PI3K/Akt/FoxO Pathway Mediates Antagonistic Toxicity in HepG2 Cells Coexposed to Deoxynivalenol and Enniatins.

The emerging mycotoxins enniatins (ENNs) and the traditional mycotoxin deoxynivalenol (DON) often co-contaminate various grain raw materials and foods. While the liver is their common target organ, the mechanism of their combined effect remains unclear. In this study, the combined cytotoxic effects of four ENNs (ENA, ENA1, ENB, and ENB1) with DON and their mechanisms were investigated using the HepG2 cell line. Additionally, a population exposure risk assessment of these mycotoxins was performed by using in vitro experiments and computer simulations. The results showed that only ENA at 1/4 IC50 and ENB1 at 1/8 IC50 coexposed with DON showed an additive effect, while ENB showed the strongest antagonism at IC50 (CI = 3.890). Co-incubation of ENNs regulated the signaling molecule levels which were disrupted by DON. Transcriptome analysis showed that ENB (IC50) up-regulated the PI3K/Akt/FoxO signaling pathway and inhibited the expression of apoptotic genes (Bax, P53, Caspase 3, etc.) via phosphorylation of FoxO, thereby reducing the cytotoxic effects caused by DON. Both types of mycotoxins posed serious health risks, and the cumulative risk of coexposure was particularly important for emerging mycotoxins.

How Fungi Biosynthesize 3-Nitropropanoic Acid: The Simplest yet Lethal Mycotoxin.

We uncovered the biosynthetic pathway of the lethal mycotoxin 3-nitropropanoic acid (3-NPA) from koji mold Aspergillus oryzae. The biosynthetic gene cluster (BGC) of 3-NPA, which encodes an amine oxidase and a decarboxylase, is conserved in many fungi used in food processing, although most of the strains have not been reported to produce 3-NPA. Our discovery will lead to efforts that improve the safety profiles of these indispensable microorganisms in making food, alcoholic beverages, and seasoning.

Effects of ferulic acid combined with light irradiation on deoxynivalenol and its production in Fusarium graminearum.

This study aimed to investigate the effects of ferulic acid (FA), a natural phenolic phytochemical, in combination with light irradiation at three wavelengths (365, 385 and 405 nm) on the concentration and toxicity of deoxynivalenol (DON), a mycotoxin produced by Fusarium graminearum. Moreover, this study examined the influence of the combination treatment on DON production in the cultured fungus. FA activated by light at a peak wavelength of 365 nm exhibited the most effective decrease in DON concentration of the tested wavelengths; a residual DON ratio of 0.23 at 24 h exposure was observed, compared with the initial concentration. The reduction in DON using 365-nm light was dependent on the concentration of FA, with a good correlation (r2 = 0.979) between the rate constants of DON decrease and FA concentration, which was confirmed by a pseudo-first-order kinetics analysis of the photoreaction with different FA concentrations (50-400 mg/L) for 3 h. The viability of HepG2 cells increased by 56.7% following in vitro treatment with a mixture containing the photoproducts obtained after treatment with 20 mg/L DON and 200 mg/L FA under 365-nm irradiation for 6 h. These results suggested that the photoreaction of FA under 365-nm irradiation induces the detoxification of DON through degradation or modification of DON. The antifungal effects of the combination (FA and 365-nm light) on F. graminearum were investigated. Conidia treated with the combination did not show additive or synergistic inhibition of fungal biomass and DON production in 7-day cultivated fungal samples compared with samples after single treatment. However, successive treatment, composed of 90 min irradiation at 365 nm and then treatment with 200 mg/L FA for 90 min in the dark, suppressed fungal growth and DON yield to 70% and 25% of the untreated sample level, respectively. This photo-technology involving the two treatment methods of 365-nm irradiation and FA addition as a food-grade phenolic acid in combination or successively, can aid in developing alternative approaches to eliminate fungal contaminants in the fields of environmental water and agriculture. However, further research is required to explore the underlying mechanisms of DON decontamination and its biosynthesis in F. graminearum.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Toxigenic Aspergillus flavus and A. carbonarius in Nuts.

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

Evaluating the human neurotoxicity and toxicological interactions impact of co-occurring regulated and emerging mycotoxins.

Mycotoxins can inflict harmful effects on diverse organs, and mounting evidence indicates their potential involvement in human neurodegenerative diseases. Given the common occurrence of these toxins in food, there is an increasing demand for a comprehensive assessment of their combined toxicity to enhance our understanding of their potential hazards. This research investigates mycotoxin exposure from widely consumed cereal-based products, including enniatin B (ENNB), sterigmatocystin (STG), aflatoxin B1 (AFB1), cyclopiazonic acid (CPZ), citrinin (CIT), and ochratoxin A (OTA). Employing the median-effect equation based on Chou and Talalay's mass-action law, we assessed their cytotoxicity in human SH-SY5Y neuronal cells. Notably, ENNB displayed the highest neurotoxicity (IC50 = 3.72 µM), followed by OTA (9.10 µM) and STG (9.99 µM). The combination of OTA + STG exhibited the highest toxicity (IC50 = 3.77 µM), while CPZ + CIT showed the least detrimental effect. Approximately 70 % of tested binary combinations displayed synergistic or additive effects, except for ENNB + STG, ENNB + AFB1, and CPZ + CIT, which showed antagonistic interactions. Intriguingly, the senary combination displayed moderate antagonism at the lowest exposure and moderate synergism at higher doses. OTA exhibited predominantly synergistic interactions, comprising approximately 90 %, a noteworthy finding considering its prevalence in food. Conversely, ENNB interactions tended to be antagonistic. The most remarkable synergy occurred in the STG and CIT combination, enabling a 50-fold reduction in CIT dosage for an equivalent toxic effect. These findings highlight the biological relevance of robust synergistic interactions, emphasizing the need to assess human exposure hazards accurately, particularly considering frequent mycotoxin co-occurrence in environmental and food settings.

Development and validation of a liquid chromatography tandem mass spectrometry method for the determination of 10 mycotoxins in beer of the Chinese market and exposure estimate.

Mycotoxins are important risk factors in beer. In this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to determine 10 mycotoxins in beer within 6 min. The method is fast, efficient, and has a simple and quick sample preparation. Validation was conducted based on the performance standards specified in Commission Decision 657/2002/EC, and the results demonstrated excellent linearity (R2 > 0.99), repeatability (RSD < 5 %), quantification limits (0.005-20.246 µg/L), and recovery rates (77 %-118 %). The prevalence of the 10 mycotoxins in 96 beers purchased from the Chinese market was analyzed, and the exposure of the Chinese population to mycotoxins through beer consumption was assessed. Deoxynivalenol (DON) was detected in 93.75 % of the beers, and the incidence of fumonisins (FBs) and zearalenone (ZEN) exceeded 50 %. Beer intake contributed significantly to the exposure of aflatoxins (AFs) and DON, especially in males. Correlation analysis between mycotoxin content in beer, raw materials, and the brewing process revealed that the brewing process significantly affected the content of DON (P < 0.001), while auxiliary materials also had a significant impact on the content of FBs and DON (P < 0.001). This study holds great significance in producing higher quality and safer beer.

A comprehensive review of biodetoxification of trichothecenes: Mechanisms, limitations and novel strategies.

Trichothecenes are Fusarium mycotoxins with sesquiterpenoid structure, which are widely occurred in grains. Due to high efficiency and environmental friendliness, biological detoxification methods have been of great interest to treat this global food and feed safety concern. This review summarized the biological detoxification methods of trichothecenes from three aspects, biosorption, biotransformation and biotherapy. The detoxification efficiency, characteristics, mechanisms and limitations of different strategies were discussed in detail. Computer-aided design will bring a new research paradigm for more efficient discovery of biodetoxifier. Integrating different detoxification approaches assisted with computational tools will become a promising research direction in the future, which will help to maximize the detoxification effect, or provide precise detoxification programs for the coexistence of various toxins at different levels in actual production. In addition, technical and regulatory issues in practical application were also discussed. These findings contribute to the exploration of efficient, applicable and sustainable methods for trichothecenes detoxification, ensuring the safety of food and feed to reduce the deleterious effects of trichothecenes on humans and animals.

Challenges and Future State for Mycotoxin Analysis: A Review From a Regulatory Perspective.

Mycotoxins are naturally occurring toxins produced by certain fungi. Exposure to mycotoxins may occur through the consumption of contaminated foods or from animals that are fed contaminated feed. To safeguard the nation's food supply, the U.S. Food and Drug Administration (FDA) utilizes a comprehensive mycotoxin program which samples and analyzes foods for surveillance and compliance purposes, including enforcing action levels. Mycotoxin analysis is at the center of the mycotoxin program, as concentration data are needed for data analysis, scientific assessments, and risk management. This review focuses on the Agency's continuous efforts to develop and incorporate fit-for-purpose analytical tools for mycotoxin analysis with particular focus on the relationship between analytical methodologies and scientific assessments. The discussion further highlights challenges and advancements in analytical methods and discusses future possibilities to develop analytical tools and preventative risk management approaches to meet the evolving regulatory needs.

Toxicodynamic of combined mycotoxins: MicroRNAs and acute-phase proteins as diagnostic biomarkers.

Mycotoxins, ubiquitous contaminants in food, present a global threat to human health and well-being. Mitigation efforts, such as the implementation of sound agricultural practices, thorough food processing, and the advancement of mycotoxin control technologies, have been instrumental in reducing mycotoxin exposure and associated toxicity. To comprehensively assess mycotoxins and their toxicodynamic implications, the deployment of effective and predictive strategies is imperative. Understanding the manner of action, transformation, and cumulative toxic effects of mycotoxins, moreover, their interactions with food matrices can be gleaned through gene expression and transcriptome analyses at cellular and molecular levels. MicroRNAs (miRNAs) govern the expression of target genes and enzymes that play pivotal roles in physiological, pathological, and toxicological responses, whereas acute phase proteins (APPs) exert regulatory control over the metabolism of therapeutic agents, both endogenously and posttranscriptionally. Consequently, this review aims to consolidate current knowledge concerning the regulatory role of miRNAs in the initiation of toxicological pathways by mycotoxins and explores the potential of APPs as biomarkers following mycotoxin exposure. The findings of this research highlight the potential utility of miRNAs and APPs as indicators for the detection and management of mycotoxins in food through biological processes. These markers offer promising avenues for enhancing the safety and quality of food products.

Rapid and simultaneous detection of five mycotoxins and their analogs with a gold nanoparticle-based multiplex immuno-strip sensor.

Mycotoxins, as secondary metabolites produced by fungi, have been the focus of researchers in various countries and are considered to be one of the major risk factors in agricultural products. There is an urgent need for a rapid, simple and high-performance method to detect residues of harmful mycotoxins in agricultural foods. We have developed a gold nanoparticle-based multiplexed immunochromatographic strip biosensor that can simultaneously detect fifteen mycotoxins in cereal samples. With this optimized procedure, five representative mycotoxins, deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), tenuazonic acid (TEA) and alternariol (AOH) were detected in the range of 0.91-4.77, 0.04-0.56, 0.11-0.68, 0.12-1.02 and 0.09-0.75 ng/mL, respectively. The accuracy and stability of these measurements were demonstrated by analysis of spiked samples with recoveries of 91.8%-115.3% and coefficients of variation <8.7%. In addition, commercially available samples of real cereals were tested using the strips and showed good agreement with the results verified by LC-MS/MS. Therefore, Our assembled ICA strips can be used for the simultaneous detection of 5 mycotoxins and their analogs (15 mycotoxins in total) in grain samples, and the results were consistent between different types of cereal foods, this multiplexed immunochromatographic strip biosensor can be used as an effective tool for the primary screening of mycotoxin residues in agricultural products.

Integrating a Multiple Isotopologue Reaction-Monitoring Technique and LC-MS/MS for Quantitation of Small Molecules: Ten Mycotoxins in Cereals as an Example.

Accurate quantification of mycotoxin in cereals is crucial for ensuring food safety and human health. However, the preparation of traditional multisample external calibration curves (MSCCs) is labor-intensive and error-prone. Here, a multiple isotopologue reaction-monitoring (MIRM)-LC-MS/MS method for accurate quantitation of ten major mycotoxins in cereals was successfully developed and validated, where a novel one-sample multipoint calibration curve (OSCC) strategy is used instead of MSCCs. The OSCC can be established by examining the correlation between the calculated theoretical isotopic abundances and the measured abundance across various MIRM channels. In comparison to the MSCC, the OSCC strategy exhibits outstanding performance including superior selectivity, accuracy (78.4-108.6%), and precision (<12.5%). Furthermore, the proposed OSCC-MIRM-LC-MS/MS method was successfully applied to investigate mycotoxin contamination in cereal samples in China. Considering the advantages of simplified workflows and improved throughput, the OSCC-MIRM-LC-MS/MS methodology holds great promise for accurately quantifying chemical contaminants in foods.

Portable self-powered electrochemical aptasensing platform for ratiometric detection of mycotoxins based on multichannel photofuel cell.

Self-powered electrochemical sensors based on photofuel cells have attracted considerable research interest because their unique advantage of not requiring an external electric source, but their application in portable and multiplexed targets assay is limited by the inherent mechanism. In this work, a portable self-powered sensor constructed with multichannel photofuel cells was developed for the ratiometric detection of mycotoxins, namely ochratoxin A (OTA) and patulin (PAT). The spatially resolved CdS/Bi2S3-modified photoanodes and a shared Prussian Blue cathode were integrated on an etched indium-tin oxide slide to fabricate the multichannel photofuel cell. The aptamers of OTA and PAT were covalently bonded to individual photoanode regions to build sensitive interfaces, and the specific recognition of analytes impaired the output performance of constructed PFC. Accordingly, ratiometric sensing of OTA and PAT was achieved by utilizing the output performance of a control PFC as a reference signal. This approach effectively eliminates the impact of light intensity on the accuracy of the detection. Under the optimal conditions, the proposed sensing chip exhibited linear ranges of 2.0-1000 nM and 5.0-500 nM for OTA and PAT, respectively. The detection limits (3 S/N) were determined to be 0.25 nM for OTA and 0.27 nM for PAT. The developed ratiometric sensing method demonstrated good selectivity and stability in the simultaneous detection of OTA and PAT. It was successfully utilized for the analysis of OTA and PAT real samples. This work provides a new perspective for construction of portable and ratiometric self-powered sensing platform.

Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin.

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 µM, which decreases at 1.5 µM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Research on Pathogenic Fungi and Mycotoxins in China (Volume II).

The presence of pathogenic fungi and contamination of mycotoxins in food and feed pose significant threats and challenging issues to food in the world [...].

Magnetic Molecularly Imprinted Polymers for Selective Extraction of Aflatoxins from Feeds.

Magnetic molecularly imprinted polymers (MMIPs) have fused molecular imprinting technology with magnetic separation technology, emerging as an innovative material capable of recognizing specific molecules and efficiently separating target substances. Their application to the extraction and purification of mycotoxins has great potential, due to the toxicity and economic impact of these contaminants. In this work, MMIP has been proposed as a sample treatment for the determination of main four aflatoxins (B1, B2, G1 and G2) in pig feed. The MMIP was formed through the integration of magnetic material (Fe3O4) with commercial molecularly imprinted polymers, avoiding the synthesis step and, therefore, simplifying the process. The analyses were carried out by high-performance liquid chromatography with fluorescence detection and the method was validated and limits of quantification (LOQs) between 0.09 and 0.47 ng/g were obtained, below the allowed or recommended levels by the European Union. Repeatability and intermediate precision showed relative standard deviations lower than 10% in all cases and trueness ranged from 92 to 111%. Finally, the proposed method was applied to 31 real pig feed samples, detecting aflatoxins with concentrations between 0.2 and 3.2 ng/g.

Association of Urinary Mycotoxins with Sperm Quality: A Case-Control Study in Southern Nigeria.

The objective of this study was to determine the association between mycotoxins and the quality of spermatozoa in Nigeria. We designed a prospective case-control study involving 136 men diagnosed with reduced sperm count and quality in five infertility clinics in southwest Nigeria and 154 normal fertile controls. Sperm analysis was conducted in accordance with the recommendations of the World Health OrganizationWHO, while Liquid Chromatography-Mass Spectrometry was used to assay three metabolites of mycotoxins (zearalenone, ochratoxin A, and deoxyvinelol) in the urine samples of cases and controls. The data were analysed with descriptive statistics and non-parametric linear regression. The results showed no overall significant difference in levels of these metabolites between the cases and control groups. In contrast, higher levels of zearalenone and ochratoxin A significantly decreased sperm motility in the cases. Similarly, an increase in the level of ochratoxin A decreased sperm morphology in the unadjusted model in the cases. We conclude that exposure to mycotoxins reduces the quality of spermatozoa (motility and morphology) in Nigerian men but may have no effect on sperm count. Efforts to reduce the exposure of men to mycotoxins are important interventions to improve sperm quality and reduce the prevalence of male infertility in the country.

Mycoviruses Increase the Attractiveness of Fusarium graminearum for Fungivores and Suppress Production of the Mycotoxin Deoxynivalenol.

RNA viruses of the genera Ambivirus, Mitovirus, Sclerotimonavirus, and Partitivirus were found in a single isolate of Fusarium graminearum. The genomes of the mitovirus, sclerotimonavirus, and partitivirus were assigned to previously described viruses, whereas the ambivirus genome putatively represents a new species, named Fusarium graminearum ambivirus 1 (FgAV1). To investigate the effect of mycoviruses on the fungal phenotype, the spontaneous loss of mycoviruses during meiosis and the transmission of mycoviruses into a new strain via anastomosis were used to obtain isogenic F. graminearum strains both with and without mycoviruses. Notable effects observed in mycovirus-harboring strains were (i) the suppression of the synthesis of trichothecene mycotoxins and their precursor trichodiene, (ii) the suppression of the synthesis of the defense compound aurofusarin, (iii) the stimulation of the emission of 2-methyl-1-butanol and 3-methyl-1-butanol, and (iv) the increased attractiveness of fungal mycelia for fungivorous collembolans. The increased attractiveness of mycovirus-infected filamentous fungi to animal predators opens new perspectives on the ecological implications of the infection of fungi with viruses.

Unveiling the Broad Substrate Specificity of Deoxynivalenol Oxidation Enzyme DepA and Its Role in Detoxifying Trichothecene Mycotoxins.

DepA, a pyrroloquinoline quinone (PQQ)-dependent enzyme isolated from Devosia mutans 17-2-E-8, exhibits versatility in oxidizing deoxynivalenol (DON) and its derivatives. This study explored DepA's substrate specificity and enzyme kinetics, focusing on DON and 15-acetyl-DON. Besides efficiently oxidizing DON, DepA also transforms 15-acetyl-DON into 15-acetyl-3-keto-DON, as identified via LC-MS/MS and NMR analysis. The kinetic parameters, including the maximum reaction rate, turnover number, and catalytic efficiency, were thoroughly evaluated. DepA-PQQ complex docking was deployed to rationalize the substrate specificity of DepA. This study further delves into the reduced toxicity of the transformation products, as demonstrated via enzyme homology modeling and in silico docking analysis with yeast 80S ribosomes, indicating a potential decrease in toxicity due to lower binding affinity. Utilizing the response surface methodology and central composite rotational design, mathematical models were developed to elucidate the relationship between the enzyme and cofactor concentrations, guiding the future development of detoxification systems for liquid feeds and grain processing. This comprehensive analysis underscores DepA's potential for use in mycotoxin detoxification, offering insights for future applications.

Hidden Hazards Revealed: Mycotoxins and Their Masked Forms in Poultry.

The presence of mycotoxins and their masked forms in chicken feed poses a significant threat to both productivity and health. This review examines the multifaceted impacts of mycotoxins on various aspects of chicken well-being, encompassing feed efficiency, growth, immunity, antioxidants, blood biochemistry, and internal organs. Mycotoxins, toxic substances produced by fungi, can exert detrimental effects even at low levels of contamination. The hidden or masked forms of mycotoxins further complicate the situation, as they are not easily detected by conventional methods but can be converted into their toxic forms during digestion. Consequently, chickens are exposed to mycotoxin-related risks despite apparently low mycotoxin levels. The consequences of mycotoxin exposure in chickens include reduced feed efficiency, compromised growth rates, impaired immune function, altered antioxidant levels, disturbances in blood biochemical parameters, and adverse effects on internal organs. To mitigate these impacts, effective management strategies are essential, such as routine monitoring of feed ingredients and finished feeds, adherence to proper storage practices, and the implementation of feed detoxification methods and mycotoxin binders. Raising awareness of these hidden hazards is crucial for safeguarding chicken productivity and health.

Human Biomonitoring Guidance Values for Deoxynivalenol Derived under the European Human Biomonitoring Initiative (HBM4EU).

The mycotoxin deoxynivalenol (DON) was one of the priority substances in the European Joint Human Biomonitoring Initiative (HBM4EU) project. In this study, to better interpret the actual internal exposure of DON in the general population and safeguard public health, human biomonitoring guidance values of DON for the general population (HBM-GVGenPop) were derived. The HBM-GVGenPop of DON was based on either the total DON (DON and its glucuronides) or DON's main metabolite (DON-15-GlcA) levels in 24-h urine samples, resulting in a HBM-GVGenPop of 0.023 µg/mL for the total DON or a HBM-GVGenPop of 0.020 µg/mL for DON-15-GlcA. The use of 24-h urine samples is recommended based on the fact that DON and its metabolites have a short elimination half-life (T1/2), and 95% of the cumulative amount was excreted within 12 h after DON intake. The T1/2 for DON, DON-15-GlcA, and total DON were estimated to be 2.55 h, 2.95 h, and 2.95 h, respectively. Therefore, a 24-h urine sample reflects almost all of the DON exposure from the previous day, and this type of sample was considered for the derivation of a HBM-GVGenPop for DON.