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Aflatoxin B1 (AFB1), a highly toxic mycotoxin, always contaminated in a variety of agricultural products. Camelid variable domain of heavy chain antibody (VHH) is a noteworthy reagent in immunoassay, owing to its excellent characteristics. Immunization of camelid animals is a straightforward strategy to produce VHHs. In this study, to avoid the dependence on the large animals, the camelized, murine antibody (cVHs) against AFB1 was prepared in vitro based on the identities between murine VH and camelid VHH and then to develop an immunoassay for AFB1. A murine anti-AFB1 VH fragment (VH-2E6) was selected for camelization through replacement of conserved hydrophobic residues in framework region 2 (FR2) (cVH-FR2), point mutation at position 103 in the FR4 region (cVH-103), and CDR3-grafted with a high AFB1-affinity VHH (cVH-Nb26). The cVH-Nb26 had a yield of 5 mg/L as refolded protein expressed from Escherichia coli and 10 mg/L expressed from Pichia pastoris. Compared with anti-AFB1 single-chain fragment variable (scFv) 2E6, cVH-Nb26 performed more than 20-fold enhancement of AFB1-binding interactions. Although the AFB1-affinity of cVH-Nb26 cannot meet the application requirement in the present form, our study provides effective strategies for preparation of camelized antibody in vitro, which could be a promising immunoreagent for AFB1 detection. Supplementary Information The online version contains supplementary material available at 10.1007/s12550-021-00433-z.

This study evaluates the influence of crop seasons, cultivation regions, and traditional agricultural practices on the occurrence of F. proliferatum and A. flavus in the rice chain in the Mekong Delta, Vietnam. A survey on pre- and post-harvest practices was performed from 2017 to 2019 in parallel with sampling. Results showed that F. proliferatum (36.3%) and A. flavus (10%) were predominantly present throughout the rice chain. These fungi frequently occurred in winter–spring and autumn–winter crops in Can Tho paddy. Especially, F. proliferatum appeared both on the field and during transportation (50–100%), while A. flavus presented at all stages (10–33%). The occurrence of F. proliferatum reduced 70–27% after drying, depended on the seasons, compared to field and transportation stages and could not be detected anymore at further stages. Applying poor pre-harvest agricultural practices such as the use of certain varieties (Jasmine, DT8 varieties), combination of fertilizers (organic–inorganic), fields with crop debris, unhygienic boats, and delayed drying time of 8–12 h or 12–28 h resulted in an increase in fungal contamination on paddy. This study provides a detailed description of fungi contamination in crop seasons, cultivation regions, and agricultural practices, which may help in understanding the fungal dynamic and allow identification of good agricultural practices to mitigate the fungal contamination and potential mycotoxin production.

This work proposes a voltammetric aptasensor to detect deoxynivalenol (DON) mycotoxin. The development steps of the aptasensor were partnered for the first time to a computational study to gain insights onto the molecular mechanisms involved into the interaction between a thiol-tethered DNA aptamer (80mer-SH) and DON. The exploited docking study allowed to find the binding region of the oligonucleotide sequence and to determine DON preferred orientation. A biotinylated oligonucleotide sequence (20mer-BIO) complementary to the aptamer was chosen to carry out a competitive format. Graphite screen-printed electrodes (GSPEs) were electrochemically modified with polyaniline and gold nanoparticles (AuNPs@PANI) by means of cyclic voltammetry (CV) and worked as a scaffold for the immobilization of the DNA aptamer. Solutions containing increasing concentrations of DON and a fixed amount of 20mer-BIO were dropped onto the aptasensor surface: the resulting hybrids were labeled with an alkaline phosphatase (ALP) conjugate to hydrolyze 1-naphthyl phosphate (1-NPP) substrate into 1-naphthol product, detected by differential pulse voltammetry (DPV). According to its competitive format, the aptasensor response was signal-off in the range 5.0-30.0 ng.mL(-1) DON. A detection limit of 3.2 ng.mL(-1) was achieved within a 1-hour detection time. Preliminary experiments on maize flour samples spiked with DON yielded good recovery values. (C) 2020 Elsevier B.V. All rights reserved.

The poultry industry in sub-Saharan Africa (SSA) is faced with feed insecurity, associated with high cost of feeds, and feed safety, associated with locally produced feeds often contaminated with mycotoxins. Mycotoxins, including aflatoxins (AFs), fumonisins (FBs), trichothecenes, and zearalenone (ZEN), are common contaminants of poultry feeds and feed ingredients from SSA. These mycotoxins cause deleterious effects on the health and productivity of chickens and can also be present in poultry food products, thereby posing a health hazard to human consumers of these products. This review summarizes studies of major mycotoxins in poultry feeds, feed ingredients, and poultry food products from SSA as well as aflatoxicosis outbreaks. Additionally reviewed are the worldwide regulation of mycotoxins in poultry feeds, the impact of major mycotoxins in the production of chickens, and the postharvest use of mycotoxin detoxifiers. In most studies, AFs are most commonly quantified, and levels above the European Union regulatory limits of 20 mu g/kg are reported. Trichothecenes, FBs, ZEN, and OTA are also reported but are less frequently analyzed. Co-occurrences of mycotoxins, especially AFs and FBs, are reported in some studies. The effects of AFs on chickens' health and productivity, carryover to their products, as well as use of mycotoxin binders are reported in few studies conducted in SSA. More research should therefore be conducted in SSA to evaluate occurrences, toxicological effects, and mitigation strategies to prevent the toxic effects of mycotoxins.

Although aflatoxin (AF) exposure has not been recognized as a major problem in the United States and other developed nations, recent global climate change may have a profound impact on distribution of toxigenic fungi growth and production of AFs in grain and groundnuts. Alterations in the contamination pattern can increase human dietary exposure, and further invoke public health concerns and associated disease risks. In this study, two populations from East and West Texas, known for their high risk of liver cancer, were examined for their AF exposure at three different time periods from 2004 to 2014. Serum samples (n=1124) were collected from participants recruited for various studies from Bexar County and Lubbock County, TX, over the span of 2004 through 2014. The exposure biomarker, serum AFB1-lysine adduct, was analyzed by HPLC-FLD and confirmed by LC-MS. Both populations showed a significant increase in detection rate, as well as median levels of serum AFB1-lysine adduct over time, from 2.35 to 4.34pg/mg albumin in East Texas (2007-2014), and 0.63-3.98pg/mg albumin in West Texas (2004-2010). This observed shift in exposure likely represents a shift in the AF contamination pattern in the State of Texas, and may warrant further studies on risk assessment for the potential etiological effects of such increased exposures.

Ruminants are generally considered to be less susceptible to the effects of mycotoxins than monogastric animals as the rumen microbiota are capable of detoxifying some of these toxins. Despite this potential degradation, mycotoxin-associated subclinical health problems are seen in dairy cows. In this research, the disappearance of several mycotoxins was determined in an in vitro rumen model and the effect of realistic concentrations of those mycotoxins on fermentation was assessed by volatile fatty acid production. In addition, two hypotheses were tested: (1) a lower rumen pH leads to a decreased degradation of mycotoxins and (2) rumen fluid of lactating cows degrade mycotoxins better than rumen fluid of non-lactating cows. Maize silage was spiked with a mixture of deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROQ-C) and zearalenone (ZEN). Fresh rumen fluid of two lactating cows (L) and two non-lactating cows (N) was added to a buffer of normal pH (6.8) and low pH (5.8), leading to four combinations (L6.8, L5.8, N6.8, N5.8), which were added to the spiked maize substrate. In this study, mycotoxins had no effect on volatile fatty acid production. However, not all mycotoxins fully disappeared during incubation. ENN B and ROQ-C disappeared only partially, whereas MPA showed almost no disappearance. The disappearance of DON, NIV, and ENN B was hampered when pH was low, especially when the inoculum of non-lactating cows was used. For ZEN, a limited transformation of ZEN to &alpha, ZEL and &beta, ZEL was observed, but only at pH 6.8. In conclusion, based on the type of mycotoxin and the ruminal conditions, mycotoxins can stay intact in the rumen.

Mycotoxins such as aflatoxins (AFs), ochratoxin A (OTA) fumonisins (FMN), deoxynivalenol (DON), zearalenone (ZEN), and patulin are stable at regular food process practices. Ozone (O-3) is a strong oxidizer and generally considered as a safe antimicrobial agent in food industries. Ozone disrupts fungal cells through oxidizing sulfhydryl and amino acid groups of enzymes or attacks the polyunsaturated fatty acids of the cell wall.Fusariumis the most sensitive mycotoxigenic fungi to ozonation followed byAspergillusandPenicillium. Studies have shown complete inactivation ofFusariumandAspergillusby O(3)gas. Spore germination and toxin production have also been reduced after ozone fumigation. Both naturally and artificially, mycotoxin-contaminated samples have shown significant mycotoxin reduction after ozonation. Although the mechanism of detoxification is not very clear for some mycotoxins, it is believed that ozone reacts with the functional groups in the mycotoxin molecules, changes their molecular structures, and forms products with lower molecular weight, less double bonds, and less toxicity. Although some minor physicochemical changes were observed in some ozone-treated foods, these changes may or may not affect the use of the ozonated product depending on the further application of it. The effectiveness of the ozonation process depends on the exposure time, ozone concentration, temperature, moisture content of the product, and relative humidity. Due to its strong oxidizing property and corrosiveness, there are strict limits for O(3)gas exposure. O(3)gas has limited penetration and decomposes quickly. However, ozone treatment can be used as a safe and green technology for food preservation and control of contaminants.

Chemical warfare including insecticidal secondary metabolites is a well-known strategy for environmental microbes to monopolize a food source. Insects in turn have evolved behavioural and physiological defences to eradicate or neutralize the harmful microorganisms. We studied the defensive repertoire of insects in this interference competition by combining behavioural and developmental assays with whole-transcriptome time-series analysis. Confrontation with the toxic filamentous fungus Aspergillus nidulans severely reduced the survival of Drosophila melanogaster larvae. Nonetheless, the larvae did not behaviourally avoid the fungus, but aggregated at it. Confrontation with fungi strongly affected larval gene expression, including many genes involved in detoxification (e.g., CYP, GST and UGT genes) and the formation of the insect cuticle (e.g., Tweedle genes). The most strongly upregulated genes were several members of the insect-specific gene family Osiris, and CHK-kinase-like domains were over-represented. Immune responses were not activated, reflecting the competitive rather than pathogenic nature of the antagonistic interaction. While internal microbes are widely acknowledged as important, our study emphasizes the underappreciated role of environmental microbes as fierce competitors.

Applying post-harvest control measures such as adding mycotoxin detoxifying agents is a frequently-used mitigation strategy for mycotoxins. EFSA states that the efficacy of these detoxifiers needs to be tested using specific biomarkers for exposure. However, the proposed biomarkers for exposure are not further optimized for specific target species. Hence, the goal of this study was a) to evaluate the most suitable biomarkers for deoxynivalenol (DON) and zearalenone (ZEN) in porcine plasma, urine and feces, and DON, aflatoxin B1 (AFB1) and ochratoxin A (OTA) in plasma and excreta of broiler chickens and b) to determine the efficacy of a candidate detoxifier, as a proof-of-concept study. Therefore, a mixture of mycotoxins was administered as a single oral bolus with or without detoxifying agent. In accordance with literature AFB1, OTA, and DON-sulphate (DON-S) proved optimal biomarkers in broilers plasma and excreta whereas, in pigs DON-glucuronide (DON-GlcA) and ZEN-glucuronide (ZEN-GlcA) proved the optimal biomarkers in plasma, DON and ZEN-GlcA in urine and, ZEN in feces. A statistically significant reduction was seen between control and treatment group for both AFB1 and DON in broiler plasma, under administration of the mycotoxin blend and detoxifier dose studied suggesting thus, beneficial bioactivity.

Mycotoxins cause adverse effects on human health. Therefore, it is of the utmost importance to confront them, particularly in agriculture and food systems. Non-thermal plasma, electron beam radiation, and pulsed light are possible novel non-thermal technologies offering promising results in degrading mycotoxins with potential for practical applications. In this paper, the available publications are reviewed—some of them report efficiency of more than 90%, sometimes almost 100%. The mechanisms of action, advantages, efficacy, limitations, and undesirable effects are reviewed and discussed. The first foretastes of plasma and electron beam application in the industry are in the developing stages, while pulsed light has not been employed in large-scale application yet.

This study aims to determine dietary exposure to aflatoxin B-1 (AFB(1)) and the sum of aflatoxins (AFT) for the Turkish population and assess the risks to human health related to aflatoxins. A total of 1473 results on five main food categories commercialised in Turkey in 2002-2019 were used in the risk assessment. In the adult population, the mean middle bound (MB) exposure levels were 0.433 ng kg(-1) b.w. per day for AFB(1) and 0.511 ng kg(-1) b.w. per day for AFT. The 95th percentile dietary exposure of AFB(1) and AFT was estimated at 1.19 and 1.29 ng kg(-1) b.w. per day, respectively. Pistachios (44.4 %) made the highest contribution to mean AFB(1) exposure in Turkish adults, followed by maize/maize flour (16.2 %), groundnuts (13.8 %), chilli (10.1 %), walnuts (4.9 %), hazelnuts (4.0 %), chocolate (3.9 %) and others (2.7 %). The margin of exposure (MOE) estimates for mean and 95th percentile exposures to AFB(1) are considerably lower than 10 000, which raises a potential health concern for Turkish adults. Scientific and Technical Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [214O671] The author gratefully acknowledges the financial support of the Scientific and Technical Research Council of Turkey (TUBITAK, project no. 214O671). WOS:000607029700007 2-s2.0-85097480037

WOS:000453279100011 Lactic acid bacteria commonly named as probiotics have a broad range of utilization area in human diet and food industry. Besides these known properties of probiotics, in recent years many researchers have focused on their anti-genotoxic called DNA-bioprotective effects. Human gets exposure with genotoxins such as mycotoxins, polycyclic aromatic hydrocarbons, and n-nitroso-compounds through diet and environmental contaminations. Aflatoxin B-1 is mycotoxin which was reported one of the most potent hepato-carcinogen and its exposure stems from human diet. In this study, we aimed to investigate DNA-bioprotective effect of Lactobacillus plantarum on human colon adenocarcinoma (Caco-2) cells against Aflatoxin B-1 (AFB(1)) with comet assay without metabolic activation. The results showed that DNA-bioprotective effect of L. plantarum did reduce the AFB(1)'s genotoxic effect on colon adenocarcinoma (Caco-2) cells. Positive control (50 iM H2O2 applied) and high dose (>10ppm) AFB(1) applied cells have the same comet tail appearance. Only visual scoring is performed. Besides negative control cells (Only PBS) and Lactobacillus plantarum+AFB(1) mixture showed the same manner with each other; no comet tail detected. These results clearly indicate that L. plantarum is capable of reduce AFB(1) safely without producing any by-products.

Ruminal microbiota of cattle are not able to detoxify all mycotoxins. In addition, detoxification can be hampered by adverse ruminal conditions (e.g., low ruminal pH). Hence, in the cattle husbandry, mycotoxin binders and modifiers could be used to prevent animal exposure to mycotoxins. In this study, an in vitro rumen model, including feed matrix, was established as first screening tool to test the efficacy of five products claiming to detoxify mycotoxins. The detoxifiers had different modes of action: (a) binding (three products), (b) enzymatic detoxification of zearalenone (ZEN, one product, ZenA), and (c) bacterial transformation of trichothecenes (one product, BBSH 797). For the mycotoxin binders, the binding to the mycotoxins enniatin B (ENN B), roquefortine C (ROQ-C), mycophenolic acid (MPA), deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) were tested at a dose recommended by the manufacturers. The in vitro model demonstrated that all binders adsorbed ENN B to a certain extent, while only one of the binders also partially adsorbed ROQ-C. The binders did not change the concentrations of the other mycotoxins in the ruminal fluid. The enzyme ZenA detoxified ZEN very quickly and prevented the formation of the more toxic metabolite &alpha, zearalenol (&alpha, ZEL), both at normal (6.8) and low ruminal pH (5.8). The addition of BBSH 797 enhanced detoxification of DON and NIV, both at normal and low ruminal pH. The in vitro rumen model demonstrated that the addition of ZenA seems to be a very promising strategy to prevent estrogenic effects of ZEN contaminated feed, and BBSH 797 is efficient in the detoxification of trichothecenes.

The assurance of food and feed safety, including the identification and effective monitoring of multiple biological and chemical hazards, is a major societal challenge, given the increasing pace at which food commodities are demanded, produced and traded across the globe. Within this context, mycotoxins are globally widespread secondary fungal metabolites, which can contaminate crops either in the field or during storage and have serious human and animal health impacts such as carcinogenic, teratogenic and hepatotoxic effects. Therefore, their presence in a wide range of foods and feeds is strictly regulated, particularly in the European Union. In order to perform effective and routine monitoring of mycotoxin levels in the field prior to further processing, during transport or during processing, rapid, simple, portable and sensitive means of screening of regulated mycotoxins are in high demand. This review focuses on (1) discussing the relevance of mycotoxins and the standard approaches for their sampling and monitoring; and (2) compiling and discussing recent advances in miniaturized analytical tools for mycotoxin detection. This provides insights into current research efforts and opportunities to develop a truly integrated and fit-for-purpose analytical tool, suitable for use at critical points of the food, feed and raw material processing and distribution chains.

Absorption, distribution, metabolism and excretion (ADME) of food-borne toxic compounds may be influenced by other compounds or constituents present in the food. The present review presents an overview of evidence currently available on food matrix-derived combination effects influencing the ADME characteristics of food-borne toxic compounds and the possible implications for risk assessment. The results obtained indicate that interactions may occur at all levels of ADME and that the interactions may decrease but also increase the bioavailability and/or toxicity of the compounds of interest. The overview also illustrates that food matrix-derived combination effects should be considered on a case-by-case basis, taking into account especially the mode of action underlying the interactions and the dose dependency of the effects. Especially food matrix-derived combination effects that proceed by a reversible mode of action, such as for example binding to biotransformation enzymes or transport proteins, may be detected at concentrations used in in vitro assays and at dose levels used in animal bioassays but may be absent at dose levels representing realistic human intake. It is concluded that although food matrix-derived combination effects may exist, their detection in in vitro assays or in animal bioassays at high dose levels may not improve risk assessment practice because interactions observed may not be maintained at low realistic levels of intake. Insight in the mode of action underlying the interactions combined with physiologically based kinetic (PBK) modelling may prove a way to obtain better insight in whether interactions detected at high dose levels will still be relevant at more realistic lower intake levels, and thus to what extent these effects should be taken into account in the risk assessment for human exposure.

WOS: 000388115200013, This study was conducted to determine whether the plasma and brain concentrations of AFB(1) are affected by the modulation of P-gp and BCRP using zosuquidar (ZQR) and prazosin (PRZ), respectively. In this study, a total of 40 healthy adult male BALB/c mice (32 +/- 3.7 g) were used. The animals were randomly divided into 5 groups, with 8 animals per group. Group 1 was used for method validation. Group 2 (AF) received intraperitoneal AFB1 at a dose of 20 mg/kg of body weight. Groups 3 (AF+PRZ), 4 (AF+ZQR), and 5 (AF+PRZ+ZQR) received 20 mg/kg of AFB(1) intraperitoneally 30 min after the intraperitoneal administration of prazosin (0.3 mg/kg), zosuquidar (25 mg/kg), and prazosin+zosuquidar (0.3 mg/kg prazosin + 25 mg/kg zosuquidar), respectively. Six hours after the administration of AFB(1), blood and brain samples were collected from the animals in Groups 2 to 5. AFB(1) concentrations were determined using an HPLC system with fluorescence detection. Individual and simultaneous administration of prazosin and zosuquidar significantly reduced the brain concentrations of AFB(1) in comparison to a single administration of AFB(1) (P

This paper summarises workshop discussions at the 5th international MYCORED meeting in Ottawa, Canada (June 2012) with over 200 participants representing academics, government and industry scientists, government officials and farming organisations (present in roughly equal proportions) from 27 countries. Workshops centred on how mycotoxins in food and feed affect value chains and trade in the region covered by the North American Free Trade Agreement. Crops are contaminated by one or more of five important mycotoxins in parts of Canada and the United States every year, and when contaminated food and feed are consumed in amounts above tolerable limits, human and animal health are at risk. Economic loss from such contamination includes reduced crop yield, grain quality, animal productivity and loss of domestic and export markets. A systematic effort by grain producers, primary, transfer, and terminal elevators, millers and food and feed processers is required to manage these contaminants along the value chain. Workshops discussed lessons learned from investments in plant genetics, fungal genomics, toxicology, analytical and sampling science, management strategies along the food and feed value chains and methods to ameliorate the effects of toxins in grain on animal production and on reducing the impact of mycotoxins on population health in developing countries. These discussions were used to develop a set of priorities and recommendations.

Ruminants are less susceptible to the effects of mycotoxins than monogastric animals as their rumen microbiota are claimed to degrade and/or deactivate at least some of these toxic compounds. However, the mycotoxin degradation is not well-known yet. For this, a sensitive, specific, and accurate analytical method is needed to determine mycotoxins in the rumen fluid. This study aims to develop and thoroughly validate an ultra-performance liquid chromatography tandem mass spectrometry method for the quantitative determination in the rumen fluid of some of the most relevant mycotoxins found in maize silage in Western Europe: deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), mycophenolic acid (MPA), roquefortine C (ROQ-C) and enniatin B (ENN B), as well as their metabolites deepoxy-deoxynivalenol (DOM-1), &alpha, zearalenol (&alpha, ZEL), &beta, zearalenol (&beta, ZEL), zearalanone (ZAN), &alpha, zearalanol (&alpha, ZAL) and &beta, zearalanol (&beta, ZAL). As feed is often present in the rumen fluid samples, the potential interaction of feed particles with the mycotoxin extraction and analysis was investigated. Extraction recovery and matrix effects were determined in the rumen fluid with and without maize silage. Differences in those parameters between rumen fluid alone and rumen fluid with maize silage highlight the importance of using matrix-matched calibration curves for the quantification of mycotoxins in rumen fluid samples. A cross-validation of the method with rumen fluid and maize silage demonstrates that this analytical method can be applied in research on rumen fluid samples to investigate the degradation of the reported mycotoxins by rumen microbiota if matrix-matched calibration is performed.

Mycotoxins can cause serious adverse effects on animal health. This may lead to great economic losses in animal husbandry. In this review, the most common methods to counteract mycotoxins are presented, including several pre- and post-harvest strategies as well as an overview of the different mycotoxin detoxifying agents. The current legislation regarding maximum, guidance or action levels of mycotoxin contamination in various feedstuffs is also mentioned. It allows the agricultural industry to interpret feed analysis results and to decide whether to undertake actions or not.

Mycotoxins are secondary metabolites produced by fungi, which may be present on a variety of crops. They are considered a major issue worldwide because of their harmful effects on animals. These contaminants lead to great economic losses, especially in pig and poultry husbandry. Over 400 mycotoxins have been identifi ed. However, only few of them have a signifi cant toxic effect and are of major concern. In this paper, the most important mycotoxins are described, including deoxynivalenol (DON), T-2 toxin (T-2), zearalenone (ZON), fumonisin B1 (FB1), ochratoxin A (OTA) and afl atoxin B1 (AFB1). For each toxin, its chemical structure, mode of action and symptoms of acute and chronic toxicity in pigs and poultry are discussed.

Made available in DSpace on 2018-11-26T17:04:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) FP7-PEOPLE-IRSES Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) This work describes the investigation of a label-free immunosensor for the detection of aflatoxin B-1 (AFB(1)). CD-trodes (electrodes obtained from recordable compact disks) were used as low-cost and disposable transducers after modification with a self-assembled monolayer (SAM) of lipoic acid. The anti-aflatoxin B1 antibody was immobilized via EDC/NHS activation, followed by blocking with bovine serum albumin and immunoassays with AFB(1). The optimization of analytical parameters and the detection were carried out using electrochemical impedance measurements. Using chemometric tools, the best conditions for the immunosensor development were defined as: anti-AFB(1) antibody at 1:2000 dilution and surface blocking with 0.5% bovine serum albumin, both incubated for 1 h, and antibody-antigen immunoreaction for 30 min. The impedimetric immunosensor showed a linear range from 5 x 10(-9) to 1 x 10(-7) mol . L-1 (1.56-31.2 ng . mL(-1)), limit of detection and limit of quantification, respectively, 3.6 x 10(-10) and 1.1 x 10(-9) mol . L-1 (0.11 and 0.34 ng.mL(-1)). The proposed immunosensor was applied to analyze peanut samples. UNESP Univ Estadual Paulista, Inst Chem, Dept Analyt Chem, Rua Francisco Degni 55, BR-14800060 Araraquara, SP, Brazil UNESP Univ Estadual Paulista, Dept Food & Nutr, Sch Pharmaceut Sci, Rodovia Araraquara Jau,Km 01 S-N,Campos Ville, BR-14800903 Araraquara, SP, Brazil UNESP Univ Estadual Paulista, Dept Clin Anal, Sch Pharmaceut Sci, Rodovia Araraquara Jau,Km 01 S-N,Campos Ville, BR-14800903 Araraquara, SP, Brazil UNESP Univ Estadual Paulista, Inst Chem, Dept Analyt Chem, Rua Francisco Degni 55, BR-14800060 Araraquara, SP, Brazil UNESP Univ Estadual Paulista, Dept Food & Nutr, Sch Pharmaceut Sci, Rodovia Araraquara Jau,Km 01 S-N,Campos Ville, BR-14800903 Araraquara, SP, Brazil UNESP Univ Estadual Paulista, Dept Clin Anal, Sch Pharmaceut Sci, Rodovia Araraquara Jau,Km 01 S-N,Campos Ville, BR-14800903 Araraquara, SP, Brazil FAPESP: 2008/07729-8 FP7-PEOPLE-IRSES: 2008-230849 CNPq: 309275/2012-1

Exposure to mycotoxins, secondary metabolites produced by fungi, may infer serious risks for animal and human health and lead to economic losses. Several approaches to reduce these mycotoxins have been investigated such as chemical removal, physical binding, or microbial degradation. This review focuses on the microbial degradation or transformation of mycotoxins, with specific attention to the actual detoxification mechanisms of the mother compound. Furthermore, based on the similarities in chemical structure between groups of mycotoxins and environmentally recalcitrant compounds, known biodegradation pathways and degrading organisms which hold promise for the degradation of mycotoxins are presented.

International audience; Produced by several species of Aspergillus, Aflatoxin B-1 (AFB(1)) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB(1) is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B-1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB(1) inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB(1) biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB(1) cluster regulation.

International audience; In Vietnam, a great number of toxic substances, including carcinogens and procarcinogens, from industrial and agricultural activities, food production, and healthcare services are daily released into the environment. In the present study, we report the development of novel yeast-based biosensor systems to determine both genotoxic carcinogens and procarcinogens by cotransformation with two plasmids. One plasmid is carrying human CPR and CYP (CYP3A4, CYP2B6, or CYP2D6) genes, while the other contains the RAD54-GFP reporter construct. The three resulting coexpression systems bearing both CPR-CYP and RAD54-GFP expression cassettes were designated as CYP3A4/CYP2B6/CYP2D6 + RAD54 systems, respectively and used to detect and evaluate the genotoxic potential of carcinogens and procarcinogens by selective activation and induction of both CPR-CYP and RAD54-GFP expression cassettes in response to DNA damage. Procarcinogens were shown to be predominantly, moderately or not bioactivated by one of the CYP enzymes and thus selectively detected by the specific coexpression system. Aflatoxin B-1 and benzo(a) pyrene were predominantly detected by the CYP3A4 + RAD54 system, while N-nitrosodimethylamine only moderately activated the CYP2B6 + RAD54 reporter system and none of them was identified by the CYP2D6 + RAD54 system. In contrast, the genotoxic carcinogen, methyl methanesulfonate, was detected by all systems. Our yeast-reporter system can be performed in 384-well microplates to provide efficient genotoxicity testing to identify various carcinogenic compounds and reduce chemical consumption to about 53% as compared with existing 96-well genotoxicity bioassays. In association with a liquid handling robot, this platform enables rapid, cost-effective, and high-throughput screening of numerous analytes in a fully automated and continuous manner without the need for user interaction.

In this study, we aimed to determined the antagonistic effects of Origanum rotundifolium essential oil against the effects of Aflatoxin B1 (AFB1) as and inducer of oxidative stress and genotoxic agent in human lymphocytes in vitro. Sister chromatid exchange (SCE) and micronucleus (MN) tests were used for genotoxic influences estimation. In both the test systems, it was observed that O. rotundifolium essential oil suppressed the mutagenic effects of AFB(1). Biochemical parameters malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were examined to determine oxidative stress. This result showed that 5 mu M concentrations of AFB(1) malondialdehyde (MDA) level increased, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities decreased. However these effects of AFB(1) seen at higher levels decreased after treatment with 1.0, 1.5 and 2.0 mu L/6mL concentrations of O. rotundifolium. The results of this experiment have clearly shown that O. rotundifolium essential oil has strong anti-genotoxic effect against AFB(1) and that this capacity could be due to anti-oxidative properties.

Cereals products for direct human consumption are rarely contaminated by moulds, unlike raw materials, which are often infected, either in the field or during storage. In this study, 27 samples of dried pasta characterised by size, packaging and marketing intended for young children consumption were collected and analysed by liquid chromatography (LC) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for Deoxynivalenol (DON), Ochratoxin A (OTA) and Aflatoxin B 1 (AFB 1 ) determination. The samples that showed the highest amounts of one of the mycotoxins were cooked for 10 min, digested with an in vitro gastrointestinal protocol and bioaccessibility values were calculated. Seven of the 27 samples exceeded from 120% to 225% the legal limit of 200 μg/kg for DON fixed for processed cereal-based baby foods by an European Regulation; all the collected samples were under the OTA legal limit (0.05 μg/kg) fixed by the European Regulation and no sample was contaminated by AFB 1 over the instrumental limit of detection of 0.10 μg/kg. The mean value of gastric bioaccessibility verified for the DON resulted of 23.1%, whereas mean duodenal bioaccessibility was 12.1%.

International audience; An original biosensor was developed for the direct conductometric detection of ochratoxin A (OTA) in olive oil samples. The biosensor is based on thermolysin (TLN) immobilization into a polyvinyl alcohol (PVA)/polyethylenimine (PEI) matrix containing gold nanoparticles (AuNPs) and cross-linked at the surface of gold interdigitated microelectrodes using glutaraldehyde. Under optimal conditions (35 min cross-linking time, working pH of 7 and temperature of 25 degrees C), the biosensor response was linear up to 60 nM OTA, with a sensitivity of 597 mu S mu M-1 and a limit of detection of 1 nM. This value was 700 times lower than the detection limit obtained using the more classical method based on enzyme cross-linking in the presence of bovine serum albumin (BSA). PVA/PEI hydrogel creates a very favorable aqueous environment for the enzyme. In addition, interactions between protonated amino groups of PEI and negative charges of both citrated AuNPs and thermolysin improve their dispersion in the polymer blend, favoring enzyme stabilization and accessibility of the substrate. No conductometric signal was observed after OTA injection in the absence of AuNPs, in agreement with the insulating properties of the cross-linked PVA/PEI hydrogel film. Incorporation of AuNPs into the TLN/BSA biomembrane helped improving the sensitivity by 5.3 but this latter remained 140 times lower than the sensitivity of TLN/AuNPs/(PVA/PEI) biosensor. The study of enzyme kinetics showed that V-i vs [OTA] plot exhibited a non-hyperbolic trend, indicating that kinetics does not display a Michaelis-Menten behavior. Biosensor response times are longer (7-48 min) comparatively to TLN/BSA biosensor with a maximal value of 25 min. This difference is due to the diffusion phenomenon through the pores of the polymer membrane. The proposed OTA biosensor was very reproducible with a relative standard deviations (RSDs) in the 3-15% range and stable over a 30-days period when stored at 4 degrees C in 20 mM phosphate buffer between two measurements. The method was further evaluated using commercial doped olive oil samples. No pretreatment of the sample was needed for testing and no matrix effect was observed. Recovery values were close to 100% demonstrating the suitability of the proposed method for OTA screening in olive oil.

This article describes the trends in analytical techniques for the determination of trichothecene mycotoxins, namely deoxynivalenol, and T-2 and HT-2 toxins in cereals and cereal products with particular emphasis on screening and rapid approaches. The driving force behind the changing methodologies is mainly attributed to legislative demands. However, for commercial and governmental testing laboratories, the need to use validated official methods is ever increasing to ensure quality assurance of results. Much research has been undertaken to improve screening assays, highlighted by the number of new methods using a variety of formats and platforms, including optical and electrochemical biosensors. Significant advances in the traditional reference methods have also been demonstrated in addition to the emergence of a variety of commercial immunoaffinity and solid-phase extraction columns for clean up. The use of liquid chromatography coupled to tandem mass spectrometry for mycotoxin detection is ever increasing, allowing simultaneous determination of many toxins in various sample matrices.

International audience; Human health effects of inhaled mycotoxins remain poorly documented, despite the large amounts present in bioaerosols. Among these mycotoxins, sterigmatocystin is one of the most prevalent. Our aim was to study the metabolism and cellular consequences of sterigmatocystin once it is in contact with the airway epithelium. Metabolites were analyzed first in vitro, using recombinant P450 1A1, 1A2, 2A6, 2A13, and 3A4 enzymes, and subsequently in porcine tracheal epithelial cell (PTEC) primary cultures at an air liquid interface. Expressed enzymes and PTECs were exposed to sterigmatocystin, uniformly enriched with C-13 to confirm the relationship between sterigmatocystin and metabolites. Induction of the expression of xenobiotic-metabolizing enzymes upon sterigmatocystin exposure was examined by real-time quantitative real-time polymerase chain reaction. Incubation of 50 mu M sterigmatocystin with recombinant P450 1A1 led to the formation of three metabolites: monohydroxy-sterigmatocystin (M1), dihydroxy-sterigmatocystin (M2), and one glutathione adduct (M3), the latter after the formation of a transient epoxide. Recombinant P450 1A2 also led to M1 and M3. P450 3A4 led to only M3. In PTEC, 1 mu M sterigmatocystin metabolism resulted in a glucuro conjugate (M4) mainly excreted at the basal side of cells. If PTEC were treated with beta-naphthoflavone prior to sterigmatocystin incubation, two other products were detected, i.e., a sulfo conjugate (M5) and a glucoro conjugate (M6) of hydroxysterigmatocystin. Exposure of PTEC for 24 h to 1 mu M sterigmatocystin induced an 18-fold increase in the mRNA levels of P450 1A1, without significantly induced 7-ethoxyresorufin O-deethylation activity. These data suggest that sterigmatocystin is mainly detoxified and is unable to produce significant amounts of reactive epoxide metabolites in respiratory cells. However, sterigmatocystin increases the P450 1A1 mRNA levels with unknown long-term consequences. These in vitro results obtained in the porcine pulmonary tract need to be confirmed in human epithelial cells.

Mycotoxin contamination in rice can create a health risk for the consumers. In this study, the measurement of 23 mycotoxins in rice samples (n = 180) was performed using a validated LC-MS/MS method. A food frequency questionnaire was used to get rice consumption data for the assessment of mycotoxin dietary exposure, before calculating the health risk in adults and children of north and south regions of the Pakistani Punjab province. The prevalence of aflatoxin B-1 (56%), aflatoxin B-2 (48%), nivalenol (28%), diacetoxyscirpenol (23%), fumonisin B-1 (42%), zearalenone (15%), HT-2 toxin (10%), deoxynivalenol (8%), and ochratoxin A (6%) was estimated in samples with a mean concentration range between 0.61 and 22.98 mu g/kg. Aflatoxin degradation by traditional Pakistani cooking recipes was evaluated and observed to be 41-63%. The dietary exposure to aflatoxins exceeded the tolerable daily intake at all levels, and ochratoxin A and zearalenone posed health risk at high contamination and high consumption levels. The margin of aflatoxin B-1 exposure ranged between 10 and 69 in adults and 10 and 62 in children. The mean cancer risk by aflatoxin B-1 exposure was 0.070 (adults) and 0.071 (children) cases/year/100,000 people in South Punjab population, and 0.122 (adults) and 0.127 (children) cases/year/100,000 people in North Punjab population. This study will provide new insights for the planning and management of mycotoxins in Pakistan.

Mycotoxins, toxic secondary metabolites of fungi, affect global agriculture so prolifically that they are virtually ubiquitous at some concentration in the average human diet. Studies of in vitro and in vivo toxicity are discussed, leading to investigations of co-exposed mycotoxins, as well as carcinogenic effects. Some of the most common and toxicologically significant mycotoxins, such as the aflatoxins, ochratoxins, fumonisins, deoxynivalenol, T-2 toxin, HT-2 toxin, patulin, zearalenone, and some ergot alkaloids are outlined. The wide variety of pathogenic mechanisms these compounds employ are shown capable of inducing a complex set of interactions. Of particular note are potential synergisms between mycotoxins with regard to carcinogenic attributable risk, indicating an important field for future study.

WOS: 000226088100007 The effect of three individual strains of Laclobacillus (L. acidophilus NCC 12, L. acidophihrs NCC 36, L. acidophilus NCC 68) and two Bifidobacterium strains (B. bifrdum Bb13, B. bifidum NCC 381) on the growth and AFB(1) production of Aspergillus flavus NRRL 2999 was determined. The commercial MRS broth and skim milk medium were used to determine the effect of tested bacteria on the growth and AFBr production of A. flavus NRRL 2999. The bacteria were not effective in preventing the growth of A. flavus NRRL 2999 both in MRS broth and skim milk. However, higher levels of AFB(1) were obtained in the presence of individual strains of Lactobacillus and Bifidobacterium. The greatest difference was observed in the AFB(1) production of A. flavus NRRL 2999 in skim milk medium compared with MRS broth.

WOS: 000221071900013 This study was carried out to investigate the effects of adding baker yeast (BY), chlortetracycline (CTC) and both BY + CTC to a control diet containing 200 ng/g of aflatoxin B I (C + AFB,)on performance, serum parameters and pathologyc alterations of broilers. A total 100chicks (Ross PM 3) were divided into five groups in individual cages and each containing 20 animals. BY, a rich source of protein and vitamin B complex, was mixed into the diets at 2.0 %, CTC was mixed into the diet at 2.5 ng/g. Feed consumption, body weight and feed efficiency were recorded weekly. Serum parameters and pathologyc alterations were determined at the end of the study. Dead animals were recorded daily. Liver changes were clearly apparent in the C+AFB(1), and C+AFB(1),+CTC most of the livers were enlarged, yellow and had pethecial hemorrhages. Canalicula cholestosis was absent in group C+AFB(1), and C+AFB(1) +CTC, but not others. When compared to the control (C) group, alkaline phosphatase (ALP), appear to be significantly increased in the C+AFB(1) and C+CTC+AFB(1) groups. Serum glutamic oxalacetic transaminase (GOT)was increased in C+AFB(1) birds. Serum alphaphetoprotein was not affected by the treatments. Feed consumption and body weight were significantly reduced in group AFB(1). Birds receiving BY + AFB(1),(,) CTC + AFB, and BY + CTC + AFB(1) had a significantly higher body weight than group C+AFB Feed efficiency was better in group CTC + AFB, than the others. The findings of this research suggest tha BY (2%) can partly counteract some of the toxic effects of AFB.

The aim of this manuscript was the development of easy-to-operate quantum dots (QDs)-based immunochemical techniques for simultaneous screening of several mycotoxins in cereals. Two different approaches for multiplex fluorescent immunosorbent assay (FLISA) were employed. In the first approach a multiwell plate in which the different wells express a different mycotoxin (deoxynivalenol, zearalenone, aflatoxin B1, T2-toxin and fumonisin B1) was considered as a multiplex because each sample was pretreated once and then will be distributed over a series of wells within the same plate (single-analyte multiplex, SAM). The entire assay allows the simultaneous determination of all compounds. For the double-analyte multiplex (DAM) two different specific antibodies were co-immobilized in one single well. Zearalenone and aflatoxin B1 were simultaneously determined, provided their conjugates are labeled with QDs which are fluorescent in different parts of the spectrum, by scanning the assay outcome at two different wavelengths. The limits of detection (LOD) for the simultaneous determination of deoxynivalenol, zearalenone, aflatoxin B1, T2-toxin and fumonisin B1 by SAM FLISA were 3.2, 0.6, 0.2, 10 and 0.4 µg kg(-1), respectively, while for the DAM FLISA they were 1.8 and 1 µg kg(-1) for zearalenone and aflatoxin B1, respectively. SAM FLISA principle was also presented in a qualitative on-site format and tested for on-site multiplex determination of four mycotoxins in cereals. The achieved cut-off values of 500, 100, 2 and 100 µg kg(-1) for deoxynivalenol, zearalenone, aflatoxin B1 and T2-toxin respectively. For simplification of multiassay results' evaluation the conjugates with QDs of different colors were used.

Satureja hortensis L. (Lamiaceae) has been used as a folk remedy to treat various such as cramps, muscle pains, nausea, indigestion, diarrhea, and infectious diseases. In this study, the antagonistic effects of essential oil of S. hortensis (SHE) were studied against aflatoxin B-1 (AFB(1)) in human lymphocytes in vitro. The analysis of the essential oil was performed by using Gas chromatography-mass spectrometry (GC-MS). Anti-genotoxic effects of the SHEs was evaluated using sister chromatid exchange (SCE), micronuclei (MN) tests against AFB1. Also level of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities used to determine the anti-oxidative effects of the SHEs. This result showed AFB(1) (5 mu M) increased the frequencies of SCE, MN and the level of MDA. AFB1 at the same concentration decreased the activities of SOD and GPx. However, different concentrations of SHE with AFB(1) decreased the frequency of SCE and MN and level of MDA and also increased the activities of SOD and GPx significantly. Especially, the 1.0, 1.5, 2.0 mu L dose of SHE are more effective than other doses. The results of this experiment have clearly shown that SHE has strong antioxidative and antigenotoxic effects, these biological activities of SHEs can be due to its component., Fatih University, Research Project Foundation [P50031301_B (2923)], The authors are thankful to the Fatih University, Research Project Foundation (Contract no. P50031301_B (2923) for financial support of this study.

Aflatoxin B-1 (AFB(1)) is a mycotoxin produced by Aspergillus spp. that can occur as a natural contaminant in foods and feeds of vegetable origin. Post-ingestion, AFB(1) can be metabolized in the liver of mammals into hydroxylated aflatoxin M-1 (AFM(1)) that is excreted with milk. Although several studies have been carried out to evaluate effects of AFB(1) on the immune system, studies regarding AFM(1) are moreover lacking. The aim of the current study was to investigate effects of AFB(1) and AFM(1) on immune function using a lymphoblastoid Jurkat T-cell line as an experimental model. Both AFB(1) and AFM(1) produced significant decreases in Jurkat cell proliferation, whereas only minor effects were noted on interleukin (IL)-2 and interferon (IFN)-gamma cytokines mRNA expression in stimulated cells that had been pre-incubated with AFB(1) and AFM(1). Particularly, AFB(1), but not AFM(1), at the highest concentration (50 mu M) induced a marked increase in IL-8 mRNA expression. The results of the current study suggested the existence of a concentration threshold for AFB(1) and AFM(1) needed to exert biological activity on cell viability and innate immunity.

The aim of this study was to investigate the effects of essential oil of Mentha longifolia L. ssp. longifolia, used since ancient times for the treatment of many digestive tract diseases, on the genotoxicity and oxidative stress by a single dose of Aflatoxin B-1 (5 mu M) in the human lymphocyte culture. The total of the 12 identified components accounted for 97.69 % by Gas chromatography (GC). Major components of the oil were cis-piperitone epoxide (26.52 %), piperitenone oxide (26.40 %), pulegone (15.66 %) and piperitenone (13.57 %). According to the results obtained from GC-MS systems, monoterpenes consist of 89.19 % of the total oil approximately while sesquiterpenes were represented in small quantities in the oil. We studied the antigenotoxic effects of essential oils of M. longifolia (MEO) by using sister chromatid exchange (SCE) and micronucleus (MN) test systems. Also, the anti-oxidative effects of MEO were evaluated by using superoxide dismutase (SOD), glutathione peroxidase (GPx) and malondialdehyde (MDA) assay. The SCE frequency and the number of MN were increased when treated with AFB(1). Especially, MEO at 1.5 and 2.0 mu 1 reduced SCE frequency and MN number in the human lymphocyte (p < 0.05). In addition, AFB(1) treatment significantly reduced the activity of SOD and GPx and elevated the level of MDA (p < 0.05). MEOs treatment led to significant attenuation of AFB(1)-induced oxidative stress by normalization of the activities of SOD and GPx and the MDA (p < 0.05). These results suggest that MEOs could provide anti-oxidative and anti-genotoxic protection for the oxidative and genotoxic agents that cause many diseases including cancer and neurodegenerative disease., Fatih University [P50031301_B (2923)], We thank to Dr. Meryem Sengul for her contribution in this study. This investigation was partially supported by Fatih University (Project no: P50031301_B (2923).

Selenium is an essential mineral for a healthy life. Appropriate doses of it may undertake a protective role in the organism. In this study, the protective role of selenium (Se4+) against aflatoxin B1 (AFB1)-induced DNA damage was determined using random amplified polymorphic DNA on two plants including Vicia faba and Zea mays. It was observed that the concentrations of 0.1, 0.2 and 0.4 ppm of AFB1 have increased polymorphism value, total chlorophyll inhibition rate (IRc, %) and total protein IR (IRp, %). Unlike protein, chlorophyll contents and genomic template stability were decreased. With the addition of different concentrations (0.8 and 80 ppm) of Se4+ to the treated samples with AFB1, the values return to normal. An 800-ppm concentration of Se4+, on the contrary, could not inhibit the toxicity of AFB1 but caused an increase in toxicity level of AFB1/enhanced the toxicity level of AFB1. Results suggested that Se4+ has an antagonistic effect against AFB1 toxicity and that the degree of antagonistic effect of Se4+ against AFB1 was related to its concentration.

An isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with a fast sample preparation using homemade clean-up cartridges was developed for simultaneous determination of co-occurring mycotoxins exemplified with aflatoxin B1 (AFB1) and T-2 toxin (T-2) in representative biomatrices of rat plasma, heart, liver, kidney, spleen, lung and brain in a total run time of 7 min. The established approach using stable internal standards of [C-13(17)]-AFB1 and [C-13(24)]-T-2 was extensively validated by determining the specificity, linearity (R-2 >= 0.9990), sensitivity (lower limit of quantitation at 0.05 ng mL(-1)), accuracy (70.9-107.7%), precision (RSD = 70.8%). Based on this methodological advance, the subsequent kinetics and tissue distribution after oral administration of 0.5 mg kg(-1) b.w. of both AFB1 and T-2 in rats were thoroughly studied. As revealed, both AFB1 and T-2 were rapidly eliminated with the half-life time (t(1/2)) in plasma of 8.44 +/- 4.02 h and 8.12 +/- 4.05 h, respectively. Moreover, AFB1 accumulated in all organs where the highest concentration was observed in liver (1.34 mu g kg(-1)), followed by kidney (0.76 mu g kg(-1)). Notably, only low levels of T-2 were observed in spleen (0.70 mu g kg(-1)) and in liver (0.15 mu g kg(-1)). The achieved data as supporting evidence would substantially promote the practical application of the proposed LC-MS/MS method for in vivo toxicokinetics and toxicity studies of co-occurring mycotoxins imitating natural incidence in rat system.