MS Method for Multi-Matrix Biomonitoring of Alternaria Toxins in Humans.

Mycotoxins, natural toxins produced by fungi, contaminate nearly 80% of global food crops. Alternaria mycotoxins, including alternariol (AOH), alternariol monomethylether (AME), and tenuazonic acid (TeA), present a health concern due to their prevalence in various plants and fruits. Exposure to these toxins exceeds the threshold of toxicological concern in some European populations, especially infants and toddlers. Despite this, regulatory standards for Alternaria toxins remain absent. The lack of toxicokinetic parameters, reference levels, and sensitive detection methods complicates risk assessment and highlights the necessity for advanced biomonitoring (HBM) techniques. This study addresses these challenges by developing and validating ultra-high performance liquid chromatography method coupled with tandem mass spectrometry to quantify AOH, AME, TeA, and their conjugates in multiple biological matrices. The validated method demonstrates robust linearity, precision, recovery (94-111%), and sensitivity across urine (LOD < 0.053 ng/mL), capillary blood (LOD < 0.029 ng/mL), and feces (LOD < 0.424 ng/g), with significantly lower LOD for TeA compared to existing methodologies. The application of minimally invasive microsampling techniques for the blood collection enhances the potential for large-scale HBM studies. These advancements represent a step toward comprehensive HBM and exposure risk assessments for Alternaria toxins, facilitating the generation of data for regulatory authorities.

Development and validation of a simultaneous quantitative analytical method for two Alternaria toxins and their metabolites in plasma and urine using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Much more attention has been paid to the contamination of Alternaria toxins because of food contamination and the threat to human health. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous detection of the prototypical alternariol, alternariol monomethylether, and the metabolites 4-oxhydryl alternariol, and alternariol monomethylether 3-sulfate ammonium salt of Alternaria toxins. The positive samples were used as matrix samples to optimize the different experimental conditions. 0.01% formic acid solution and acetonitrile were used as the mobile phase, and analytes were scanned in negative electron spray ionization under multiple reaction monitoring, and quantitative determination by isotope internal standard method. Application of this method to samples of human plasma and urine showed the detection of the above analytes. The results showed that the recoveries were from 80.40% to 116.4%, intra-day accuracy was between 0.6% and 8.0%, and inter-day accuracy was between 1.1% and 12.1%. The limit of detection of the four analytes ranged from 0.02 to 0.6 µg/L in urine, and 0.02 to 0.5 µg/L in plasma, respectively. Thus, the developed method was rapid and accurate for the simultaneous detection of analytes and provided a theoretical basis for the risk assessment of Alternaria toxins for human exposure.

Frequent Dietary Multi-Mycotoxin Exposure in UK Children and Its Association with Dietary Intake.

Mycotoxins are potent fungal toxins that frequently contaminate agricultural crops and foods. Mycotoxin exposure is frequently reported in humans, and children are known to be particularly at risk of exceeding safe levels of exposure. Urinary biomonitoring is used to assess overall dietary exposure to multiple mycotoxins. This study aims to quantify multi-mycotoxin exposure in UK children and to identify major food groups contributing to exposure. Four repeat urine samples were collected from 29 children (13 boys and 16 girls, aged 2.4-6.8 years), and food diaries were recorded to assess their exposure to eleven mycotoxins. Urine samples (n = 114) were hydrolysed with ß-glucuronidase, enriched through immunoaffinity columns and analysed by LC-MS/MS for deoxynivalenol (DON), nivalenol (NIV), T-2/HT-2 toxins, zearalenone (ZEN), ochratoxin A (OTA) and aflatoxins. Food diaries were analysed using WinDiet software, and the daily intake of high-risk foods for mycotoxin contamination summarised. The most prevalent mycotoxins found in urine samples were DON (95.6% of all samples), OTA (88.6%), HT-2 toxin (53.5%), ZEN (48.2%) and NIV (26.3%). Intake of total cereal-based foods was strongly positively associated with urinary levels of DON and T-2/HT-2 and oat intake with urinary T-2/HT-2. Average daily mycotoxin excretion ranged from 12.10 µg/d (DON) to 0.03 µg/d (OTA), and co-exposure to three or more mycotoxins was found in 66% of samples. Comparing mycotoxin intake estimates to tolerable daily intakes (TDI) demonstrates frequent TDI exceedances (DON 34.2% of all samples, T-2/HT-2 14.9%, NIV 4.4% and ZEN 5.2%). OTA was frequently detected at low levels. When mean daily OTA intake was compared to the reference value for non-neoplastic lesions, the resulting Margin of Exposure (MoE) of 65 was narrow, indicating a health concern. In conclusion, this study demonstrates frequent exposure of UK children to multiple mycotoxins at levels high enough to pose a health concern if exposure is continuous.

Assessment of Mycotoxin Exposure and Associated Risk in Pregnant Dutch Women: The Human Biomonitoring Approach.

Mycotoxins are toxic secondary metabolites produced by various fungi that can contaminate food crops, which, in turn, may lead to human exposure. Chronic exposure to mycotoxins can cause adverse health effects including reproductive and developmental toxicity. Pregnant women and their foetuses present a vulnerable group for exposure to mycotoxins that can cross the placenta. Human biomonitoring of mycotoxins provides a real-life approach to estimate internal exposure. In this pilot study, 24-h urine samples from 36 pregnant Dutch women were analysed for aflatoxin M1 (AFM1), total deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM-1), total zearalenone (ZEN), total α-zearalenol (α-ZEL), total ß-zearalenol (ß-ZEL) and total zearalanone (ZAN), where 'total' refers to mycotoxins and their conjugated forms. Serum samples from these women were analysed for fumonisin B1 (FB1) and ochratoxin A (OTA). All samples were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most prevalent mycotoxins were total DON, total ZEN and OTA, with a detection frequency of 100%. DOM-1, total α-ZEL and total ß-ZEL were detected but to a lesser extent, while AFM1, total ZAN and FB1 were undetected. Median concentrations were 4.75 µg total DON/L, 0.0350 µg DOM-1/L, 0.0413 µg total ZEN/L, 0.0379 µg total α-ZEL/L, 0.0189 µg total ß-ZEL/L, and 0.121 µg OTA/L. The calculated median concentration for total ZEN and its metabolites was 0.105 µg/L. Based on two separate risk assessment approaches, total DON exposure in this group was considered to be of low concern. Similarly, exposure to total ZEN and its metabolites in this group was of low concern. For OTA, the risk of non-neoplastic effects was of low concern based on exposure in this group, and the risk of neoplastic effects was of low concern in the majority of participants in this group. The findings of this pilot study confirm the presence of mycotoxins in the urine and serum of pregnant Dutch women, with total DON, total ZEN, and OTA most frequently detected. Exposure to all measured mycotoxins was considered to be of low concern in this group, except for exposure to OTA, which was of low concern for the majority of participants. The study's findings offer valuable insights but should be confirmed using a larger and more diverse sample of the Dutch general population.

The screening method for 39 phytotoxins and mycotoxins in blood and urine with liquid chromatography-high resolution mass spectrometry.

BACKGROUND: Poisonings caused by plant toxins and mycotoxins occur frequently, which do great harm to human health and social public health safety. When a poisoning incident occurs, biological samples are commonly be used to conduct the detection of toxic substances and their metabolites for targeted clinical treatment and incident analysis. OBJECTIVE: To establish an efficient and accurate analysis method of 39 phytotoxins and mycotoxins in blood and urine by high performance liquid chromatography quadrupole tandem orbitrap mass spectrometry (HPLC-Orbitrap MS). METHOD: After 3 mL of methanol being added to 1 mL blood and urine respectively for extraction and protein precipitation, the supernatant was injected into HPLC-Orbitrap MS for analysis. The phytotoxins and mycotoxins were separated by Hypersil GOLD PFP column with gradient elution using methanol-5 mmol/L ammonium acetate as mobile phase. The data were collected in ESI positive ion mode using Full MS/dd-MS2 for mass spectrometry detection. RESULT: The mass database of 39 phytotoxins and mycotoxins was developed, and accurate qualitative analysis can be obtained by matching with the database using the proposed identification criteria. Limit of detections (LODs) were 1.34 × 10-4 âˆ¼ 1.92 ng/mL and 1.92 × 10-4 âˆ¼ 9.80 ng/mL for blood and urine samples, respectively. Limits of quantification (LOQ) of toxins in blood and urine ranged from 4.47 × 10-4 âˆ¼ 6.32 ng/mL and 6.39 × 10-4 âˆ¼ 32.67 ng/mL, respectively. Intra-day relative standard deviations (RSDs) were 0.79 % âˆ¼ 10.90 %, and inter-day RSDs were 1.08 % âˆ¼ 18.93 %. The recoveries can reach 90 % âˆ¼ 110 % with matrix matching calibration curves. CONCLUSION: The established method is simple and rapid to operate, which can complete the sample analysis within 30 min, providing technical support for clinical poisoning treatment and public health poisoning analysis.

Simultaneous detection of mycotoxins and pesticides in human urine samples: A 24-h diet intervention study comparing conventional and organic diets in Spain.

Pesticides and mycotoxins, prominent chemical hazards in the food chain, are commonly found in plant-based foods, contributing to their pervasive presence in the human body, as evidenced by biomonitoring programs. Despite this, there is limited knowledge about their co-occurrence patterns. While intervention studies have demonstrated that organic diets can significantly reduce pesticide levels, their impact on mycotoxin exposure has been overlooked. To address this gap, this study pursued two objectives: first, to characterize the simultaneous presence of mycotoxins and pesticides in human urine samples by means of the control of the biomarkers of exposure, and second, to investigate the influence of consuming organic foods on these co-exposure patterns. A pilot study involving 20 healthy volunteers was conducted, with participants consuming either exclusively organic or conventional foods during a 24-h diet intervention in autumn 2021 and spring 2022 to account for seasonal variability. Participants provided detailed 24-h dietary records, and their first-morning urine samples were collected, minimally treated and analysed using LC-Q-ToF-MS by means of a multitargeted method in order to detect the presence of these residues. Results indicated that among the 52 screened compounds, four mycotoxins and seven pesticides were detected in over 25% of the samples. Deoxynivalenol (DON) and the non-specific pesticide metabolite diethylphosphate (DEP) exhibited the highest frequency rates (100%) and concentration levels. Correlations were observed between urine levels of mycotoxins (DON, ochratoxin alpha [OTα], and enniatin B [ENNB]) and organophosphate pesticide metabolites DEP and 2-diethylamino-6-methyl-4-pyrimidinol (DEAMPY). The pilot intervention study suggested a reduction in ENNB and OTα levels and an increase in ß-zearalenol levels in urine after a short-term replacement with organic food. However, caution is advised due to the study's small sample size and short duration, emphasizing the need for further research to enhance understanding of the human chemical exposome and refine chemical risk assessment.

Biomonitoring of Dietary Mycotoxin Exposure and Associated Impact on the Gut Microbiome in Nigerian Infants.

Mycotoxins are toxic chemicals that adversely affect human health. Here, we assessed the influence of mycotoxin exposure on the longitudinal development of early life intestinal microbiota of Nigerian neonates and infants (NIs). Human biomonitoring assays based on liquid chromatography tandem mass spectrometry were applied to quantify mycotoxins in breast milk (n = 68) consumed by the NIs, their stool (n = 82), and urine samples (n = 15), which were collected longitudinally from month 1-18 postdelivery. Microbial community composition was characterized by 16S rRNA gene amplicon sequencing of stool samples and was correlated to mycotoxin exposure patterns. Fumonisin B1 (FB1), FB2, and alternariol monomethyl ether (AME) were frequently quantified in stool samples between months 6 and 18. Aflatoxin M1 (AFM1), AME, and citrinin were quantified in breast milk samples at low concentrations. AFM1, FB1, and ochratoxin A were quantified in urine samples at relatively high concentrations. Klebsiella and Escherichia/Shigella were dominant in very early life stool samples (month 1), whereas Bifidobacterium was dominant between months 3 and 6. The total mycotoxin levels in stool were significantly associated with NIs' gut microbiome composition (PERMANOVA, p < 0.05). However, no significant correlation was observed between specific microbiota and the detection of certain mycotoxins. Albeit a small cohort, this study demonstrates that mycotoxins may influence early life gut microbiome composition.

HLA gene variations and mycotoxin toxicity: Four case reports.

Mycotoxins are produced by certain molds that can cause many health effects. We present four human cases of prolonged consistent mycotoxins exposure linked to genetic variations in human leukocyte antigen (HLA) alleles. The HLA-DR/DQ isotype alleles are linked to mycotoxins susceptibility due to the lack of proper immune response; individuals with these alleles are poor eliminators of mycotoxins from their system. Four subjects with variations in their HLA-DR alleles were exposed to mycotoxins from living in mold-infested houses and experienced persistent mold-related symptoms long after moving out from the mold-infested houses and only exposed to the levels of molds found in the ambient air. From one of the subjects, two urine samples were collected ~ 18 months apart after the cessation of exposure. Urinary elimination rate was extremely slow for two of the mycotoxins (ochratoxin A [OTA] and mycophenolic acid [MPA]) detected in both samples. In 18 months, decline in OTA level was only ~ 3-fold (estimated t½ of ~ 311 days) and decline in MPA level was ~ 11-fold (estimated t½ of ~ 160 days), which was ~ 10- and ~ 213-fold slower than expected in individuals without HLA-DR alleles, respectively. We estimated that ~ 4.3 and ~ 2.2 years will be required for OTA and MPA to reach < LLQ in urine, respectively. Three other subjects with variations in HLA-DR alleles were members of a family who lived in a mold-infested house for 4 years. They kept experiencing mold-related issues >2 years after moving to a non-mold-infested house. Consistent exposure was confirmed by the presence of several mycotoxins in urine >2 years after the secession of higher than background (from outdoor ambient air) exposure. This was consistent with the extremely slow elimination of mycotoxins from their system. Variations in HLA-DR alleles can, consequently, make even short periods of exposure to chronic exposure scenarios with related adverse health effects. It is, therefore, important to determine genetic predisposition as a reason for prolonged/lingering mold-related symptoms long after the cessation of higher than background exposure. Increased human exposure to mycotoxins is expected from increased mold infestation that is anticipated due to rising CO2, temperature, and humidity from the climate change with possibly increased adverse health effects, especially in individuals with genetic susceptibility to mold toxicity.

Associated factors with mycotoxin exposure in Spanish population.

Human exposure to mycotoxins is a global concern since filamentous fungi can contaminate food and feed from crops to ready-to-eat meals. Human urine biomonitoring is a widely used technique to evaluate mycotoxins exposure, as an alternative to food correlation studies. The aim of this study is to describe human exposure to mycotoxins and to investigate the associated sociodemographic, lifestyle and dietary variables. Participants were 540 women from the Valencia (Spain) cohort of the Spanish Childhood and Environment Project (INMA). A validated multi-mycotoxin method using HPLC-Q-TOF-MS was applied to determine the concentration of ten selected mycotoxins: Enniatin A, Enniatin B, Enniatin A1, Enniatin B1, Beauvericine, Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2 and Ochratoxin A. A simultaneous untargeted screening of mycotoxins and their metabolites has been performed. Mycotoxins associations were assessed by bivariate and multivariate regression models using participants' sociodemographic, lifestyle and dietary data collected through questionnaires. Mycotoxins were detected in 81% of urine samples. The method quantified mycotoxins concentrations in up to 151 samples. Most quantified mycotoxins were: Enniatin B [% of detection (concentration range)] = 26% (1.0-39.7 ng/mg) and Enniatin B1 = 7% (0.5-14.4 ng/mg). Besides the ten-targeted mycotoxins, other mycotoxins and metabolites were studied, and higher incidence was observed for Deepoxy-deoxynivalenol (45%), Ochratoxin B (18%) and Ochratoxin α (17%). Higher mycotoxins concentrations were associated with rural areas as well as with participants belonged to lower social class, beer, light sodas and fruit juice consumers. On the contrary, higher processed meat intake was related to lower mycotoxins' levels. Studies are required to better evaluate the exposure to mycotoxins from food and their environmental relationships.

Human biomonitoring of deoxynivalenol (DON) - Assessment of the exposure of young German adults from 1996 - 2021.

The mycotoxin deoxynivalenol (DON) is a frequently found contaminant in cereals and cereal-based products. As a German contribution to the European Joint Programme HBM4EU, we analysed the total DON concentration (tDON) in 24-h urine samples from the German Environmental Specimen Bank (ESB). In total, 360 samples collected in 1996, 2001, 2006, 2011, 2016, and 2021 from young adults in Muenster (Germany), were measured by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after enzymatic deconjugation of the glucuronide metabolites. tDON was found in concentrations above the lower limit of quantification (0.3 µg/L) in 99% of the samples. Medians of the measured concentrations and the daily excretion were 4.3 µg/L and 7.9 µg/24 h, respectively. For only nine participants, urinary tDON concentrations exceeded the provisional Human biomonitoring guidance value (HBM GV) of 23 µg/L. Urinary tDON concentrations were significantly higher for male participants. However, 24-h excretion values normalized to the participant's body weight did not exhibit any significant difference between males and females and the magnitude remained unchanged over the sampling years with exception of the sampling year 2001. Daily intakes were estimated from excretion values. Exceedance of the tolerable daily intake (TDI) of 1 µg/kg bw per day was observed for less than 1% of all participants. TDI exceedances were only present in the sampling year 2001 and not in more recent sampling years while exceedance of the HBM guidance value was also observed in 2011 and 2021.

Maternal exposure to multiple mycotoxins and adverse pregnancy outcomes: a prospective cohort study in rural Bangladesh.

There is limited and inconsistent evidence, primarily from cross-sectional studies, linking mycotoxins to adverse birth outcomes. This study investigates the potential role of maternal dietary exposure to multiple mycotoxins in the development of several adverse pregnancy and birth outcomes. We analyzed data from 436 singleton pregnancies enrolled in a prospective cohort study in the rural Habiganj district, Bangladesh, between July 2018 and November 2019. Thirty-five urinary mycotoxin biomarkers were quantified using liquid chromatography coupled with tandem mass spectrometry and used to estimate dietary mycotoxin exposure. Multivariable regression models, adjusted for potential confounding and clustering, were fitted to assess the associations between maternal exposure to frequently occurring mycotoxins (ochratoxin A-OTA, citrinin- CIT, and Deoxynivalenol- DON) and pregnancy loss, preterm birth (PTB), low birth weight (LBW), born small-for-gestational-age (SGA) and small-vulnerable newborn. The results indicate that only in 16 of 436 pregnancies (4%) were urine samples free from all investigated mycotoxins. Biomarkers for six major mycotoxins were detected in the urine samples. OTA (95%), CIT (61%), and DON (6%) were most frequently detected, with at least two mycotoxins co-occurring in the majority of women (63%). There was evidence that maternal dietary intake of OTA was associated with higher odds of having an LBW baby, with the odds increasing in a dose-dependent manner. We found no evidence of associations between pregnancy loss, PTB, SGA, small-vulnerable newborns, and maternal dietary exposure to OTA, CIT, and DON, albeit with large confidence intervals, so findings are consistent with protective as well as large harmful effects. Exposure to multiple mycotoxins during pregnancy is widespread in this rural community and represents a health risk for mothers and babies. Tailored public health policies and interventions must be implemented to reduce mycotoxin exposure to the lowest possible level.

Assessment of multiple mycotoxin exposure and its association with food consumption: a human biomonitoring study in a pregnant cohort in rural Bangladesh.

Aflatoxins (AFs), ochratoxin A (OTA), citrinin (CIT), fumonisin B1 (FB1), zearalenone (ZEN), and deoxynivalenol (DON) are mycotoxins that may contaminate diets, especially in low-income settings, with potentially severe health consequences. This study investigates the exposure of 439 pregnant women in rural Bangladesh to 35 mycotoxins and their corresponding health risks and links their exposure to certain foods and local stimulants. Overall, 447 first-morning urine samples were collected from pregnant women between July 2018 and November 2019. Mycotoxin biomarkers were quantified by DaS-HPLC-MS/MS. Urinary concentration of frequently occurring mycotoxins was used to estimate dietary mycotoxin exposure. Median regression analyses were performed to investigate the association between the consumption of certain foods and local stimulants, and urinary concentration of frequently occurring mycotoxins. Only in 17 of 447 urine samples (4%) were none of the investigated mycotoxins detected. Biomarkers for six major mycotoxins (AFs, CIT, DON, FB1, OTA, and ZEN) were detected in the urine samples. OTA (95%), CIT (61%), and DON (6%) were most frequently detected, with multiple mycotoxins co-occurring in 281/447 (63%) of urine samples. Under the lowest exposure scenario, dietary exposure to OTA, CIT, and DON was of public health concern in 95%, 16%, and 1% of the pregnant women, respectively. Consumption of specific foods and local stimulants-betel nut, betel leaf, and chewing tobacco-were associated with OTA, CIT, and DON urine levels. In conclusion, exposure to multiple mycotoxins during early pregnancy is widespread in this rural community and represents a potential health risk for mothers and their offspring.

High-Throughput Determination of Major Mycotoxins with Human Health Concerns in Urine by LC-Q TOF MS and Its Application to an Exposure Study.

Human biomonitoring constitutes a suitable tool to assess exposure to toxins overcoming the disadvantages of traditional methods. Urine constitutes an accessible biological matrix in biomonitoring studies. Mycotoxins are secondary metabolites produced naturally by filamentous fungi that produce a wide range of adverse health effects. Thus, the determination of urinary mycotoxin levels is a useful tool for assessing the individual exposure to these food contaminants. In this study, a suitable methodology has been developed to evaluate the presence of aflatoxin B2 (AFB2), aflatoxin (AFG2), ochratoxin A (OTA), ochratoxin B (OTB), zearalenone (ZEA), and α-zearalenol (α-ZOL) in urine samples as exposure biomarkers. For this purpose, different extraction procedures, namely, the Solid Phase Extraction (SPE); Dispersive Liquid-Liquid Microextraction (DLLME); and Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) methods were assessed, followed by Liquid Chromatography coupled to Quadrupole Time of Flight Mass Spectrometry with Electrospray Ionization (LC-ESI-QTOF-MS) determination. Then, the proposed methodology was applied to determine mycotoxin concentrations in 56 human urine samples from volunteers and to estimate the potential risk of exposure. The results obtained revealed that 55% of human urine samples analyzed resulted positive for at least one mycotoxin. Among all studied mycotoxins, only AFB2, AFG2, and OTB were detected with incidences of 32, 41, and 9%, respectively, and levels in the range from

Assessment of Human Exposure to Five Alternaria Mycotoxins in China by Biomonitoring Approach.

This biomonitoring study was conducted to investigate the concentration levels of five Alternaria mycotoxins in urine samples from 269 healthy volunteers living in the Yangtze River Delta, China. Alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA) and tentoxin (TEN) were detected in 38.3%, 48.7%, 63.9% and 23.4% of urine samples with the concentrations ranging from 0.057 to 45.8 ng/mL, 0.020 to 0.802 ng/mL, 0.050 to 80.6 ng/mL and 0.021 to 0.939 ng/mL, respectively. Altenuene (ALT) was not detected in any urine sample. Based on the urinary concentrations, the probable daily intake (PDI) values of Alternaria mycotoxins were calculated, and 100%, 99.2-100%, 0.372% and 1.12% of participants exceeded the threshold of toxicological concern (TTC) values for AOH, AME, TeA and TEN, respectively. This study revealed high potential health risks related to the contaminations of major Alternaria mycotoxins in China and highlighted the necessity for more toxicological studies to provide better basis for further comprehensive risk assessments.

Assessment of Mycotoxin Exposure in a Rural County of Chile by Urinary Biomarker Determination.

Aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), and deoxynivalenol (DON) are frequent mycotoxins that may cause carcinogenic, mutagenic, estrogenic, or gastrointestinal effects. The aim of this study was to assess the exposure to and risk from AFB1, OTA, ZEN, and DON in 172 participants of the Maule Cohort (MAUCO) by a biomarker analysis in urine and to associate their exposure with food consumption and occupation. Mycotoxins in the first morning urine were analyzed by solid-phase extraction and quantified by Ultra-High-Performance Liquid Chromatography with a mass-mass detector. Participants' information regarding food consumption, occupation, and other characteristics was obtained from a baseline and 2-year follow-up survey of the cohort. The prevalence and mean levels of mycotoxins in the urine were as follows: DON 63%, 60.7 (±78.7) ng/mL; AFB1 8%, 0.3 (±0.3) ng/mL; α-zearalenol (α-ZEL) 4.1%, 41.8 (±115) ng/mL; ß-ZEL 3.5%, 17.4 (±16.1) ng/mL; AFM1 2%, 1.8 (±1.0) ng/mL; OTA 0.6% (1/172), 1.3 ng/mL; and ZEN 0.6%, 1.1 ng/mL. These results were translated into exposures of DON, ZEN, and aflatoxins of public health concern. Participants who consumed coffee and pepper the day before had a significantly greater presence of DON (OR: 2.3, CI95 1.17-4.96) and total ZEL (OR: 14.7, CI95 3.1-81.0), respectively, in their urine. Additionally, we observed associations between the habitual consumption of beer and DON (OR: 2.89, CI95 1.39-6.42). Regarding the levels of mycotoxins and the amount of food consumed, we found correlations between DON and nuts (p = 0.003), total ZEL and cereals (p = 0.01), and aflatoxins with capsicum powder (p = 0.03) and walnuts (p = 0.03). Occupation did not show an association with the presence of mycotoxins in urine.

Determination of Urinary Mycotoxin Biomarkers Using a Sensitive Online Solid Phase Extraction-UHPLC-MS/MS Method.

In the course of assessing the human exposure to mycotoxins, biomarker-based approaches have proven to be important tools. Low concentration levels, complex matrix compositions, structurally diverse analytes, and the large size of sample cohorts are the main challenges of analytical procedures. For that reason, an online solid phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry (online SPE-UHPLC-MS/MS) method was developed, allowing for the sensitive, robust, and rapid analysis of 11 relevant mycotoxins and mycotoxin metabolites in human urine. The included spectrum of analytes comprises aflatoxin M1 (AFM1), altenuene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), citrinin (CIT) and its metabolite dihydrocitrinone (DH-CIT), fumonisin B1 (FB1), ochratoxin A (OTA), and zearalenone (ZEN) as well as α- and ß-zearalenol (α- and ß-ZEL). Reliable quantitation was achieved by means of stable isotope dilution, except for ALT, AME and AOH using matrix calibrations. The evaluation of method performance displayed low limits of detection in the range of pg/mL urine, satisfactory apparent recovery rates as well as high accuracy and precision during intra- and interday repeatability. Within the analysis of Zimbabwean urine samples (n = 50), the applicability of the newly developed method was shown. In addition to FB1 being quantifiable in all analyzed samples, six other mycotoxin biomarkers were detected. Compared to the occurrence rates obtained after analyzing the same sample set using an established dilute and shoot (DaS) approach, a considerably higher number of positive samples was observed when applying the online SPE method. Owing to the increased sensitivity, less need of sample handling, and low time effort, the herein presented online SPE approach provides a valuable contribution to human biomonitoring of mycotoxin exposure.

[Determination of amanita peptide toxins in human urine by TurboFlow online clean-up-liquid chromatography-tandem mass spectrometry].

Amanita peptide toxins are cyclic polypeptide mushroom toxins that can cause acute liver damage. The fatality rate associated with these toxins is very high. Monitoring the concentration of amanita peptide toxins in human urine can provide valuable information for early clinical diagnosis and treatment. Therefore, a TurboFlow online clean-up-liquid chromatography-triple quadrupole mass spectrometry (TF-LC-MS/MS) method was established for the simultaneous quantitative determination of five amanita peptide toxins (α-amanitin, ß-amanitin, γ-amanitin, phallacidin, and phalloidin) in human urine. After pre-treatment with high-speed centrifugation, urine samples were analyzed using TF-LC-MS/MS. The main factors influencing purification efficiency, including the TF column, loading solution, eluting solution, transfer flow, and transfer time, were optimized in this study. Under the optimized experimental conditions, the analytes were purified using a TurboFlowTM Cyclone column (50 mm×0.5 mm) and separated on a Hypersil GOLD C18 column (100 mm×2.1 mm) using the mobile phases of methanol and 4 mmol/L aqueous ammonium acetate solution with gradient elution. The analytes were detected in selected reaction monitoring (SRM) mode via positive electrospray ionization. Matrix-matched external standard calibration was used for quantitation. The linear range of the method ranged from 1.0 µg/L to 50.0 µg/L for all five amanita peptide toxins, with correlation coefficients (r2) higher than 0.997. The limits of detection were 0.15-0.3 µg/L and the limits of quantification (LOQs) were 0.5-1.0 µg/L for the five amanita peptide toxins in urine. The intra-day and inter-day recoveries of amanita peptide toxins were 87.0%-108.6% and 86.8%-112.7%, respectively, at the spiked levels of 2.0, 5.0, and 10.0 µg/L. The intra-day and inter-day relative standard deviations (RSDs) were less than 14.5%. The method is accurate, rapid, sensitive, easy to operate, and can satisfy the requirements of public health emergency testing or clinical poisoning testing.

Biomonitoring of Multiple Mycotoxins in Urine by GC-MS/MS: A Pilot Study on Patients with Esophageal Cancer in Golestan Province, Northeastern Iran.

A pilot study to investigate the occurrence of 10 mycotoxins (deoxynivalenol, DON; 3-acetyldeoxynivalenol, 3-ADON; 15-acetyldeoxynivalenol, 15-ADON; fusarenon-X, FUS-X; diacetoxyscirpenol, DAS; nivalenol, NIV; neosolaniol, NEO; zearalenone, ZON; zearalanone, ZAN; T-2 toxin, T-2; and HT-2 toxin, HT-2) in esophageal cancer patients was performed with the urinary biomarkers approach in Golestan, Iran. Urine multimycotoxin analysis was performed by dispersive liquid-liquid microextraction and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis, and values were normalized with urinary creatinine (µg/g). Four mycotoxins, namely NEO (40%), HT-2 (17.6%), DON (10%), and HT-2 (5.8%), were detected in the analyzed urine samples. DON was only detected in the control group (5.09 µg/g creatinine), while T-2 (44.70 µg/g creatinine) was only present in the esophageal cancer group. NEO and HT-2 were quantified in both control and case groups, showing average of positive samples of 9.09 and 10.45 µg/g creatinine for NEO and 16.81 and 29.09 µg/g creatinine for HT-2, respectively. Mycotoxin co-occurrence was observed in three samples as binary (NEO/HT-2 and T-2/HT-2) and ternary (DON/NEO/HT-2) combinations, reaching total concentrations of 44.58, 79.13, and 30.04 µg/g creatinine, respectively. Further investigations are needed to explore a causal association between mycotoxin contamination and esophageal cancer. For this pilot study in Golestan, the low sample size was a very limiting factor.

Case-Control Study of Nodding Syndrome in Acholiland: Urinary Multi-Mycotoxin Screening.

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.