Impact of Environmental Conditions on the Concentrations of Trichothecenes, Their Glucosides, and Emerging Fusarium Toxins in Naturally Contaminated, Irradiated, and Fusarium langsethiae Inoculated Oats.

Trichothecenes produced by Fusarium species are commonly detected in oats. However, the ratios of the concentrations of free trichothecenes and their conjugates and how they are impacted by different interacting environmental conditions are not well documented. This study aims to examine the effect of water activity (0.95 and 0.98 aw) and temperature (20 and 25 °C) stress on the production of T-2 and HT-2 toxins, deoxynivalenol and their conjugates, as well as diacetoxyscirpenol (DAS). Multiple mycotoxins were detected using liquid chromatography-tandem mass spectrometry from 64 contaminated oat samples. The highest concentrations of HT-2-glucoside (HT-2-Glc) were observed at 0.98 aw and 20 °C, and were higher than other type A trichothecenes in the natural oats' treatments. However, no statistical differences were found between the mean concentrations of HT-2-Glc and HT-2 toxins in all storage conditions analysed. DAS concentrations were generally low and highest at 0.95 aw and 20 °C, while deoxynivalenol-3-glucoside levels were highest at 0.98 aw and 20 °C in the naturally contaminated oats. Emerging mycotoxins such as beauvericin, moniliformin, and enniatins mostly increased with a rise in water activity and temperature in the naturally contaminated oats treatment. This study reinforces the importance of storage aw and temperature conditions in the high risk of free and modified toxin contamination of small cereal grains.

Comparative Performance of Rapid Diagnostics for the Detection of T-2 and HT-2 Toxins in Oats.

The contamination of oat crops by trichothecene mycotoxins, T-2 and HT-2 is an ongoing threat to our food safety. Within the industry, there are increasing concerns about the continued and growing presence of these mycotoxins occurring in oat crops due to climate change, farming practices and the handling of crops post-harvest. To safeguard human health, monitoring these mycotoxins in foodstuffs is paramount to ensure human exposure is limited. To achieve this, effective testing regimes must be established within the industry, consisting not only of rapid, reliable, and accurate analytical methods but also efficient sampling strategies. Four commercial rapid diagnostic kits were assessed against liquid chromatography coupled to mass spectrometry and included three lateral flow devices and one enzyme-linked immunosorbent assay. One-way ANOVA showed a p-value of 0.45 indicating no significant difference between the methods assessed. Qualitative analysis revealed test kits 1, 2, 3, and 4 showed false negative/false positive rates of 1.1/2.2, 7.6/0, 2.2/0, and 6.5/0 percent, respectively. Test Kit 1, the Neogen Reveal® Q+ MAX for T-2/HT-2 Kit provided the most reliable, accurate and cost-effective results. Furthermore, its ease of use and no requirement for technical skill makes it applicable for on-site testing.

A model framework to communicate the risks associated with aflatoxins.

Risk communication is defined as the interactive exchange of information and opinions concerning risk, risk-related factors and risk perceptions amongst all the stakeholders of food safety throughout the risk analysis process. The interactive exchange of information occurs at three different levels i.e. informed level, dialogue level and engagement level. For an effective food safety risk communication (FSRC), it is important that the information should adhere to the core principles of risk communication which are transparency, openness, responsiveness and timeliness. Communication of a food safety risk within all the components of risk communication strategy constitutes a complex network of information flow that can be better understood with the help of a framework. Therefore, a model framework to communicate the risks associated with aflatoxins (AFs) dietary intake has been developed with the aim of (a) creating general awareness amongst public and (b) involving industry stakeholders in the prevention and control of risk. The framework has been motivated by the learnings and best practices outlined in the identified technical guidance documents for risk communication. Risk assessors, risk managers, industry stakeholders and general public have been identified as the major stakeholders for the present framework. Amongst them, industry stakeholders and general public has been selected as the major target audience for risk managers. Moreover, population residing in low- and middle-income countries (LMIC) has been identified as the main target group to reach.

T-2 and HT-2 Toxins: Toxicity, Occurrence and Analysis: A Review.

One of the major classes of mycotoxins posing serious hazards to humans and animals and potentially causing severe economic impact to the cereal industry are the trichothecenes, produced by many fungal genera. As such, indicative limits for the sum of T-2 and HT-2 were introduced in the European Union in 2013 and discussions are ongoing as to the establishment of maximum levels. This review provides a concise assessment of the existing understanding concerning the toxicological effects of T-2 and HT-2 in humans and animals, their biosynthetic pathways, occurrence, impact of climate change on their production and an evaluation of the analytical methods applied to their detection. This study highlights that the ecology of F. sporotrichioides and F. langsethiae as well as the influence of interacting environmental factors on their growth and activation of biosynthetic genes are still not fully understood. Predictive models of Fusarium growth and subsequent mycotoxin production would be beneficial in predicting the risk of contamination and thus aid early mitigation. With the likelihood of regulatory maximum limits being introduced, increased surveillance using rapid, on-site tests in addition to confirmatory methods will be required. allowing the industry to be proactive rather than reactive.

Assessment of Cold Atmospheric Pressure Plasma (CAPP) Treatment for Degradation of Antibiotic Residues in Water.

The presence of antibiotic residues in water is linked to the emergence of antibiotic resistance globally and necessitates novel decontamination strategies to minimize antibiotic residue exposure in both the environment and food. A holistic assessment of cold atmospheric pressure plasma technology (CAPP) for ß-lactam antibiotic residue removal is described in this study. CAPP operating parameters including plasma jet voltage, gas composition and treatment time were optimized, with highest ß-lactam degradation efficiencies obtained for a helium jet operated at 6 kV. Main by-products detected indicate pH-driven peroxidation as a main mechanism of CAPP-induced decomposition of ß-lactams. No in vitro hepatocytotoxicity was observed in HepG2 cells following exposure to treated samples, and E. coli exposed to CAPP-degraded ß-lactams did not exhibit resistance development. In surface water, over 50% decrease in antibiotic levels was achieved after only 5 min of treatment. However, high dependence of treatment efficiency on residue concentration, pH and presence of polar macromolecules was observed.

Understanding Current Methods for Sampling of Aflatoxins in Corn and to Generate a Best Practice Framework.

Aflatoxin contamination in corn is a significant issue, posing substantial health threats to humans and animals. Aflatoxin testing protects consumer health, ensures the safe global trade of corn, and verifies compliance with legislation; however, effective sampling procedures are essential to ensure reliable results. While many sampling procedures exist, there is no evidence to indicate which is the best approach to ensure accurate detection. Using scientific and gray literature sources, this review analyzed sampling procedures to determine an optimum approach to guide the development of standard practices. Results revealed that sampling is the major source of error in the accurate assessment of aflatoxin levels in food and crucial for obtaining reliable results. To guarantee low variability and sample bias-increased sample size and sampling frequency, the use of automatic dynamic sampling techniques, adequate storage, and homogenization of aggregate samples for analysis are advised to ensure a representative sample. However, there is a lack of evidence to support this or indicate the current utilization of the reviewed procedures. Inadequate data prevented the recommendation of sample sizes or frequency for optimum practice, and thus, further research is required. There is an urgent need to make sampling procedures fit-for-purpose to obtain accurate and reliable aflatoxin measurements.

The Efficacy of Additives for the Mitigation of Aflatoxins in Animal Feed: A Systematic Review and Network Meta-Analysis.

The contamination of animal feed with aflatoxins is an ongoing and growing serious issue, particularly for livestock farmers in tropical and subtropical regions. Exposure of animals to an aflatoxin-contaminated diet impairs feed efficiency and increases susceptibility to diseases, resulting in mortality, feed waste, and increased production costs. They can also be excreted in milk and thus pose a significant human health risk. This systematic review and network meta-analysis aim to compare and identify the most effective intervention to alleviate the negative impact of aflatoxins on the important livestock sector, poultry production. Eligible studies on the efficacy of feed additives to mitigate the toxic effect of aflatoxins in poultry were retrieved from different databases. Additives were classified into three categories based on their mode of action and composition: organic binder, inorganic binder, and antioxidant. Moreover, alanine transaminase (ALT), a liver enzyme, was the primary indicator. Supplementing aflatoxin-contaminated feeds with different categories of additives significantly reduces serum ALT levels (p < 0.001) compared with birds fed only a contaminated diet. Inorganic binder (P-score 0.8615) was ranked to be the most efficient in terms of counteracting the toxic effect of aflatoxins, followed by antioxidant (P-score 0.6159) and organic binder (P-score 0.5018). These findings will have significant importance for farmers, veterinarians, and animal nutrition companies when deciding which type of additives to use for mitigating exposure to aflatoxins, thus improving food security and the livelihoods of smallholder farmers in developing countries.

The Occurrence and Co-Occurrence of Regulated, Emerging, and Masked Mycotoxins in Rice Bran and Maize from Southeast Asia.

Raw feed materials are often contaminated with mycotoxins, and co-occurrence of mycotoxins occurs frequently. A total of 250 samples i.e., rice bran and maize from Cambodia, Laos, Myanmar, and Thailand were analysed using state-of-the-art liquid chromatography-mass spectrometry (LC-MS/MS) for monitoring the occurrence of regulated, emerging, and masked mycotoxins. Seven regulated mycotoxins - aflatoxins, ochratoxin A, fumonisin B1, deoxynivalenol, zearalenone, HT-2, and T-2 toxin were detected as well as some emerging mycotoxins, such as beauvericin, enniatin type B, stachybotrylactam, sterigmatocystin, and masked mycotoxins, specifically zearalenone-14-glucoside, and zearalenone-16-glucoside. Aspergillus and Fusarium mycotoxins were the most prevalent compounds identified, especially aflatoxins and fumonisin B1 in 100% and 95% of samples, respectively. Of the emerging toxins, beauvericin and enniatin type B showed high occurrences, with more than 90% of rice bran and maize contaminated, whereas zearalenone-14-glucoside and zearalenone-16-glucoside were found in rice bran in the range of 56-60%. Regulated mycotoxins (DON and ZEN) were the most frequent mycotoxin combination with emerging mycotoxins (BEA and ENN type B) in rice bran and maize. This study indicates that mycotoxin occurrence and co-occurrence are common in raw feed materials, and it is critical to monitor mycotoxin levels in ASEAN's feedstuffs so that mitigation strategies can be developed and implemented.

Agronomic Factors Influencing the Scale of Fusarium Mycotoxin Contamination of Oats.

Seven agronomic factors (crop season, farming system, harvest date, moisture, county, oat variety, and previous crop) were recorded for 202 oat crops grown across Ireland, and samples were analysed by LC-MS/MS for four major Fusarium mycotoxins: deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin and HT-2 toxin. Type A trichothecenes were present in 62% of crops, with 7.4% exceeding European regulatory limits. DON (6.4%) and ZEN (9.9%) occurrences were relatively infrequent, though one and three samples were measured over their set limits, respectively. Overall, the type of farming system and the previous crop were the main factors identified as significantly influencing mycotoxin prevalence or concentration. Particularly, the adherence to an organic farming system and growing oats after a previous crop of grass were found to decrease contamination by type A trichothecenes. These are important findings and may provide valuable insights for many other types of cereal crops as Europe moves towards a much greater organic-based food system.

Low Doses of Mycotoxin Mixtures below EU Regulatory Limits Can Negatively Affect the Performance of Broiler Chickens: A Longitudinal Study.

Several studies have reported a wide range of severe health effects as well as clinical signs, when livestock animals are exposed to high concentration of mycotoxins. However, little is known regarding health effects of mycotoxins at low levels. Thus, a long-term feeding trial (between May 2017 and December 2019) was used to evaluate the effect of low doses of mycotoxin mixtures on performance of broiler chickens fed a naturally contaminated diet. In total, 18 successive broiler performance trials were carried out during the study period, with approximately 2200 one-day-old Ross-308 chicks used for each trial. Feed samples given to birds were collected at the beginning of each trial and analysed for multi-mycotoxins using a validated LC-MS/MS method. Furthermore, parameters including feed intake, body weight and feed efficiency were recorded on a weekly basis. In total, 24 mycotoxins were detected in samples analysed with deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FBs), apicidin, enniatins (ENNs), emodin and beauvericin (BEV), the most prevalent mycotoxins. Furthermore, significantly higher levels (however below EU guidance values) of DON, ZEN, FBs, BEV, ENNs and diacetoxyscirpenol (DAS) were detected in 6 of the 18 performance trials. A strong positive relationship was observed between broilers feed efficiency and DON (R2 = 0.85), FBs (R2 = 0.53), DAS (R2 = 0.86), ZEN (R2 = 0.92), ENNs (R2 = 0.60) and BEV (R2 = 0.73). Moreover, a three-way interaction regression model revealed that mixtures of ZEN, DON and FBs (p = 0.01, R2 = 0.84) and ZEN, DON and DAS (p = 0.001, R2 = 0.91) had a statistically significant interaction effect on the birds' feed efficiency. As farm animals are often exposed to low doses of mycotoxin mixtures (especially fusarium mycotoxins), a cumulative risk assessment in terms of measuring and mitigating against the economic, welfare and health impacts is needed for this group of compounds.

Comparative In Vitro Assessment of a Range of Commercial Feed Additives with Multiple Mycotoxin Binding Claims.

Contamination of animal feed with multiple mycotoxins is an ongoing and growing issue, as over 60% of cereal crops worldwide have been shown to be contaminated with mycotoxins. The present study was carried out to assess the efficacy of commercial feed additives sold with multi-mycotoxin binding claims. Ten feed additives were obtained and categorised into three groups based on their main composition. Their capacity to simultaneously adsorb deoxynivalenol (DON), zearalenone (ZEN), fumonisin B1 (FB1), ochratoxin A (OTA), aflatoxin B1 (AFB1) and T-2 toxin was assessed and compared using an in vitro model designed to simulate the gastrointestinal tract of a monogastric animal. Results showed that only one product (a modified yeast cell wall) effectively adsorbed more than 50% of DON, ZEN, FB1, OTA, T-2 and AFB1, in the following order: AFB1 > ZEN > T-2 > DON > OTA > FB1. The remaining products were able to moderately bind AFB1 (44-58%) but had less, or in some cases, no effect on ZEN, FB1, OTA and T-2 binding (<35%). It is important for companies producing mycotoxin binders that their products undergo rigorous trials under the conditions which best mimic the environment that they must be active in. Claims on the binding efficiency should only be made when such data has been generated.

A holistic study to understand the detoxification of mycotoxins in maize and impact on its molecular integrity using cold atmospheric plasma treatment.

The need for safe and quality food, free from the presence of hazardous contaminants such as mycotoxins is an on-going and complex challenge. Cold atmospheric pressure plasma (CAPP) has the potential to contribute to achieving this goal. Decontamination efficacy of CAPP against six of the most common mycotoxins found in foods and feedstuffs was assessed herein. Concentration reduction of up to 66% was achieved in maize for both aflatoxin B1 and fumonisin B1. Degradation products were detected only in the case of aflatoxin B1 and zearalenone and were tested on human hepatocarcinoma cells with no increase in cytotoxicity observed. Analysis of treated maize revealed substantial changes to small molecular mass components of the matrix. While CAPP shows promise in terms of mycotoxin detoxification important questions concerning potential changes to the nutritional and safety status of the food matrix require further investigations.

Assessing the combined toxicity of the natural toxins, aflatoxin B1, fumonisin B1 and microcystin-LR by high content analysis.

As human co-exposure to natural toxins through food and water is inevitable, risk assessments to safeguard health are necessary. Aflatoxin B1 and fumonisin B1, frequent co-contaminants of maize and microcystin-LR, produced in freshwater by cyanobacteria are all naturally occurring potent toxins that threaten human health. Populations in the poorest regions of the world may suffer repeated simultaneous exposure to these contaminants. Using High Content Analysis, multiple cytotoxicity endpoints were measured for the individual toxins and mixtures in various cell lines. Results highlighted that significant cytotoxic effects were observed for aflatoxin B1 in all cell lines while no cytotoxic effects were observed for fumonisin B1 or microcystin-LR. Aflatoxin B1/microcystin-LR was cytotoxic in the order HepG2 > Caco-2 > MDBK. Fumonisin B1/microcystin-LR affected MDBK cells. The ternary mixture was cytotoxic to all cell lines. Most combinations were additive, however antagonism was observed for binary and ternary mixtures in HepG2 and MDBK cell lines at low and high concentrations. Synergy was observed in all cell lines, including at low concentrations. The combination of these natural toxins may pose a significant risk to populations in less developed countries. Furthermore, the study highlights the complexity around trying to regulate for human exposure to multiple contaminants.

Uptake and accumulation of Microcystin-LR based on exposure through drinking water: An animal model assessing the human health risk.

Harmful Algal Blooms (HABs) in freshwater systems and intensified aquaculture have increased the risk to human health through exposure to cyanotoxins such as microcystin-LR (MC-LR). To understand the uptake and processing of MC-LR in humans, the pig was chosen as an animal model. This was assessed by repeated exposure for 13 weeks of eight animals dosed daily with MC-LR at 0.04 µg/kg bw, repeated with six animals over five weeks at a dose 50 times higher at 2 µg/kg bw. An analytical method was developed for MC-LR in porcine serum and also to analyse levels of free MC-LR in harvested porcine tissues, with Lemieux Oxidation employed to determine bound MC-LR in these tissues. MC-LR was not detected in the serum of treated animals from either experiment but free MC-LR was observed in the large intestine and kidney from two animals from the higher dosed group at levels of 1.4 and 1.9 µg/kg dry weight (dw) respectively. The results indicated 50% of higher dosed animals accumulated bound MC-LR in liver tissue, averaging 26.4 µg, approximately 1.1% of the dose administered. These results point to the potential uptake and accumulation of MC-LR in human liver tissue exposed chronically to sub-acute doses.

Advanced LC-MS-based methods to study the co-occurrence and metabolization of multiple mycotoxins in cereals and cereal-based food.

Liquid chromatography (LC) coupled with mass spectrometry (MS) is widely used for the determination of mycotoxins in cereals and cereal-based products. In addition to the regulated mycotoxins, for which official control is required, LC-MS is often used for the screening of a large range of mycotoxins and/or for the identification and characterization of novel metabolites. This review provides insight into the LC-MS methods used for the determination of co-occurring mycotoxins with special emphasis on multiple-analyte applications. The first part of the review is focused on targeted LC-MS approaches using cleanup methods such as solid-phase extraction and immunoaffinity chromatography, as well as on methods based on minimum cleanup (quick, easy, cheap, effective, rugged, and safe; QuEChERS) and dilute and shoot. The second part of the review deals with the untargeted determination of mycotoxins by LC coupled with high-resolution MS, which includes also metabolomics techniques to study the fate of mycotoxins in plants.

ß-methylamino-L-alanine (BMAA) is not found in the brains of patients with confirmed Alzheimer's disease.

Controversy surrounds the proposed hypothesis that exposure to ß-methylamino-L-alanine (BMAA) could play a role in various neurodegenerative conditions including Alzheimer's disease (AD). Here we present the results of the most comprehensive scientific study on BMAA detection ever undertaken on brain samples from patients pathologically confirmed to have suffered from AD, and those from healthy volunteers. Following the full validation of a highly accurate and sensitive mass spectrometric method, no trace of BMAA was detected in the diseased brain or in the control specimens. This contradicts the findings of other reports and calls into question the significance of this compound in neurodegenerative disease. We have attempted to explain the potential causes of misidentification of BMAA in these studies.

Development and Validation of a Lateral Flow Immunoassay for the Rapid Screening of Okadaic Acid and All Dinophysis Toxins from Shellfish Extracts.

A single-step lateral flow immunoassay was developed and validated to detect okadaic acid (OA) and dinophysis toxins (DTXs), which cause diarrhetic shellfish poisoning. The performance characteristics of the test were investigated, in comparison to reference methods (liquid chromatography tandem mass spectrometry and/or bioassay), using both spiked and naturally contaminated shellfish. A portable reader was used to generate a qualitative result, indicating the absence or presence of OA-group toxins, at concentrations relevant to the maximum permitted level (MPL). Sample homogenates could be screened in 20 min (including extraction and assay time) for the presence of free toxins (OA, DTX1, DTX2). DTX3 detection could be included with the addition of a hydrolysis procedure. No matrix effects were observed from the species evaluated (mussels, scallops, oysters, and clams). Results from naturally contaminated samples (n = 72) indicated no false compliant results and no false noncompliant results at <50% MPL. Thus, the development of a new low-cost but highly effective tool for monitoring a range of important phycotoxins has been demonstrated.

Gremlin1 preferentially binds to bone morphogenetic protein-2 (BMP-2) and BMP-4 over BMP-7.

Gremlin (Grem1) is a member of the DAN family of secreted bone morphogenetic protein (BMP) antagonists. Bone morphogenetic protein-7 (BMP-7) mediates protective effects during renal fibrosis associated with diabetes and other renal diseases. The pathogenic mechanism of Grem1 during diabetic nephropathy (DN) has been suggested to be binding and inhibition of BMP-7. However, the precise interactions between Grem1, BMP-7 and other BMPs have not been accurately defined. In the present study, we show the affinity of Grem1 for BMP-7 is lower than that of BMP-2 and BMP-4, using a combination of surface plasmon resonance and cell culture techniques. Using kidney proximal tubule cells and HEK (human embryonic kidney)-293 cell Smad1/5/8 phosphorylation and BMP-dependent gene expression as readouts, Grem1 consistently demonstrated a higher affinity for BMP-2>BMP-4>BMP-7. Cell-associated Grem1 did not inhibit BMP-2- or BMP-4-mediated signalling, suggesting that Grem1-BMP-2 binding occurred in solution, preventing BMP receptor activation. These data suggest that Grem1 preferentially binds to BMP-2 and this may be the dominant complex in a disease situation where levels of Grem1 and BMPs are elevated.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures.

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4 min injection assay to detect total microcystins in water samples below the WHO recommended limit (1 µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5 ng/mL for extracellular toxins and 0.05 ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

Microcystins: measuring human exposure and the impact on human health.

CONTEXT: Freshwater cyanobacterial toxins, microcystins, may be a contributing factor to the development of hepatocellular cancer and colorectal cancer. OBJECTIVES: This review summarizes the toxicity data, exposure routes and the methodologies available to determine exposure to elucidate the relationship to liver and colorectal cancer. METHODS: Literature searches were conducted using Medline, PubMed and Web of Science. RESULTS: There is evidence of human poisonings resulting from exposure to microcystins, however current methods rely on targeted approaches only suitable for acute exposure. No methods exist for the determination of chronic exposure to microcystins. CONCLUSIONS: With the growing evidence of exposure to microcystins and the possible links to cancer, methods to measure medium to long-term human exposure are needed. The identification and validation of candidate biomarkers are key to undertaking urgently required epidemiological studies.