Dietary mycotoxin exposure and human health risks: A protocol for a systematic review.

BACKGROUND: Mycotoxins are toxic fungal secondary metabolites that contaminate a wide spectrum of essential foods worldwide, such as grain-based products, nuts and spices, causing adverse health effects pertaining to their carcinogenic, nephrotoxic and hepatotoxic nature, among others. AIM: The aim of this systematic review (SR) is to systematically search for, appraise and synthesize primary research evidence to identify what is known about dietary mycotoxin-related health effects and what remains unknown, as well as the uncertainty around findings and the recommendations for the future. SEARCH STRATEGY AND ELIGIBILITY CRITERIA: Search strategies, as well as eligibility criteria were structured according to a predefined PECO (population, exposure, comparison, and outcome) research question and developed in an iterative scoping process. Several bibliographic databases, including Embase, Cochrane Library, Pubmed, Web of Science Core Collection and Scopus, will be searched. Primary research on any measured or modelled dietary exposure to a single or multiple mycotoxins, and adverse human health outcomes (i.e. cancer, non-carcinogenic diseases, and reproductive & developmental adverse outcomes) will be included, and references will be imported into Covidence. In vitro, ex vivo, in silico, animal and review studies, as well as expert's opinions, secondary literature, conference abstracts, presentations, posters, book chapters, dissertations and studies involving non-dietary mycotoxin exposure, will be excluded. STUDY SELECTION: Two independent reviewers will screen titles and abstracts, and review full-texts. Any disagreements will be resolved by a third reviewer based on two-third majority. DATA EXTRACTION: Data from retained eligible studies will be extracted by the principal reviewer, and peer-checked by a second reviewer. STUDY QUALITY ASSESSMENT: Eligible studies will be evaluated for risk of bias (Overall High-Quality Assessment Tool, OHAT) and certainty of evidence (Grading of Recommendations Assessment, Development and Evaluation, GRADE). EVIDENCE SYNTHESIS: A detailed summary of the included studies will be provided within a tabular format and narratively discussed. Heat maps will be constructed to provide information on available knowledge (gaps), and a meta-analysis may be performed based on the variability in predefined PECO elements and depending on the heterogeneity of studies. CONCLUSION: This protocol describes the methodology for the conduct of a SR on mycotoxin-related human health risks, that could guide future research and inform regulatory decisions, as emphasized by the European Commission within the field of regulatory risk assessment for emerging chemicals.

Burden of disease associated with dietary exposure to carcinogenic aflatoxins in Portugal using human biomonitoring approach.

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 (B1, B2, G1, G2 and M1). 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% (AFB1), 16% (AFB2), 1% (AFG1), 2% (AFG2) and 19% (AFM1) 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.

Exposure assessment of Portuguese population to multiple mycotoxins: The human biomonitoring approach.

Mycotoxins constitute a relevant group of food contaminants with several associated health outcomes such as estrogenic, immunotoxic, nephrotoxic and teratogenic effects. Although scarce data are available in Portugal, human biomonitoring studies have been globally developed to assess the exposure to mycotoxins at individual level. In order to overcome this lack of data, the present study concerned the analysis of mycotoxins in 24h urine and first-morning urine paired samples from 94 participants enrolled within the scope of the National Food, Nutrition, and Physical Activity Survey of the Portuguese General Population (2015-2016). Following a salt-assisted matrix extraction, urine samples were analysed by liquid chromatography-mass spectrometry for the simultaneous determination of 37 urinary mycotoxins' biomarkers and data obtained used to estimate the probable daily intake as well as the risk characterization applying the Hazard Quotient approach. Results revealed the exposure of Portuguese population to zearalenone, deoxynivalenol, ochratoxin A, alternariol, citrinin and fumonisin B1 through the quantification in 24h urine and first-morning urine paired samples. Risk characterization data revealed a potential concern to some reported mycotoxins since the reference intake values were exceeded by some of the considered participants. Alternariol was identified for the first time in urine samples from a European country; however, risk characterization was not performed due to lack of reference intake value. These results confirmed mycotoxins as part of the human exposome of the Portuguese population reinforcing the need for further studies regarding the determinants of exposure.

Natural occurrence of mycotoxins in maize and sorghum in Togo.

Mycotoxins are fungal secondary metabolites frequently affecting agronomical crops and consequently imposing a major challenge for food safety and public health. In this study, a total of 67 raw cereals (55 maize and 12 sorghum) were collected from the market of Togo. The samples were investigated on the occurrence of 21 mycotoxins using state-of-the-art high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The most frequent occurring mycotoxins were fumonisins (88 and 67% for maize and sorghum respectively) with concentrations ranging from 101 to 1838 µg/kg for maize and 81.5 to 361 µg/kg for sorghum, respectively. Aflatoxin B1 was detected in 38% of the maize samples with maximum contamination levels of 256 µg/kg, and 25% of the sorghum samples (range 6-16 µg/kg). The concentrations of aflatoxins were high in maize, with some cases exceeding the maximum legislative limits (EU) for unprocessed maize placed on the market. In addition to these high contamination levels, the co-occurrence of three classes of mycotoxins (i.e., aflatoxins, fumonisins, and trichothecenes) was observed in this study. For the first time, the multi-mycotoxins occurrence in agronomical crops in Togo was reported.

Processing of mycotoxin contaminated waste streams through anaerobic digestion.

Food and feed stocks heavily contaminated with mycotoxins are rendered unfit for consumption and therefore discarded as waste. Due to the lack of guidelines and in accordance with the prudent avoidance principle, these waste streams are often incinerated. For better valorization, these streams could be used as input for anaerobic digestion. However, the degradation of multiple mycotoxins during anaerobic digestion and their effect on the methane production is currently unknown. In batch tests spiked with mycotoxins, aflatoxin B1, ochratoxin A, deoxynivalenol, zearalenone and T-2 toxin were degraded for more than 90%. For mesophile and thermophile digestion respectively, fumonisin B1 was degraded for 70% and 85%, and most ergot alkaloids for 64% and 98%. Neither biogas production, nor methane production were influenced by the presence of the mycotoxins. Subsequently, semi-continuous reactors fed with contaminated maize resulted in more than 99% degradation for all mycotoxins after 1.8 hydraulic retention time with stable biogas production and process parameters. This study shows that mycotoxin contaminated organic waste can be safely valorized to methane while the digestate is void of mycotoxin residues.

Evaluation Of Mycotoxin Content In Soybean (Glycine max L.) Grown In Rwanda.

Soybean is a critical food and nutritional security crop in Rwanda. Promoted by the Rwandan National Agricultural Research System for both adults and as an infant weaning food, soybean is grown by approximately 40% of households. Soybean may be susceptible to the growth of mycotoxin-producing moulds; however, data has been contradictory. Mycotoxin contamination is a food and feed safety issue for grains and other field crops. This study aimed to determine the extent of mycotoxin contamination in soybean, and to assess people's awareness on mycotoxins. A farm-level survey was conducted in 2015 within three agro-ecological zones of Rwanda suitable for soybean production. Soybean samples were collected from farmers (n=300) who also completed questionnaires about pre-and post-harvest farm practices, and aflatoxin awareness. The concentration of total aflatoxin in individual soybean samples was tested by enzymelinked immunosorbent assay (ELISA) using a commercially-available kit. Other mycotoxins were analyzed using liquid chromatography-mass spectrometry (LCMS/MS) on 10 selected sub samples. Only 7.3% of the respondents were aware of aflatoxin contamination in foods, but farmers observed good postharvest practices including harvesting the crop when the pods were dry. Using enzyme-linked immunosorbent assay (ELISA), only one sample had a concentration (11 µg/kg) above the most stringent EU maximum permitted limit of 4 µg/kg. Multi-mycotoxins liquid chromatography-mass spectrometry (LC-MS/MS) results confirmed that soybeans had low or undetectable contamination; only one sample contained 13µg/kg of sterigmatocystine. The soybean samples from Rwanda obtained acceptably low mycotoxin levels. Taken together with other studies that showed that soybean is less contaminated by mycotoxins, these results demonstrate that soybean can be promoted as a nutritious and safe food. However, there is a general need for educating farmers on mycotoxin contamination in food and feed to ensure better standards are adhered to safeguard the health of the consumers regarding these fungal secondary metabolites.

Aphids transform and detoxify the mycotoxin deoxynivalenol via a type II biotransformation mechanism yet unknown in animals.

Biotransformation of mycotoxins in animals comprises phase I and phase II metabolisation reactions. For the trichothecene deoxynivalenol (DON), several phase II biotransformation reactions have been described resulting in DON-glutathiones, DON-glucuronides and DON-sulfates made by glutathione-S-transferases, uridine-diphosphoglucuronyl transferases and sulfotransferases, respectively. These metabolites can be easily excreted and are less toxic than their free compounds. Here, we demonstrate for the first time in the animal kingdom the conversion of DON to DON-3-glucoside (DON-3G) via a model system with plant pathogenic aphids. This phase II biotransformation mechanism has only been reported in plants. As the DON-3G metabolite was less toxic for aphids than DON, this conversion is considered a detoxification reaction. Remarkably, English grain aphids (Sitobion avenae) which co-occur with the DON producer Fusarium graminearum on wheat during the development of fusarium symptoms, tolerate DON much better and convert DON to DON-3G more efficiently than pea aphids (Acyrthosiphon pisum), the latter being known to feed on legumes which are no host for F. graminearum. Using a non-targeted high resolution mass spectrometric approach, we detected DON-diglucosides in aphids probably as a result of sequential glucosylation reactions. Data are discussed in the light of an eventual co-evolutionary adaptation of S. avenae to DON.

Immunochemical approach for zearalenone-4-glucoside determination.

Zearalenone-4-ß-D-glucopyranoside (zearalenone-4-glucoside) detection techniques, based on a combination of acidic or enzymatic hydrolysis of the masked mycotoxin to the parent form (i.e. zearalenone), and immunochemical determination of zearalenone-4-glucoside as a difference between the zearalenone concentration after and before cleavage of the glycosidic bond were developed. The limit of detection for zearalenone-4-glucoside, achieved for the enzyme linked immunosorbent assay, was 3 µg kg(-1); the cut-off level for the sum of zearalenone and zearalenone-4-glucoside determination by a qualitative gel-based immunoassay was 50 µg kg(-1). Trifluoromethanesulfonic acid was checked for acidic hydrolysis and resulted in approximately 70% of glycosidic bond cleavage in optimal conditions. Seven different glycoside hydrolases were tested during the design of the enzymatic hydrolysis technique. Enzymatic hydrolysis combined with enzyme linked immunosorbent assay and gel-based immunoassay determinations was applied for the determination of zearalenone-4-glucoside or the sum of zearalenone and zearalenone-4-glucoside in cereal samples. The chosen enzyme (glucosidase from Aspergillus niger) allowed to cleave 102% of zearalenone-4-glucoside in standard solutions and 85% in cereal samples. Liquid chromatography coupled to tandem mass spectrometry was used as confirmatory method. As a result, good correlations between immunochemical techniques and the chromatographic data were obtained. The developed technique is suitable for simultaneous immunochemical determination of zearalenone and its masked form, zearalenone-4-glucoside.

Development and validation of an LC-MS/MS method for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin and some masked metabolites in different cereals and cereal-derived food.

An LC-MS/MS method was developed and validated for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin, HT-2-toxin and metabolites, including 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol-3-glucoside, α-zearalenol, ß-zearalenol, zearalenone-4-glucoside, α-zearalenol-4-glucoside, ß-zearalenol-4-glucoside and zearalenone-4-sulfate in maize, wheat, oats, cornflakes and bread. Extraction was performed with acetonitrile/water/acetic acid (79/20/1, v/v/v) followed by a hexane defatting step. After filtration, the extract was evaporated and the residue was redissolved in mobile phase for injection. The mobile phase, which consisted of a mixture of methanol and water with 10 mM ammonium acetate, was adjusted to pH 3 with glacial acetic acid. A sample clean-up procedure was not included because of the low recoveries of free and masked mycotoxins and their differences in polarity. The method allowed the simultaneous determination of 13 Fusarium mycotoxins in a one-step chromatographic run using a Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer. The method was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, expanded measurement uncertainty and specificity. The limits of detection varied from 5 to 13 ng g⁻¹; those for the limit of quantification from 10 to 26 ng g⁻¹. The results of the performance characteristics of the developed LC-MS/MS method were in good agreement with the criteria mentioned in Commission Regulation (EC) No. 401/2006. Thirty samples of a variety of food and feed matrices were sampled and analysed between July 2010 and January 2011.