A new physical and biological strategy to reduce the content of zearalenone in infected wheat kernels: the effect of cold needle perforation, microorganisms, and purified enzyme.

With the aim of reintroducing wheat grains naturally contaminated with mycotoxins into the food value chain, a decontamination strategy was developed in this study. For this purpose, in a first step, the whole wheat kernels were pre-treated using cold needle perforation. The pore size was evaluated by scanning electron microscopy and the accessibility of enzymes and microorganisms determined using fluorescent markers in the size range of enzymes (5 nm) and microorganisms (10 µm), and fluorescent microscopy. The perforated wheat grains, as well as non-perforated grains as controls, were then incubated with selected microorganisms (Bacillus megaterium Myk145 and B. licheniformis MA572) or with the enzyme ZHD518. The two bacilli strains were not able to significantly reduce the amount of zearalenone (ZEA), neither in the perforated nor in the non-perforated wheat kernels in comparison with the controls. In contrast, the enzyme ZHD518 significantly reduced the initial concentration of ZEA in the perforated and non-perforated wheat kernels in comparison with controls. Moreover, in vitro incubation of ZHD518 with ZEA showed the presence of two non-estrogenic degradation products of ZEA: hydrolysed zearalenone (HZEA) and decarboxylated hydrolysed ZEA (DHZEA). In addition, the physical pre-treatment led to a reduction in detectable mycotoxin contents in a subset of samples. Overall, this study emphasizes the promising potential of combining physical pre-treatment approaches with biological decontamination solutions in order to address the associated problem of mycotoxin contamination and food waste reduction.

Infant exposure to ochratoxin A, zearalenone, and deoxynivalenol from the consumption of milk formula and baby cereal in Chile.

Ochratoxin A (OTA), zearalenone (ZEN), and deoxynivalenol (DON) are mycotoxins whose exposure is associated with various adverse health effects, including cancer and renal disorders, estrogenic effects, and immunosuppressive and gastrointestinal disorders, respectively. Infants (<2 years) are the most vulnerable group to mycotoxins, representing a unique combination of restricted food consumption types, low body weight, lower ability to eliminate toxins, and more future years to accumulate toxins. This study aimed to estimate the infant́s exposure to OTA, DON, and ZEN due to the consumption of milk formula and baby cereals in Chile. Milk formula samples (n = 41) and baby cereals (n = 30) were collected and analyzed using commercial ELISA kits for OTA, DON, and ZEA determination. Exposure was assessed by the Estimated Daily Intake (EDI) approach (mean and worst-case scenario, WCS) with the levels found in a modified Lower Bound (mLB) and Upper Bound (UB); ideal consumption (<6m, 7-12 m, and 13-24 m); adjusted by the weight of each group. The risk was estimated by comparing the EDI with a reference tolerable daily intake or by the margin of exposure (MOE) in the case of OTA. DON and OTA occurrence in infant formula were 34 % and 41 %, respectively. The co-occurrence between these mycotoxins was 22 %. Mycotoxin contents were below LOQ values except for OTA determined in one sample (0.29 ng/ml). No milk formulae were contaminated with ZEN. In the case of baby cereals, the occurrences were 17 % for OTA, 30 % for DON, and 7 % for ZEN, all below LOQ. Co-occurrence was seen in two samples between ZEN and OTA. According to exposure calculations, the MOE for OTA was less than 10,000 in all models for milk formula between 0 to 12 months of age and in the UB and WCS for cereal consumption. Health concerns were observed for DON in the WCS and UB for milk consumption in all ages and only in the UB WCS for cereal consumption. Considering the high consumption of milk formula in these age groups, regulation of OTA and other co-occurring mycotoxins in infant milk and food is strongly suggested.

Rapid, on-site quantitative determination of mycotoxins in grains using a multiple time-resolved fluorescent microsphere immunochromatographic test strip.

The label probe plays a crucial role in enhancing the sensitivity of lateral flow immunoassays. However, conventional fluorescent microspheres (FMs) have limitations due to their short fluorescence lifetime, susceptibility to background fluorescence interference, and inability to facilitate multi-component detection. In this study, carboxylate-modified Eu(III)-chelate-doped polystyrene nanobeads were employed as label probes to construct a multiple time-resolved fluorescent microsphere-based immunochromatographic test strip (TRFM-ICTS). This novel TRFM-ICTS facilitated rapid on-site quantitative detection of three mycotoxins in grains: Aflatoxin B1 (AFB1), Zearalenone (ZEN), and Deoxynivalenol (DON). The limit of detection (LOD) for AFB1, ZEN, and DON were found to be 0.03 ng/g, 0.11 ng/g, and 0.81 ng/g, respectively. Furthermore, the TRFM-ICTS demonstrated a wide detection range for AFB1 (0.05-8.1 ng/g), ZEN (0.125-25 ng/g), and DON (1.0-234 ng/g), while maintaining excellent selectivity. Notably, the test strip exhibited remarkable stability, retaining its detection capability even after storage at 4 °C for over one year. Importantly, the detection of these mycotoxins relied solely on simple manual operations, and with a portable reader, on-site detection could be accomplished within 20 min. This TRFM-ICTS presents a promising solution for sensitive on-site mycotoxin detection, suitable for practical application in various settings due to its sensitivity, accuracy, simplicity, and portability.

Magnetic aptamer copper nanoclusters fluorescent biosensor for the visual detection of zearalenone based on docking-aided rational tailoring.

To address the food safety issues caused by toxins, we established a fluorescent copper nanocluster biosensor based on magnetic aptamer for the visual and quantitative detection of ZEN. Specifically, we utilized the docking-aided rational tailoring (DART) strategy to analyze intermolecular force and interaction sites between zearalenone (ZEN) and the aptamer, and optimize the long-chain aptamer step by step to enhance the binding affinity by 3.4 times. The magnetic bead-modified aptamer underwent conformational changes when competing with complementary sequences to bind with ZEN. Then, the released complementary sequences will be amplified in template-free mode with the presence of the terminal deoxynucleotidyl transferase (TdT), and generating T-rich sequences as the core sequences for the luminescence of copper nanoclusters. The luminescence could be visualized and quantitatively detected through ultraviolet irradiation. The proposed label-free aptasensor exhibited high sensitivity and specificity, with a low limit of detection (LOD) of 0.1 ng/mL.

Fabrication of amino- and hydroxyl dual-functionalized magnetic microporous organic network for extraction of zearalenone from traditional Chinese medicine prior to the HPLC determination.

Efficient enrichment of trace zearalenone (ZEN) from the complex traditional Chinese medicine (TCM) samples is quite difficult, but of great significance for TCM quality control. Herein, we reported a novel magnetic solid phase extraction (MSPE) strategy for ZEN enrichment using the amino- and hydroxyl dual-functionalized magnetic microporous organic network (Fe3O4@MON-NH2-OH) as an advanced adsorbent combined with the high-performance liquid chromatography (HPLC) determination. Efficient extraction of ZEN was achieved via the possible hydrogen bonding, hydrophobic, and π-π interactions between Fe3O4@MON-NH2-OH and ZEN. The adsorption capacity of Fe3O4@MON-NH2-OH for ZEN was 215.0 mg g-1 at the room temperature, which was much higher than most of the reported adsorbents. Under the optimal condition, the developed Fe3O4@MON-NH2-OH-MSPE-HPLC method exhibited wide linear range (5-2500 µg L-1), low limits of detection (1.4-35 µg L-1), less adsorbent consumption (5 mg), and large enhancement factor (95) for ZEN. The proposed method was successfully applied to detect trace ZEN from 10 kinds of real TCM samples. Conclusively, this work demonstrates the Fe3O4@MON-NH2-OH can effectively extract trace ZEN from the complex TCM matrices, which may open up a new way for the application of MONs in the enrichment and extraction of trace contaminants or active constituents from the complex TCM samples.

Development of a corn flour certified reference material for the accurate determination of zearalenone.

A certified reference material (CRM, KRISS 108-01-002) for zearalenone in corn flour was developed to assure reliable and accurate measurements in testing laboratories. Commercially available corn flour underwent freeze-drying, pulverization, sieving, and homogenization. The final product was packed in amber bottles, approximately 14 g per unit, and preserved at -70 °C. 13C18-Zearalenone was used as an internal standard (IS) for the certification of zearalenone by isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC‒MS/MS) and for the analysis of α-zearalenol, ß-zearalenol, and zearalanone by LC‒MS/MS. The prepared CRM was sufficiently homogeneous, as the among-unit relative standard deviation for each mycotoxin ranged from 2.2 to 5.7 %. Additionally, the stability of the mycotoxins in the CRM was evaluated under different temperature conditions and scheduled test periods, including storage at -70°C, -20°C, and 4°C and room temperature for up to 12 months, 6 months, and 1 month, respectively. The content of each target mycotoxin in the CRM remained stable throughout the monitoring period at each temperature. Zearalenone content (153.6 ± 8.0 µg/kg) was assigned as the certified value. Meanwhile, the contents of α-zearalenol (1.30 ± 0.17 µg/kg), ß-zearalenol (4.75 ± 0.33 µg/kg), and zearalanone (2.09 ± 0.16 µg/kg) were provided as informative values.

Development of a microfluidic device to enrich and detect zearalenone in food using quantum dot-embedded molecularly imprinted polymers.

Mycotoxins are secondary metabolites of certain moulds, prevalent in 60-80% of food crops and many processed products but challenging to eliminate. Consuming mycotoxin-contaminated food and feed can lead to various adverse effects on humans and livestock. Therefore, testing mycotoxin residue levels is critical to ensure food safety. Gold standard analytical methods rely on liquid chromatography coupled with optical detectors or mass spectrometers, which are high-cost with limited capacity. This study reported the successful development of a microfluidic "lab-on-a-chip" device to enrich and detect zearalenone in food samples based on the fluorescence quenching effect of quantum dots and selective affinity of molecularly imprinted polymers (MIPs). The dummy template and functional polymer were synthesized and characterized, and the detailed microfluidic chip design and optimization of the flow conditions in the enrichment module were discussed. The device achieved an enrichment factor of 9.6 (±0.5) in 10 min to quantify zearalenone spiked in food with high recoveries (91-105%) at 1-10 mg kg-1, covering the concerned residue levels in the regulations. Each sample-to-answer test took only 20 min, involving 3 min of manual operation and no advanced equipment. This microfluidic device was mostly reusable, with a replaceable detection module compatible with fluorescence measurement using a handheld fluorometer. To our best knowledge, the reported device was the first application of an MIP-based microfluidic sensor for detecting mycotoxin in real food samples, providing a novel, rapid, portable, and cost-effective tool for monitoring mycotoxin contamination for food safety and security.

Impact of predicted climate change environmental conditions on the growth of Fusarium asiaticum strains and mycotoxins production on a wheat-based matrix.

Fusarium asiaticum is a predominant fungal pathogen causing Fusarium Head Blight (FHB) in wheat and barley in China and is associated with approximately £201 million in annual losses due to grains contaminated with mycotoxins. F. asiaticum produces deoxynivalenol and zearalenone whose maximum limits in cereals and cereals-derived products have been established in different countries including the EU. Few studies are available on the ecophysiological behaviour of this fungal pathogen, but nothing is known about the impact of projected climate change scenarios on its growth and mycotoxin production. Therefore, this study aimed to examine the interacting effect of i) current and increased temperature (25 vs 30 °C), ii) drought stress variation (0.98 vs 0.95 water activity; aw) and iii) existing and predicted CO2 concentrations (400 vs 1000 ppm) on fungal growth and mycotoxin production (type B trichothecenes and zearalenone) by three F. asiaticum strains (CH024b, 82, 0982) on a wheat-based matrix after 10 days of incubation. The results showed that, when exposed to increased CO2 concentration (1000 ppm) there was a significant reduction of fungal growth compared to current concentration (400 ppm) both at 25 and 30 °C, especially at 0.95 aw. The multi-mycotoxin analysis performed by LC-MS/MS qTRAP showed a significant increase of deoxynivalenol and 15-acetyldeoxynivalenol production when the CH024b strain was exposed to elevated CO2 compared to current CO2 levels. Zearalenone production by the strain 0982 was significantly stimulated by mild water stress (0.95 aw) and increased CO2 concentration (1000 ppm) regardless of the temperature. Such results highlight that intraspecies variability exist among F. asiaticum strains with some mycotoxins likely to exceed current EU legislative limits under prospected climate change conditions.

Construction of a self-reporting molecularly-imprinted electrochemical sensor based on CuHCF modified by rGNR-rGO for the detection of zearalenone.

A self-reporting molecularly-imprinted electrochemical sensor is prepared for the detection of Zearalenone (ZEA). Firstly, the reduced graphene nanoribbons and reduced graphene oxide (rGNR-rGO) were simultaneously modified onto a glassy carbon electrode (GCE) to improve the sensor's sensitivity. After electrodepositing copper nanoparticles onto the rGNR-rGO/GCE, cyclic voltammetry scanning was performed in potassium ferrocyanide solution, and copper hexacyanoferrate (CuHCF) was deposited onto rGNR-rGO/GCE to further improve the sensor's sensitivity while giving it self-reporting capability. Then, molecularly-imprinted polymer films were prepared on the CuHCF/rGNR-rGO/GCE to ensure the selectivity of the sensor. It is found that the linear range of ZEA detection by the constructed sensor is 0.25-500 ng·mL -1, with a detection limit of 0.09 ng·mL -1. This sensor shows the merits of good selectivity, high sensitivity and accurate detection, providing a great possibility for the precise detection of low concentration ZEA in food.

Aggregation-induced emission nanoparticles facilitating multicolor lateral flow immunoassay for rapid and simultaneous detection of aflatoxin B1 and zearalenone.

Herein we developed a multicolor lateral flow immunoassay (LFIA) test strip for rapid and simultaneous quantitative detection of aflatoxin B1 (AFB1) and zearalenone (ZEN). Three differently colored aggregation-induced emission nanoparticles (AIENPs) were designed as LFIA signal tags, with red and green AIENPs for targeting AFB1 and ZEN at the test line, and yellow AIENPs for indicating the validity of the test strip at the control (C) line. After surface functionalization with antibodies, the developed AIENP-based multicolor LFIA allows simultaneous and accurate quantification of AFB1 and ZEN using an independent C-line assisted ratiometric signal output strategy. The detection limits of AFB1 and ZEN were 6.12 and 26 pg/mL, respectively. The potential of this method for real-world applications was well demonstrated in corn and wheat. Overall, this multicolor LFIA shows great potential for field screening of multiple mycotoxins and can be extended to rapid and simultaneous monitoring of other small molecule targets.

Dual function of magnetic nanocomposites-based SERS lateral flow strip for simultaneous detection of aflatoxin B1 and zearalenone.

Aflatoxin B1 (AFB1) and zearalenone (ZEN) are two mycotoxins that often co-occur in corn. A surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-LFIA) that can simultaneously detect AFB1 and ZEN in corn samples was developed employing the core-interlayer-satellite magnetic nanocomposites (Fe3O4@PEI/AuMBA@AgMBA) as dual-functional SERS tags. Under the optimal conditions, the detection ranges of AFB1 and ZEN in corn samples were 0.1-10 µg/kg and 4-400 µg/kg, respectively. Moreover, the test results for two mycotoxins in contaminated corn samples employing the suggested SERS-LFIA was in line with those of the HPLC technique. In view of its satisfactory sensitivity, accuracy, precision and short testing time (20 min), the developed system has a promising application prospect in the on-site simultaneous detection of AFB1 and ZEN.

Exposure assessment of children to dietary mycotoxins: A pilot study conducted in Ribeirão Preto, São Paulo, Brazil.

Exposure to mycotoxins through food is a major health concern, especially for youngsters. This study performed a preliminary investigation on children's exposure to dietary mycotoxins in Ribeirão Preto, Brazil. Sampling procedures were conducted between August and December 2022, to collect foods (N = 213) available for consumption in the households of children (N = 67), including preschoolers (aged 3-6 years, n = 21), schoolers (aged 7-10 years, n = 15), and adolescents (aged 11-17 years, n = 31) cared in the Vila Lobato Community Social Medical Center of Ribeirão Preto. Ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was used to determine concentrations of the mycotoxins in foods. Mycotoxins measured in all foods comprised aflatoxins (AFs), fumonisins (FBs), zearalenone (ZEN), T-2 toxin, deoxynivalenol (DON) and ochratoxin A (OTA). Higher incidence and levels were found for FBs, ZEN, and DON in several commonly consumed foods. Furthermore, 32.86 % foods had two to four quantifiable mycotoxins in various combinations. The mean estimated daily intake (EDI) values were lower than the tolerable daily intake (TDI) for AFs, FBs, and ZEN, but higher than the TDI (1.0 µg/kg bw/day) for DON, hence indicating a health risk for all children age groups. Preschoolers and adolescents were exposed to DON through wheat products (EDIs: 2.696 ± 7.372 and 1.484 ± 2.395 µg/kg body weight (bw)/day, respectively), while schoolers were exposed through wheat products (EDI: 1.595 ± 1.748 µg/kg bw/day) and rice (EDI: 1.391 ± 1.876 µg/kg bw/day). The results indicate that wheat-based foods and rice may be risky to children, implying the need for stringent measures to avoid DON contamination in these products.

Natural compounds mitigate mycotoxins-induced neurotoxicity by modulating oxidative tonus: in vitro and in vivo insights - a review.

This review explores the repercussions of mycotoxin contamination in food and feed, emphasising potential threats to agriculture, animal husbandry and public health. The primary objective is to make a comprehensive assessment of the neurotoxic consequences of mycotoxin exposure, an aspect less explored in current literature. Emphasis is placed on prominent mycotoxins, including aflatoxins, fumonisins, zearalenone (ZEA) and ochratoxins, known for inducing acute and chronic diseases such as liver damage, genetic mutation and cancer. To elucidate the effects, animal studies were conducted, revealing an association between mycotoxin exposure and neurological damage. This encompasses impairments in learning and memory, motor alterations, anxiety and depression. The underlying mechanisms involve oxidative stress, disrupting the balance between reactive oxygen species (ROS) and antioxidant capacity. This oxidative stress is linked to neuronal damage, brain inflammation, neurochemical imbalance, and subsequent behavioural changes. The review underscores the need for preventive measures against mycotoxin exposure. While complete avoidance is ideal, exploration into the potential use of antioxidants as a viable solution is discussed, given the widespread contamination of many food products. Specifically, the protective role of natural compounds, such as polyphenols, is highlighted, showcasing their efficacy in mitigating mycotoxicosis in the central nervous system (CNS), as evidenced by findings in various animal models. In summary, countering mycotoxin-induced neurotoxicity requires a multifaceted approach. The identified natural compounds show promise, but their practical use hinges on factors like bioavailability, toxicity and understanding their mechanisms of action. Extensive research is crucial, considering the diverse responses to different mycotoxins and neurological conditions. Successful implementation relies on factors such as the specific mycotoxin(s) involved and achievable effective concentrations. Further research and clinical trials are imperative to establish the safety and efficacy of these compounds in practical applications.

Programmable DNA tweezers-SDA for ultra-sensitive signal amplification fluorescence sensing strategy.

BACKGROUND: DNA tweezers, classified as DNA nanomachines, have gained prominence as multifunctional biosensors due to their advantages, including a straightforward structure, response mechanism, and high programmability. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. Some small molecules, such as mycotoxins, often require more sensitive detection due to their extremely high toxicity. Therefore, more effective signal amplification strategies are needed to further enhance the sensitivity of DNA tweezers in biosensing. RESULTS: We designed programmable DNA tweezers that detect small-molecule mycotoxins and miRNAs through simple sequence substitution. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. We introduced the Strand Displacement Amplification (SDA) technique to address this limitation, proposing a strategy of novel programmable DNA tweezers-SDA ultrasensitive signal amplification fluorescence sensing. We specifically investigate the effectiveness of this approach concerning signal amplification for two critical mycotoxins: aflatoxin B1 (AFB1) and zearalenone (ZEN). Results indicate that the detection ranges of AFB1 and ZEN via this strategy were 1-10,000 pg mL -1 and 10-100,000 pg mL -1, respectively, with corresponding detection limits of 0.933 pg mL -1 and 1.07 pg mL -1. Compared with the DNA tweezers direct detection method for mycotoxins, the newly constructed programmable DNA tweezers-SDA fluorescence sensing strategy achieved a remarkable 104-fold increase in the detection sensitivity for AFB1 and ZEN. SIGNIFICANCE: The constructed programmable DNA tweezers-SDA ultrasensitive signal-amplified fluorescence sensing strategy exhibits excellent detection performance for mycotoxins. The superb versatility of this strategy allows the developed method to be easily used for detecting other analytes by simply replacing the aptamer and cDNA, which has incredible potential in various fields such as food safety screening, clinical diagnostics, and environmental analysis.

Pore Size Adjustment Strategy for the Fabrication of Molecularly Imprinted Covalent Organic Framework Nanospheres at Room Temperature for Selective Extraction of Zearalenone in Cereal Samples.

Covalent organic frameworks (COFs) are attractive adsorbents for sample pretreatment due to their unique structure and properties. However, the selectivity of COFs for the extraction of hazardous compounds is still limited due to the lack of specific interactions between COFs and targets. Herein, we report a pore size adjustment strategy for room-temperature synthesis of molecularly imprinted COF (MICOF) for selective extraction of zearalenone (ZEN) in complex food samples. The three-dimensional building block tetra(4-aminophenyl) methane was used as a functional monomer, while dialdehyde monomers with different numbers of benzene ring were used to adjust the pore size of MICOF to match with the size of ZEN molecules. The prepared MICOF gave the largest adsorption capacity of 177.2 mg g-1 and the highest imprinting factor of 10.1 for ZEN so far. MICOF was used as the adsorbent for dispersed solid-phase extraction in combination with high-performance liquid chromatography for the determination of trace ZEN in cereals. The high selectivity of the developed method allows simple aqueous standard calibration for the matrix effect-free determination of ZEN in food samples. The limit of detection and the recoveries of the developed method were 0.21 µg kg-1 and 93.7-101.4%, respectively. The precision for the determination of ZEN was less than 3.8% (RSD, n = 6). The developed method is promising for the selective determination of ZEN in complex matrices.

Colorimetric and chemiluminescent enzyme immunoassays based on the alkaline phosphatase-tagged single-chain variable fragment fusion tracer for detecting zearalenone in agro-products.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin and seriously threatens food safety, which requires rapid and sensitive detection methods for monitoring ZEN in agro-products. Herein, an alkaline phosphatase-tagged single-chain variable fragment fusion protein (ALP-scFv) was used as a bifunctional tracer to develop a colorimetric enzyme immunoassay (CEIA) and a chemiluminescent enzyme immunoassay (CLEIA) for ZEN. In addition, the interactions between scFv and ZEN were exploited by computer-assisted simulation, and four key amino acid sites were preliminarily identified. After optimization, the CEIA and CLEIA exhibited a limit of detection of 0.02 and 0.006 ng/mL, respectively. Furthermore, both methods showed favorable accuracy in recovery experiments and good selectivity in cross reactions. Moreover, the detection results of the actual samples from both methods correlated well with those from high-performance liquid chromatography. Overall, the ALP-scFv fusion tracer-based CEIA and CLEIA are demonstrated as reliable tools for ZEN detection in food.

Equine Mycotoxins.

The main mycotoxins involved in adverse equine health issues are aflatoxins, fumonisins, trichothecenes, and probably ergovaline (fescue grass endophyte toxicosis). Most exposures are through contaminated grains and grain byproducts, although grasses and hays can contain mycotoxins. Clinical signs are often nonspecific and include feed refusal, colic, diarrhea, and liver damage but can be dramatic with neurologic signs associated with equine leukoencephalomalacia and tremorgens. Specific antidotes for mycotoxicosis are rare, and treatment involves stopping the use of contaminated feed, switching to a "clean" feed source, and providing supportive care.

Estrogen receptor α interaction of zearalenone and its phase I metabolite α-zearalenol in combination with soy isoflavones in hERα-HeLa-9903 cells.

Risk assessment primarily relies on toxicological data of individual substances, with limited information on combined effects. Recent in vitro experiments using Ishikawa cells, an endometrial carcinoma cell line expressing both estrogen receptor isoforms, demonstrated interactive effects of phyto- and mycoestrogens. The mycoestrogens, zearalenone (ZEN), and α-zearalenol (α-ZEL) exhibited significantly enhanced estrogenic responses in the presence of isoflavones (ISF), depending on substance ratios and concentrations. This study investigated the impact of phyto- and mycoestrogen combinations on estrogenic response following OECD guideline 455, utilizing hERα-HeLa-9903 cells. Test substances included mycoestrogens (ZEN and α-ZEL) and isoflavones (genistein (GEN), daidzein (DAI), and S-equol (EQ), a gut microbial metabolite of DAI). Mycoestrogens were tested in the range of 0.001 to 100 nM, while isoflavones were used at concentrations 1000 times higher based on relevant occurrence ratios. Results showed that ZEN and α-ZEL induced ERα-dependent luciferase expression in concentrations above 1 nM and 0.01 nM, respectively. However, ISF caused a superinduction of the luciferase signal above 1 µM. A superinduction is characterized by an unusually strong or heightened increase in the activity of the luciferase enzyme. This signal is not affected by the estrogen receptor antagonist 4-hydroxytamoxifen (4-OH-TAM), which was additionally used to verify whether the increase of signal is a true reflection of receptor activation. This superinduction was observed in all combinations of ZEN and α-ZEL with ISFs. Contrary to the luciferase activity findings, RT-qPCR experiments and a stability approach revealed lower real ERα activation by ISFs than measured in the ONE-Glo™ luciferase test system. In conclusion, the OECD protocol 455 appears unsuitable for testing ISFs due to their superinduction of luciferase and interactions with the test system, resulting in experimental artifacts. Further studies are necessary to explore structure-activity relationships within polyphenols and clarify the test system's applicability.

Role of ferroptosis in food-borne mycotoxin-induced toxicities.

Contamination by toxic substances is a major global food safety issue, which poses a serious threat to human health. Mycotoxins are major class of food contaminants, mainly including aflatoxins (AFs), zearalenone (ZON), deoxynivalenol (DON), ochratoxin A (OTA), fumonisins (FBs) and patulin (PAT). Ferroptosis is a newly identified iron-dependent form of programmed or regulated cell death, which has been found to be involved in diverse pathological conditions. Recently, a growing body of evidence has shown that ferroptosis is implicated in the toxicities induced by certain types of food-borne mycotoxins, which provides novel mechanistic insights into mycotoxin-induced toxicities and paves the way for developing ferroptosis-based strategy to combat against toxicities of mycotoxins. In this review article, we summarize the key findings on the involvement of ferroptosis in mycotoxin-induced toxicities and propose issues that need to be addressed in future studies for better utilization of ferroptosis-based approach to manage the toxic effects of mycotoxin contamination.

Sensitive fluorescence ELISA for the detection of zearalenone based on self-assembly DNA nanocomposites and copper nanoclusters.

Zearalenone (ZEN), produced by Fusarium species, is a potential risk to human health. Traditional enzyme-linked immunosorbent assay (ELISA) is restricted due to low sensitivity for the detection of ZEN. Herein, enzyme nanocomposites (ALP-SA-Bio-ssDNA, ASBD) were prepared with the self-assembly strategy based on streptavidin-labeled alkaline phosphatase (SA-ALP) and dual-biotinylated ssDNA (B2-ssDNA). The enzyme nanocomposites improved the loading amount of ALP and catalyzed more ascorbic acid 2-phosphate to generate ascorbic acid (AA). Subsequently, Cu2+ could be reduced to copper nanoclusters (CuNCs) having strong fluorescence signal by AA with poly T. Benefiting from the high enzyme load of nanocomposites and the strong signal of CuNCs, the fluorescence ELISA was successfully established for the detection of ZEN. The proposed method exhibited lower limit of detection (0.26 ng mL-1) than traditional ELISA (1.55 ng mL-1). The recovery rates ranged from 92.00% to 108.38% (coefficient of variation < 9.50%) for the detection of zearalenone in corn and wheat samples. In addition, the proposed method exhibited no cross reaction with four other mycotoxins. This proposed method could be used in trace detection for food safety.