Potential Health Risk Associated with Mycotoxins in Oat Grains Consumed in Spain

Spain is a relevant producer of oats (Avena sativa), but to date there has been no study on the occurrence/co-occurrence of mycotoxins in oats marketed in Spain. The present study is addressed to overcome this lack of knowledge. One hundred oat kernel samples were acquired across different Spanish geographic regions during the years 2015–2019 and analyzed for mycotoxin content using an ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC–ESI–MS/MS) method and matrix-matched calibration. The focus was on the regulated mycotoxins although other relevant mycotoxins were considered. The percentage of incidence (levels ≥ limit of detection), mean and range (ng/g) of mycotoxins were as follows: zearalenone (66%, mean 39.1, range 28.1–153), HT-2 toxin (47%, mean 37.1, range 4.98–439), deoxynivalenol, (34%, mean 81.4, range 19.1–736), fumonisin B1 (29%, mean 157.5, range 63.2–217.4), and T-2 toxin, (24%, mean 49.9, range 12.3–321). Fumonisin B2, 3-acetyldeoxynivalenol, aflatoxins B1, B2, and G2, and ochratoxin A were also detected at low levels, but aflatoxin G1 was not. The maximum limits established by the European Commission for unprocessed oats were not exceeded, except for zearalenone (in one sample), and the sum of aflatoxins (in two samples). Mycotoxin co-occurrence at quantifiable levels in the same sample (two to five combinations) was found in 31% of samples. The most common mixtures were those of HT-2 + T-2 toxins alone or together with deoxynivalenol and/or zearalenone.     

免疫亲和净化-液相色谱-串联质谱测定中成药中14个真菌毒素

目的:建立同时测定中成药中黄曲霉毒素B1、B2、G1、G2,HT-2毒素,T-2毒素,赭曲霉毒素A,伏马毒素B1、B2,玉米赤霉烯酮,α-玉米赤霉烯醇,β-玉米赤霉烯醇,α-玉米赤霉醇以及β-玉米赤霉醇共14个真菌毒素的测定方法。方法:采用PBS溶液和70%甲醇-PBS溶液依次提取样品中真菌毒素,提取液经多功能免疫亲和柱净化,0.1%吐温-PBS溶液和水依次淋洗,甲醇洗脱,氮气流下浓缩定容,测定时采用Waters Xterra C18MS(100 mm×2.1 mm,3.5μm)分析柱,0.3 mL·min-1的流速,梯度洗脱,质谱测定采用多反应监测(MRM)模式。结果:14个真菌毒素的最小检出浓度(LOQ)为1.0～5.0μg·kg-1;4个中成药基质的3个不同水平的添加回收率(n=6)为75.7%～97.9%,RSD为5.3%～13.8%。结论:本方法采用了多功能免疫亲和柱净化,质谱定性定量,加快了检测速度,降低了中成药复杂基体的干扰,方法检测限满足了国内外中成药多个真菌毒素限量控制要求。     

Lipid peroxidation and glutathione levels in porcine kidney PK15 cells after individual and combined treatment with fumonisin B(1), beauvericin and ochratoxin A

Individual and combined effects of the mycotoxins fumonisin B(1), beauvericin and ochratoxin A on cell viability, lipid peroxidation (TBARS) and intracellular glutathione (GSH) were studied on porcine kidney epithelial cells (PK15). Cells were treated with 0.05, 0.5 and 5 microg/ml of each mycotoxin or the combinations of two or all three applied in equal concentrations for 24 and 48 hr. Changes in cell viability, GSH and TBARS levels showed that the cytotoxic effects of these mycotoxins were concentration- and time-dependent. After 24 hr, cell viability was significantly decreased by the exposure to 5 microg/ml of fumonisin B(1) (25%), beauvericin (30%) and ochratoxin A (35%), as compared to controls. Only ochratoxin A (5 microg/ml) increased TBARS (56%), with further significant increase (85%) after 48 hr exposure. Fumonisin B(1) and beauvericin significantly increased TBARS (57% and 80%, respectively) only when the highest dose was applied for 48 hr. After 24 hr, GSH was significantly decreased (18%) by ochratoxin A (0.05 microg/ml), whereas fumonisin B(1) and beauvericin significantly decreased GSH at the concentration of 0.5 microg/ml. Combined treatment with fumonisin B(1), beauvericin and ochratoxin A resulted mostly in additive effects especially after a 24-hr exposure, although synergistic as well as antagonistic interactions could not be excluded depending on toxin concentrations and time of exposure. This is the first report on beauvericin-induced effects on lipid peroxidation and GSH in animal cells.     

Multiple Mycotoxins in Kenyan Rice

Multiple mycotoxins were tested in milled rice samples (n = 200) from traders at different milling points within the Mwea Irrigation Scheme in Kenya. Traders provided the names of the cultivar, village where paddy was cultivated, sampling locality, miller, and month of paddy harvest between 2018 and 2019. Aflatoxin, citrinin, fumonisin, ochratoxin A, diacetoxyscirpenol, T2, HT2, and sterigmatocystin were analyzed using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Deoxynivalenol was tested using enzyme-linked immunosorbent assay (ELISA). Mycotoxins occurred in ranges and frequencies in the following order: sterigmatocystin (0–7 ppb; 74.5%), aflatoxin (0–993 ppb; 55.5%), citrinin (0–9 ppb; 55.5%), ochratoxin A (0–110 ppb; 30%), fumonisin (0–76 ppb; 26%), diacetoxyscirpenol (0–24 ppb; 20.5%), and combined HT2 + T2 (0–62 ppb; 14.5%), and deoxynivalenol was detected in only one sample at 510 ppb. Overall, low amounts of toxins were observed in rice with a low frequency of samples above the regulatory limits for aflatoxin, 13.5%; ochratoxin A, 6%; and HT2 + T2, 0.5%. The maximum co-contamination was for 3.5% samples with six toxins in different combinations. The rice cultivar, paddy environment, time of harvest, and millers influenced the occurrence of different mycotoxins. There is a need to establish integrated approaches for the mitigation of mycotoxin accumulation in the Kenyan rice.     

Assessment of Multi-Mycotoxin Exposure in Southern Italy by Urinary Multi-Biomarker Determination

Human exposure assessment to deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone (ZEA) and ochratoxin A (OTA) can be performed by measuring their urinary biomarkers. Suitable biomarkers of exposure for these mycotoxins are DON + de-epoxydeoxynivalenol (DOM-1), aflatoxin M1 (AFM1), FB₁, ZEA + α-zearalenol (α-ZOL) + β-zearalenol (β-ZOL) and OTA, respectively. An UPLC-MS/MS multi-biomarker method was used to detect and measure incidence and levels of these biomarkers in urine samples of 52 volunteers resident in Apulia region in Southern Italy. The presence of ZEA + ZOLs, OTA, DON, FB1 and AFM1 were detected in 100%, 100%, 96%, 56% and 6%, of samples, respectively. All samples contained biomarkers of two or more mycotoxins. The mean concentrations of biomarkers ranged from 0.055 ng/mL (FB1) to 11.89 ng/mL (DON). Urinary biomarker concentrations were used to estimate human exposure to multiple mycotoxin. For OTA and DON, 94% and 40% of volunteers, respectively exceeded the tolerable daily intake (TDI) for these mycotoxins. The estimated human exposure to FB1 and ZEA was largely below the TDI for these mycotoxins for all volunteers.     

Ochratoxin A in the morning and afternoon portions of urine from Coimbra and Valencian populations.

The widespread contamination of foodstuffs and beverages by mycotoxins, such as ochratoxin A (OTA), has made the monitoring of human contamination levels essential. By using a sensitive, accurate and speedy method that combines extraction with 5% NaHCO(3), immunoaffinity column clean-up and HPLC with fluorescence detection, the human exposure to OTA through urine analysis can be monitored. This method is less invasive than blood monitoring and has the potential to be a good marker of human exposure. The limit of quantification of the method was 0.007 ng/mL of urine, with recoveries of OTA, from urine samples spiked at levels between 0.02 and 0.1 ng/mL, higher than 91% with RSD lower than 15.5%. This study evaluated OTA contamination levels in human urine sample fractions, collected in the morning and afternoon, in two populations, one from Coimbra city, in Portugal, and another from the Valencian community, in Spain. In the Coimbra population, 60 samples from 30 healthy individuals were analyzed, levels of OTA in 13 morning samples and 14 afternoon samples having been detected, with concentrations ranging from 0.011 to 0.208 and 0.008 to 0.11 ng/mL respectively. In the Valencia population, 62 samples from 31 healthy individuals were analyzed, with OTA being detected in 25 morning samples and 26 afternoon samples. The concentrations varied between 0.007 and 0.124 ng/mL in the morning samples, and 0.008 and 0.089 ng/mL in the afternoon samples. Significant differences were found between the morning levels of OTA from both populations (P=0.033). For afternoon samples, significant differences were not found, P value=0.163.     

Efficacy of a commercial mycotoxin binder in alleviating effects of ochratoxin A, fumonisin B1, moniliformin and zearalenone in adult mink

The addition of nutritionally inert adsorbents to mycotoxin-contaminated animal feed has been a popular approach to decreasing toxicity in animals and carryover of mycotoxins from contaminated feed to animal by-products. Some studies suggest that esterified glucomannan derived from the cell wall of Saccharomyces cerevisiae is effective in reducing the bioavailability of at least some of the mycotoxins occurring in contaminated feed. Because cereal grains are important components of ranch mink diets, mycotoxicoses in mink is a potential problem faced by mink ranchers. We conducted a series of studies to determine if inclusion of a commercially available esterified glucomannan in ranch mink feed was effective in alleviating clinical signs indicative of exposure to ochratoxin A, fumonisin B1, moniliformin or zearalenone in adult mink. In 4 separate trials, mink were fed diets that contained 2.5, 5 or 10 mg ochratoxin A/kg feed, 200 mg fumonisin B1/kg feed, 20 mg moniliformin/kg feed, or 30 mg zearalenone/kg feed with or without 2 g esterified glucomannan/kg feed. Male mink fed diets containing ochratoxin A had significantly decreased feed intake as well as renal lesions characteristic of exposure to that mycotoxin. Inclusion of the esterified glucomannan did not ameliorate these effects. Male mink exposed to fumonisin B1 had increased urinary sphinganine concentration, which was not significantly reduced by the mycotoxin adsorbent. Male mink that consumed monilformin-contaminated diets had characteristic ultrastructural changes in the heart that were not reduced in severity by the esterified glucomannan. Female mink exposed to zearalenone had increased uterine weight, which was not reversed by inclusion of commercial mycotoxin binder in the contaminated feed. The results of this study suggest that a commercial esterified glucomannan was generally ineffective in alleviating effects indicative of exposure to ochratoxin A, fumonisin B1, monilformin and zearalenone in mink.     

Sorption of ochratoxin A from aqueous solutions using β-cyclodextrin-polyurethane polymer

The ability of a cyclodextrin-polyurethane polymer to remove ochratoxin A from aqueous solutions was examined by batch rebinding assays. The results from the aqueous binding studies were fit to two parameter models to gain insight into the interaction of ochratoxin A with the nanosponge material. The ochratoxin A sorption data fit well to the heterogeneous Freundlich isotherm model. The polymer was less effective at binding ochratoxin A in high pH buffer (9.5) under conditions where ochratoxin A exists predominantly in the dianionic state. Batch rebinding assays in red wine indicate the polymer is able to remove significant levels of ochratoxin A from spiked solutions between 1-10 μg·L(-1). These results suggest cyclodextrin nanosponge materials are suitable to reduce levels of ochratoxin A from spiked aqueous solutions and red wine samples.     

Rapid Multiple Immunoenzyme Assay of Mycotoxins

Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin–polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.     

Ochratoxin A and some of its derivatives modulate radical formation of porcine blood monocytes and granulocytes

Modulating effects of ochratoxin A (OTA) and some of its derivatives on viability and oxidative burst activity of porcine monocytes and granulocytes have been studied. The formation of free oxygen radicals by monocytes was suppressed by OTA and ochratoxin C (OTC) at concentrations between 10 and 1000 ng/ml. Intracellular radical formation of granulocytes was in part already significantly reduced at 1 ng/ml of these mycotoxins. Conversely, the intracellular formation of radicals in monocytes of individual pigs was stimulated by the toxins at 1-100 ng/ml. A biologically active fraction of the crude toxin (RE2) which had been identified as OTC had a stronger effect than all other derivatives of ochratoxin A. Whether these modulating effects of OTA and OTC on phagocyte functions are of significance in the pathogenesis of infectious diseases, needs to be studied in more detail. In this context, the occurrence of OTC in food and feeds should be examined more closely.     

Biomonitoring of concurrent mycotoxin exposure among adults in Sweden through urinary multi-biomarker analysis

Mycotoxin producing moulds may contaminate numerous agricultural commodities either before harvest or during storage. A varied diet consisting of different foods may therefore be contaminated with a range of mycotoxins. The aim of the present study was to study concurrent exposure to mycotoxins through urinary multi-biomarker analysis, as well as its possible associations with the diet.Urinary samples from 252 adults, participating in the Swedish national dietary survey Riksmaten 2010–11, were collected together with a 4-day diet record. Concurrent mycotoxin exposure was studied using a multi-biomarker LC-MS/MS method. The results revealed that exposure to mycotoxins is common and concurrent exposure to more than one toxin was found in 69% of the study population. However, when comparing the number of toxins detected with the reported consumption data it was difficult to distinguish food patterns which would indicate an increased risk of exposure to many mycotoxins simultaneously.This is the first study to investigate concurrent mycotoxin exposure and urinary levels of fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), nivalenol (NIV), ochratoxin A (OTA), zearalenone (ZEA), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL) and de-epoxydeoxynivalenol (DOM-1) among adults in Sweden.     

Efflux at the Blood-Brain Barrier Reduces the Cerebral Exposure to Ochratoxin A,Ochratoxin α, Citrinin and Dihydrocitrinone

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations.     

Studies in the use of magnetic microspheres for immunoaffinity extraction of paralytic shellfish poisoning toxins from shellfish

Paralytic shellfish poisoning (PSP) is a potentially fatal human health condition caused by the consumption of shellfish containing high levels of PSP toxins. Toxin extraction from shellfish and from algal cultures for use as standards and analysis by alternative analytical monitoring methods to the mouse bioassay is extensive and laborious. This study investigated whether a selected MAb antibody could be coupled to a novel form of magnetic microsphere (hollow glass magnetic microspheres, brand name Ferrospheres-N) and whether these coated microspheres could be utilized in the extraction of low concentrations of the PSP toxin, STX, from potential extraction buffers and spiked mussel extracts. The feasibility of utilizing a mass of 25 mg of Ferrospheres-N, as a simple extraction procedure for STX from spiked sodium acetate buffer, spiked PBS buffer and spiked mussel extracts was determined. The effects of a range of toxin concentrations (20-300 ng/mL), incubation times and temperature on the capability of the immuno-capture of the STX from the spiked mussel extracts were investigated. Finally, the coated microspheres were tested to determine their efficiency at extracting PSP toxins from naturally contaminated mussel samples. Toxin recovery after each experiment was determined by HPLC analysis. This study on using a highly novel immunoaffinity based extraction procedure, using STX as a model, has indicated that it could be a convenient alternative to conventional extraction procedures used in toxin purification prior to sample analysis.     

Natural Co-Occurrence of Multiple Mycotoxins in Unprocessed Oats Grown in Ireland with Various Production Systems

The natural co-occurrence of 42 mycotoxins was investigated in unprocessed oat grains grown in Ireland. The sample set included a total of 208 oat crops harvested during 2015–2016 and produced using conventional, organic, or gluten free farming systems. A range of different toxins was identified, including the major type A (neosolaniol, HT-2 and T-2 toxins, T-2 triol, and T-2-glucoside, co-occurring in 21 samples) and B trichothecenes (deoxynivalenol, nivalenol, and deoxynivalenol-3-glucoside), enniatins (B1, B, and A1, co-occurring in 12 samples), as well as beauvericin, alternariol, mycophenolic acid, and sterigmatocystin. The influences of sowing season, year, and production system were investigated, eventually indicating that the latter factor may have a higher impact than others on the production of certain mycotoxins in oats. The most frequently quantified compounds were HT-2 (51%) and T-2 (41%) toxins, with gluten free oats containing significantly lower concentrations of HT-2 compared to conventionally produced oats. Although the prevalence and concentrations of mycotoxin found in oat samples in this study should be substantially reduced by processing. However, as mycotoxin occurrence is clearly influenced by multiple factors, controlled field trials should be carried out to define optimal agronomic practices and mitigate mycotoxin production. Furthermore, this work highlights the need for regularly testing cereal-based foods with multi-residue analytical methods with wider specificities than the traditionally screened and regulated toxins, to generate knowledge on the occurrence of several mycotoxins that are, to date, rarely investigated.     

An Immunoenzymatic Method for the Determination of Ochratoxin A in Biological Liquids (Colostrum and Cow’s Milk)

Ochratoxins are mycotoxins that have been extensively studied lately due to the multiple toxic effects such as nephrotoxicity, hepatotoxicity, and carcinogenicity. These toxins contaminate plant and animal foods and after ingestion they reach into body fluids. The method of competitive direct enzyme immunoassay, in the solid phase, was validated through the determination of specific parameters (performance, linearity, recovery percentage, limit of detection, limit of quantification). The validated method was used to determine ochratoxin A in colostrum and cow’s milk. The method applied for the determination of ochratoxin A was linear for the concentration range of 0.0–0.5 ng/mL, the value for the regression coefficient (r) was 0.9838. Ochratoxin A was present in 91.67% of the colostrum and in 93.33% of cow’s milk samples. The linearity of the method, demonstrated for very low concentrations of analyte, the detection limit as well as the limit of quantification recommend the method for the determinations of micro-pollutants from foods, including biological fluids.     

Effects of the mycotoxins citrinin and ochratoxin a on hepatic mixed-function oxidase and adenosinetriphosphatase in neonatal rats

The effects of citrinin, ochratoxin A, or a combination of the two mycotoxins on the hepatic monooxygenase system and on hepatic and renal adenosinetriphosphatase (ATPase) activities were examined in neonatal rats exposed to a single treatment of one or both toxins. Animals received (po) 25 mg/kg citrinin, 1 mg/kg ochratoxin A, or 25 mg/kg citrinin plus 1 mg/kg ochratoxin A within 24 h of birth. Pups were killed 12 d later. Citrinin or ochratoxin A alone did not affect hepatic ATPase. Renal oligomycin-sensitive Mg2+-ATPase was inhibited to the same degree by ochratoxin A and the combination treatment. A synergistic effect of the two mycotoxins was observed on renal Na+-K+-ATPase. Significant effects, due to the mycotoxin interaction, were also observed on cytochrome P-450 content, NADPH-dependent dehydrogenase, and NADPH-cytochrome c reductase.     

A Two-Year Occurrence of Fusarium T-2 and HT-2 Toxin in Croatian Cereals Relative of the Regional Weather

To investigate into the T-2 and HT-2 toxin occurrence, 240 samples of unprocessed cereals (maize, wheat, barley, and oats) were sampled from different fields located in three Croatian regions during 2017–2018. In all samples, sum concentrations of T-2/HT-2 toxin were determined using the ELISA method, while the LC-MS/MS was used as a confirmatory method for both mycotoxins in positive samples (>LOD) and the establishment of T-2 over HT-2 toxin ratios. The results showed oats to be the most contaminated cereal, with T-2/HT-2 toxins detected in 70.0% of samples, followed by barley (40.9%), maize (26.8%) and wheat (19.2%), with the mean T-2/HT-2 ratio ranging from 1:2.7 in maize to 1:4.4 in oats. Sum T-2/HT-2 concentrations in two maize samples were higher than the indicative level recommended by the European Commission, necessitating subsequent investigations into the conditions under which these poorly investigated mycotoxins are produced. Statistically significantly (p < 0.05) higher concentrations of T-2/HT-2 toxin were determined in oats throughout study regions as compared to those found in wheat, but not maize and barley, while the concentrations of these mycotoxins were related to the regional weather in Croatia.     

Modulating effects of fumonisin B1 and ochratoxin A on leukocytes and messenger cytokines of the human immune system

Fumonisin B1 and ochratoxin A are mycotoxins of importance to public health and agro-economics. Although much is known about their cellular toxicity and carcinogenesis in animals, there are no reports of adverse effects on immune cells (leukocytes) or on the immune modulation of the molecular messengers (cytokines) in humans. This study was designed, therefore, to determine and compare the morphological effects of fumonisin B1 and ochratoxin A on lymphocytes and neutrophils harvested from the circulation of healthy volunteer subjects and patients with oesophageal and breast carcinomas. Both fumonisin B1 and ochratoxin A reduced the number of viable lymphocytes and neutrophils harvested from the circulation of volunteer subjects carcinoma patients in a dose-dependent manner. Leukocyte secretion of cytokines on exposure to the mycotoxins was evaluated by immunocytochemical methods. Expression of granulocyte-colony stimulating factor (G-CSF), tumour necrosis factor (TNF-alpha) and chemokine (CX3CR1) receptors were determined on the circulating leukocytes and the immunolabelling visualized by brightfield-and electron-microscopy. Cytokine levels were determined in the circulation of healthy volunteer subjects and in patients with oesophageal and breast carcinomas since they reflect the status of the immune system in humans. The findings of this study on immunocytes (leukocytes) and the immune molecular messengers (cytokines) suggest that fumonisin B1 and ochratoxin A have an immuno-suppressive effect in humans, in particular patients with cancer by impairing immune surveillance.     

Synergistic effects of fumonisin B1 and ochratoxin A: are in vitro cytotoxicity data predictive of in vivo acute toxicity?

Contamination of food and feeds by mycotoxins is a major problem of human and animals health concern which is also extremely detrimental to economy. Mycotoxins producing moulds may produce a diversity of toxins such as aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins, tremorgenic toxins and ergot alkaloids. Although toxicological, environmental and epidemiological studies have addressed the problem of these toxins one by one, more than one mycotoxin are found usually in the same contaminated commodities. That rises the incommensurable problem of multi-toxicosis in which the respective metabolites are also involved. These mycotoxins bear potential toxicity leading to acute and chronic effects in humans and animals, depending on species. The mechanisms that lead to toxic effects, such as immune toxicity, and carcinogenicity are complexe. The risk assessment for humans potentially exposed to multi-mycotoxins suffers very much from the lack of adequate food consumption data. Furthermore, for a given mycotoxin synergism and antagonism with other mycotoxins found in the same food commodities are not taken into account. The case of combination of ochratoxin A (OTA) and fumonisin B1 (FB1) has been addressed in the present paper with the purpose of predicting the in vivo toxicity using a simple in vitro test, i.e. neutral red uptake, in three different cell-lines, C6 glioma cells, Caco-2 cells and Vero cells. Using the equation of [ATLA 27 (1999) 957], in vivo toxicity (LD50) is in adequation with the in vitro data, (IC50 values) for both toxins as well as for the combination of 10 microM OTA and variable concentrations of FB1 (10-50 microM). A synergistic effect is prouved in vitro that is in line with some in vivo data from the literature. Such simple in vitro test may thus help predicting in vivo toxicity of combinations of mycotoxins naturally occurring in foodstuffs.     

Mould contamination and co-occurrence of mycotoxins in maize grain in Croatia

Maize grain samples (n=15) collected during the autumn of 2002 were analyzed for the presence of moulds and mycotoxins fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA), and ochratoxin A (OTA). Mycological analysis showed that all samples were contaminated with Fusarium spp. and Penicillium spp., while Aspergillus spp. were found in 5 samples. F. proliferatum and F. verticilloides, the producers of fumonisins, were found in 14 and 8 samples, respectively, while F. graminearum, the producer of ZEA, was present in all samples. The most frequent mycotoxins were FB1 (15/15) and ZEA (12/15), followed by OTA (7/15), while FB2 was found in only two samples. Seven samples were contaminated with two mycotoxins, seven with three, and one sample with only one mycotoxin. The concentrations (mean+/-SD) of FB1, ZEA, and OTA in positive samples were 459.5+/-314.6, 1.70+/-0.80, and 1.40+/-0.55 microg kg-1, respectively, and the concentrations of FB2 in two samples were 68.4 and 3084.0 microg kg-1. In general, such low mycotoxin concentrations are not a significant source of exposure to humans, but they may contribute to exposure from other commodities. A few samples with extreme values indicate that strict control is needed.