Biomarkers of Exposure to Zearalenone in In Vivo and In Vitro Studies.

The measurement of human exposure to mycotoxins is necessary for its association with adverse health effects. This exposure is usually estimated from contamination levels of foodstuffs, which are the primary source of toxin exposure, and data on food consumption patterns. However, variations in contamination level, intestinal absorption, toxin distribution, and excretion lead to individual variations in toxin exposure that can be more readily measured with a biomarker. This review deals with the latest literature information about ZEN biomarkers in humans, animals, and cell line cultures. Their presence in urine, biomarkers that have effects in the kidney, liver, reproductive system and blood and biomarkers of cell response have been reported. It has highlighted the importance of determining α-zearalenol and ß-zearalenol biomarkers to estimate the probable dietary intake (PDI) of a specific population or to characterize the severity of exposure to ZEN in animals or cell lines. α-ZEL and ß-ZEL are cytotoxic by inhibiting cell proliferation, total protein and DNA syntheses, in this sense, an induction of expression proteins Hsp27 and Hsp70 was observed, and an increase in gene expression (TLR4, NF-kBp65, TNF-α, IL-1ß, IL-6, IL-8, MGMT, α-GST, Hsp70, Nrf2, L-Fabp, HO-1, MAPK8), the determination of which indicates an oxidative stress effect. The integrity of the cell or tissue membrane is assessed by lactate dehydrogenase (LDH), which increase at exposure of ZEN (84.2 µM), and the proportions of some fatty acids of the renal tissue membrane were increased at treatments with ZEN. This review allows starting future studies of animal and population exposure in parallel with those of health effects works.

Development of an Extraction Method of Aflatoxins and Ochratoxin A from Oral, Gastric and Intestinal Phases of Digested Bread by In Vitro Model.

Validated extraction methods from in vitro digestion phases are necessary to obtain a suitable bioaccessibility study of mycotoxins in bakery products. The bakery industry produces bread with different ingredients to enrich the nutritional properties of this product and protect it from fungal growth. This bread can be contaminated by AFB1, AFB2, AFG1, AFG2 and OTA, so an extraction method was developed to analyse these five legislated mycotoxins in digested phases of two types of bread, one with wheat and the other with wheat and also enriched with Cucurbita Maxima Pepo at 20%. The studied "in vitro" digestion model consists of oral, gastric and duodenal phases, each one with different salt solutions and enzymes, that can affect the extraction and most probably the stability of the mycotoxins. The proposed method is a liquid-liquid extraction using ethyl acetate by extract concentration. These analytes and components have an important effect on the matrix effect (MEs) in the analytical equipment, therefore, validating the method and obtaining high sensitivity will be suitable. In the proposed method, the highest MEs were observed in the oral phase of digested pumpkin bread (29 to 15.9 %). Regarding the accuracy, the recoveries were above 83% in the digested duodenal wheat bread and above 76 % in the digested duodenal pumpkin wheat bread. The developed method is a rapid, easy and optimal option to apply to oral, gastric and duodenal phases of digested bread contaminated at a level of established maximum levels by European legislation (RC. 1881/2006) for food.

Evaluation of Mycotoxins in Infant Breast Milk and Infant Food, Reviewing the Literature Data.

In this review, an analysis focusing on mycotoxin determination in infant breast milk and infant food has been summarised for the last fifteen years of research focused on the intended population group of 1-9 months. The objective was to know the level of exposure of the child population to an estimated daily intake (EDI) of mycotoxins from the consumption of habitual foods. The EDI was compared with the tolerable daily intake (TDI) established by EFSA to estimate risk. In breast milk, the high prevalence and levels were for samples from Africa (Egypt and Tanzania) with aflatoxin M1 (1.9 µg/L and 10%), and Asia (Iran) with ochratoxin-A (7.3 µg/L and 100%). In infant formulas, high incidences and values were for samples with aflatoxin M1 from Burkina Faso (167 samples, 84%, 87 µg/kg). In cereal products, the highest incidence was for DON from the United States (96 samples), and the highest value was an Italian sample (0.83 µg/kg of enniatin B). In fruit products, patulin was the most detected in Italian (78) and Spanish (24) samples. The highest risk was observed in breast milk during the first month of age, the highest EDI for aflatoxin M1 was reported for Egypt (344-595 ng/kg bw/day) and ochratoxin-A for Iran (97-167ng/kg bw/day), representing a public health problem.

Evaluating the impact of supercritical-CO2 pressure on the recovery and quality of oil from "horchata" by-products: Fatty acid profile, α-tocopherol, phenolic compounds, and lipid oxidation parameters.

The effect of supercritical carbon dioxide (SC-CO2) (10-40 MPa) and conventional extraction (CE) to recover oil from by-products obtained during "horchata" production was assessed. To evaluate both extraction techniques, the fatty acid composition, polyphenols, α-tocopherol, antioxidant capacity and lipid oxidation parameters of the extracts obtained were analysed. A linear relationship between extraction pressure and oil yield was observed. However, the highest oil yield was obtained under conventional extraction. The by-products from "horchata" presented a profile where monounsaturated fatty acids (MUFA) were the predominant, representing ≈ 70% of total fatty acids. The amount of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) was higher and MUFA lower at 10 MPa samples compared to the oils extracted using SC-CO2 at 20, 30 or 40 MPa, where no differences were detected. The content of α-tocopherol was significantly higher after SC-CO2 treatments compared to conventional extraction, independently of the applied treatment. On the other hand, the values of phenolic compounds and total antioxidant activity (TAC) increased as the pressure conditions of the SC-CO2 extraction increased, presenting a linear adjustment of the data. Regarding lipid oxidation, the lower oxidation indexes were obtained when the SC-CO2 pressure increased. Finally, our results confirmed that the application of SC-CO2 could be a potential alternative to conventional extraction in order to obtain oils from "horchata" by-products rich in high-added value compounds without the use of organic solvents which can be toxic.

Phenolic profile of oils obtained from "horchata" by-products assisted by supercritical-CO2 and its relationship with antioxidant and lipid oxidation parameters: Triple TOF-LC-MS-MS characterization.

In this study, the effect of different supercritical CO2 (SC-CO2) pressures (10-40 MPa) on phenolic compounds extraction in oils obtained from "horchata" by-products was evaluated, and the results were compared to those obtained after conventional oil extraction (CE). Moreover, the relationship between the individual phenolic compounds and the total antioxidant capacity as well as oil oxidative quality parameters was compared. The phenolic profile and contents were largely influenced by extracting conditions. The main phenolic compound obtained by SC-CO2 was the isohydroxymatairesinol, particularly at 30 and 40 MPa, while 3-vinylphenol was the predominant compound in oils extracted by CE procedure. Increasing SC-CO2 extraction pressures enhanced the extraction of phenolic compounds, along with improving the antioxidant capacity and oxidative quality of extracted oil. The principal component analysis indicated that the main phenolic compounds associated with TEAC values were those extracted by SC-CO2, which were inversely correlated to oxidative indexes.

Thermal and non-thermal preservation techniques of tiger nuts' beverage "horchata de chufa". Implications for food safety, nutritional and quality properties.

"Horchata de chufa" is a traditional Spanish beverage produced from tiger nuts (Cyperus esculentus L.). Due to its richness in nutritional compounds, it is highly perishable and its conservation by pasteurization and/or adding preservatives is required. Although efficient, conventional thermal treatment for pasteurization induces changes in the nutritional and sensory properties. Replacing conventional pasteurization by non-thermal technologies such as pulsed electric fields, ultraviolet, and high pressure, combined with moderate temperatures (<40°C) allows a reduction of energy consumption, along with the preservation of the most thermo-sensitive molecules. Accordingly, this review deals with the description of the most relevant non-thermal technologies applied to preserve "horchata" beverage in order to extend the shelf life and inactivate pathogenic microorganisms as well as to preserve the nutritional and quality properties of this food beverage.

Development of microextraction techniques in combination with GC-MS/MS for the determination of mycotoxins and metabolites in human urine.

Simple and highly efficient sample preparation procedures, namely, dispersive liquid-liquid microextraction and salting-out liquid-liquid extraction for the analysis of ten Fusarium mycotoxins and metabolites in human urine were compared. Various parameters affecting extraction efficiency were carefully evaluated. Under optimal extraction conditions, salting-out liquid-liquid extraction showed a better accuracy (84-96%) and precision (<14%) than dispersive liquid-liquid microextraction. Hence, a multibiomarker method based on salting-out liquid-liquid extraction followed by gas chromatography with tandem mass spectrometry was proposed. Satisfactory results in terms of validation were achieved. The method resulted in low limits of detection and quantitation within the range of 0.12-4 and 0.25-8 µg/L, respectively. The method accuracy and precision were evaluated at three spiking levels (8, 25 and 100 µg/L) and the recoveries were in a range from 70 to 120% with relative standard deviations lower than 15%. Matrix effect was evaluated and matrix-matched calibrations were used for quantitation purpose. The developed method was applied in 12 human urine samples as a pilot study before and after sample treatment with ß-glucuronidase before the analysis to quantify the mycotoxin conjugates. Total deoxynivalenol (free + conjugated) was found in 83% of samples at an average concentration in positive samples of 31.6 µg/L.

Development of a GC-MS/MS strategy to determine 15 mycotoxins and metabolites in human urine.

The widespread mycotoxins contamination of food commodities has made the monitoring of their levels essential. To overcome the disadvantages of the indirect approach by food analysis, detection of mycotoxin as biomarkers in urine provides a useful and specific data for exposure assessment to these food contaminants. In this work, a sensitive, rapid and accurate method based on gas chromatography-tandem mass spectrometry procedure to determine 15 mycotoxins and metabolites in human urine was optimized and validated taking into consideration the guidelines specified in Commission Decision 2002/657/EC and 401/2006/EC. A salting-out assisted acetonitrile-based extraction was used for sample preparation. The extraction recoveries were in a range of 72-109%, with intra-day relative standard deviation and inter-day relative standard deviation lower than 10% and 13%, respectively for all mycotoxins at 50, 100 and 200 µg/L spiking levels. The limits of quantitation ranged from 0.25 to 8 µg/L. Matrix effect was evaluated and matrix-matched calibration was used for quantitation. The proposed procedure was applied to 10 urine samples collected from children. Mycotoxins were quantified in 30% of samples.

Exposure assessment approach through mycotoxin/creatinine ratio evaluation in urine by GC-MS/MS.

In this pilot survey human urine samples were analyzed for presence of 15 mycotoxins and some of their metabolites using a novel urinary multi-mycotoxin GC-MS/MS method following salting-out liquid-liquid extraction. Fifty-four urine samples from children and adults residents in Valencia were analyzed for presence of urinary mycotoxin and expressed in gram of creatinine. Three out of 15 mycotoxins were detected namely, HT-2 toxin, nivalenol and deoxynivalenol (DON). 37 samples showed quantifiable values of mycotoxins. Co-occurrence of these contaminants was also observed in 20.4% of assayed samples. DON was the most frequently detected mycotoxin (68.5%) with mean levels of 23.3 µg/g creatinine (range: 2.8-69.1 µg/g creatinine). The levels of urinary DON were used to carry out an exposure assessment approach. 8.1% of total subjects were estimated to exceed the DON provisional maximum tolerable daily intake (PMTDI) (1 µg/kg b.w.). Two out of 9 exposed children exceeded the DON PMTDI thus, making them the most exposed based on the urinary results.

Quantitative determination of trichothecenes in breadsticks by gas chromatography-triple quadrupole tandem mass spectrometry.

Breadsticks are pencil-sized sticks of dry bread widely consumed as a pre-meal appetiser. They are basically wheat-based snacks, which makes them a good matrix to evaluate mycotoxin contamination, since wheat is very susceptible to fungal attack. In this sense, the fast, selective and sensitive gas chromatography-triple quadrupole tandem mass spectrometry (GC-QqQ-MS/MS) method proposed here allows simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol, fusarenon-X, diacetoxyscirpenol, nivalenol, neosolaniol, HT-2 and T-2 toxin in breadsticks after QuEChERS extraction and clean-up. The performance of the method was assessed with respect to European Commission Regulations by studying the selectivity and specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, matrix effect, accuracy, precision and trueness. Satisfactory results in terms of validation parameters were obtained for all selected mycotoxins (recovery range of 70-110%, RSD < 10%, LOQ <40 µg kg(-1)). The trueness of the method was supported by using certified reference material (DON 1062 ± 110 µg kg(-1)). The method was successfully used to evaluate the occurrence of the studied Fusarium toxins in 61 breadstick samples. A total of 64% of the samples showed mycotoxin contamination, DON being the most frequently detected toxin. Nonetheless, mean levels obtained were far below the maximum levels permitted by European Union legislation. An additional goal was to carry out a risk-characterisation approach to DON by comparing probable daily intake and provisional maximum tolerable daily intake (PMTDI).

Presence of mycotoxins in sorghum and intake estimation in Tunisia.

Sorghum samples (n = 60) from Tunisian markets were analysed for the occurrence of 22 of both traditional and emerging mycotoxins. Samples were extracted with a QuEChERS-like method and mycotoxins were detected by LC-MS/MS. This method was validated and adequate analytical parameters were obtained. All samples had contamination with mycotoxins and several samples had higher contamination levels than European Union legislative limits (MLs). The most frequently found mycotoxins were ENB (100%), OTA (98%), ENA1 (63%), ENB1 (56%), BEA (48%), AFB1 (38%) and STG (33%). Mean contaminations were 30.7, 1.93, 33.2, 51.0, 15.4, 1.49 and 20.5 µg kg(-1), respectively. While two samples were contaminated with FB2 and FB3 at mean values of 16.2 and 45.9 µg kg(-1), respectively, one sample was contaminated with AFB2 and ZEA at levels of 0.82 and 45.0 µg kg(-1), respectively. The results were used to estimate the daily intake of mycotoxins through sorghum consumption with regard to normal consumers (low-risk population) and high consumers such as babies (high-risk consumers) who are facing an alarming situation.

A survey of trichothecenes, zearalenone and patulin in milled grain-based products using GC-MS/MS.

An analytical protocol based on QuEChERS and gas chromatography-tandem mass spectrometry (GC-MS/MS) was successfully applied for the determination of trichothecenes, patulin and zearalenone in 182 milled grain-based samples. The analytical method was validated following the SANCO 1495/2011 document. LOQs were lower than 10µgkg(-1) for the selected mycotoxins. Recoveries of fortified cereals ranged between 76-108% and 77-114% at 20 and 80µgkg(-1), respectively, with relative standard deviation lower than 9%. More than 60% of the samples analysed showed deoxynivalenol contamination, followed by HT-2 toxin and nivalenol with frequencies of 12.1% and 10.4%, respectively. Co-occurrence of mycotoxins was also present in major cereals. A risk characterisation was carried out based on probable daily intake (PDI) and tolerable daily intake (TDI). Despite PDI of the average consumers were below TDI, special attention should be paid in high consumers as well as other susceptible population.

Validation of a confirmatory method for the determination of macrolides in liver and kidney animal tissues in accordance with the European Union regulation 2002/657/EC.

This study proposes a simple multiresidue liquid chromatography-diode array detector (LC-DAD) method capable of determining seven macrolide antibiotics in samples of liver and kidney animals at concentrations lower than those allowed by current legislation. Samples were prepared by homogenizing the tissue with EDTA-McIlvaine's buffer and extracted with an Oasis HLB cartridge. The consumption of organic solvent during extraction was minimum. The analytes were detected by LC-DAD and also by liquid chromatography-mass spectrometry with electrospray ionization (LC-(ESI)MS). The method was specific, stable and robust enough for the required purposes. The DAD method was validated in accordance with the European Commission Decision 657/2002. Recovery data were also satisfactory with values higher than 67% for most macrolide antibiotics extracted from liver and kidney samples spiked at 200 microg/kg, the lowest MRL established for the macrolides studied. The relative standard deviations (RSD (%), (n=3)) were lower than 13% and 15% for intra-day and inter-day assays. The method was applied to investigate the occurrence of the studied macrolides in 31 beef and kidney animal samples. The results obtained by LC-DAD for positive samples were compared to those obtained by LC-(ESI)MS. Therefore, the method with simpler instrumentation than a LC-(ESI)MS can be used as a control method and the results of the validation process demonstrate that this method is suitable for application in a European Union program for monitoring residues of veterinary drugs.

Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin.

Zearalenone (ZEA) is a mycotoxin produced mainly by fungi belonging to the genus Fusarium in foods and feeds. It is frequently implicated in reproductive disorders of farm animals and occasionally in hyperoestrogenic syndromes in humans. There is evidence that ZEA and its metabolites possess oestrogenic activity in pigs, cattle and sheep. However, ZEA is of a relatively low acute toxicity after oral or interperitoneal administration in mice, rat and pig. The biotransformation for ZEA in animals involves the formation of two metabolites alpha-zearalenol (alpha-ZEA) and beta-zearalenol (beta-ZEA) which are subsequently conjugated with glucuronic acid. Moreover, ZEA has also been shown to be hepatotoxic, haematotoxic, immunotoxic and genotoxic. The exact mechanism of ZEA toxicity is not completely established. This paper gives an overview about the acute, subacute and chronic toxicity, reproductive and developmental toxicity, carcinogenicity, genotoxicity and immunotoxicity of ZEA and its metabolites. ZEA is commonly found on several foods and feeds in the temperate regions of Europe, Africa, Asia, America and Oceania. Recent data about the worldwide contamination of foods and feeds by ZEA are considered in this review. Due to economic losses engendered by ZEA and its impact on human and animal health, several strategies for detoxifying contaminated foods and feeds have been described in the literature including physical, chemical and biological process. Dietary intakes of ZEA were reported from few countries from the world. The mean dietary intakes for ZEA have been estimated at 20 ng/kgb.w./day for Canada, Denmark and Norway and at 30 ng/kgb.w./day for the USA. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established a provisional maximum tolerable daily intake (PMTDI) for ZEA of 0.5 microg/kg of body weight.

Accelerated solvent extraction of ochratoxin a from rice samples.

In this paper, accelerated solvent extraction (ASE) for the analysis of ochratoxin A (OTA) is applied for the first time. Optimization of the method is given for the extraction of OTA from rice samples. Several parameters such as type of solvent, temperature, pressure, static time, and cell size were investigated thoroughly to find the optimal extraction conditions. The optimum ASE operating conditions were methanol as extraction solvent, 1500 psi, 40 degrees C, 5 min of static time, 50% flush volume, 60 s of purge, 1 cycle, and 11 mL cell size. The total extraction time was approximately 15 min. OTA was determined by liquid chromatography coupled with a fluorescence detector and confirmed by methyl ester derivatization. The analytical performance of the method was monitored with quality control parameters. Finally, the optimized method was used to evaluate 12 rice samples, 1 of which was positive with an OTA content of 4.17 ng/g. The proposed method offers the possibility of a fast and simple process to obtain a quantitative extraction of OTA.