Preliminary survey of the occurrence of mycotoxins in cereals and estimated exposure in a northwestern region of Mexico.

Mycotoxins have several toxicological implications. In the present study, we evaluate the presence of aflatoxin B1 (AFB1), ochratoxin A (OTA), and fumonisin (FB1) in paddy rice, polished rice, and maize from the fields and markets in Nayarit State (Mexico). The results indicated the presence of AFB1 in 21.21% of paddy rice samples and 11.11% of market maize samples. OTA was present in only 3.03% (one sample) of paddy rice samples. FB1 was detected in 87.50% and 88.88% of maize samples from field and market, respectively. The estimated human exposure was calculated for FB1 using the probable daily intake (PDI), which suggested that FB1 could contribute to the development of diseases through the consumption of contaminated maize. Positive samples indicated that some rice and maize samples were not suitable for human consumption. Further efforts are needed to continue monitoring mycotoxins and update national legislation on mycotoxins accordingly.

Mycotoxins occurrence and fungal populations in different types of silages for dairy cows in Spain.

Silages constitute a major component of the feed ration for dairy cows, being a potential source of mycotoxins due to the possible contamination by filamentous fungi capable of producing these toxic compounds. In this study, samples of different kinds of silages collected from farms located in four regions of Spain, were analysed to evaluate the occurrence of aflatoxins (AFs) and Fusarium mycotoxins. Lactic acid bacteria and fungal populations as well as pH and water activity were also studied. Penicillium, Geotrichum and Monascus were the main fungi identified in all the silages examined. The incidence of AFs was low (10 % of positive samples). Fusarium mycotoxins were detected in 40 % of the samples and fumonisins (FBs) were the most commonly detected. Maize silage was the most heavily contaminated type of silage. Levels of mycotoxins detected in positive samples did not exceed the EU guidance values. The lack of relationship between Fusarium counts and its mycotoxin concentrations suggested that mycotoxin production possibly occurred pre-ensiling or immediately post-ensiling. Outcomes showed that mould growth and mycotoxin contamination in silages should be regularly monitored in order to minimize the exposure of dairy cows to contaminated feed.

Usefulness of the analytical control of aflatoxins in feedstuffs for dairy cows for the prevention of aflatoxin M1 in milk.

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1) and can be excreted in milk of cows after consuming aflatoxin (AF)-contaminated feed. The aim of this research was to assess the levels of total AFs in samples of feedingstuff for dairy cows (n = 193) and the levels of AFM1 in raw bulk tank milk samples (n = 375), in order to estimate the ratio of "AFB1 feed input" versus "AFM1 milk output" in four specific regions of Spain. Moreover, the correlation between the raw materials used as ingredients of the total mixed ration (TMR) and the presence of AFs was studied. About one-third (34.7%) of the feed samples were positive for total AFs in a range of 0.05-6.45 µg/kg, and 12.4% were positive for AFB1. AFM1 was detected in 18.9% of bulk milk samples, with concentrations ranging from 0.009 to 1.36 µg/kg. While none of the feed samples exceeded the European Union (EU) maximum content for AFB1 in feedingstuff for dairy animals of 5 µg/kg, three bulk milk samples exceeded the EU maximum level for AFM1 in milk of 50 ng/kg. The transfer ratio AFB1/AFM1, which was derived from AFB1 levels in feed, AFM1 levels in bulk tank milk, feed intake, and milk yield data, was 0.6-6%, which corresponded well with the range of published carry-over data for aflatoxins. Statistical analyses showed that the main sources of AFB1 in TMR were maize silage, bagasse, soya bean husk, maize, alfalfa hay, cotton seed and compound feed, thus special attention should be paid in controlling these raw materials when used in TMR preparation. Although the analysis of AFs in feed did not correlate with the presence of AFM1 in milk, monitoring feedstuffs is a useful tool in order to try and minimise AF-contamination of milk.

A review of the mycotoxin adsorbing agents, with an emphasis on their multi-binding capacity, for animal feed decontamination.

Contamination of animal feed with mycotoxins still occurs very often, despite great efforts in preventing it. Animal feeds are contaminated, at low levels, with several mycotoxins, particularly with those produced by Aspergillus and Fusarium genera (Aflatoxin B1, Ochratoxin A, Zearalenone, Deoxynivalenol and Fumonisina B1). In animal feed, to date, only Aflatoxin B1 is limited through EU regulation. Consequently, mycotoxins cause serious disorders and diseases in farm animals. In 2009, the European Union (386/2009/EC) approved the use of mycotoxin-detoxifying agents, as feed additives, to prevent mycotoxicoses in farm animals. The present review gives an overview of the problem of multi-mycotoxin contamination of feed, and aims to classify mycotoxin adsorbing agents (minerals, organic, and synthetic) for feed decontamination, focusing on adsorbents with the ability to bind to multiple mycotoxins, which should have a more effective application in farms but they are still little studied in scientific literature.

Exploring polyamine metabolism of Alternaria alternata to target new substances to control the fungal infection.

Polyamines are essential for all living organisms as they are involved in several vital cell functions. The biosynthetic pathway of polyamines and its regulation is well established and, in this sense, the ornithine descarboxylase (ODC) enzyme acts as one of the controlling factors of the entire pathway. In this work we assessed the inhibition of the ODC with D, l-α-difluoromethylornithine (DFMO) on Alternaria alternata and we observed that fungal growth and mycotoxin production were reduced. This inhibition was not completely restored by the addition of exogenous putrescine. Actually, increasing concentrations of putrescine on the growth media negatively affected mycotoxin production, which was corroborated by the downregulation of pksJ and altR, both genes involved in mycotoxin biosynthesis. We also studied the polyamine metabolism of A. alternata with the goal of finding new targets that compromise its growth and its mycotoxin production capacity. In this sense, we tested two different polyamine analogs, AMXT-2455 and AMXT-3016, and we observed that they partially controlled A. alternata's viability in vitro and in vivo using tomato plants. Finding strategies to design new fungicide substances is becoming a matter of interest as resistance problems are emerging.

LaeA and VeA are involved in growth morphology, asexual development, and mycotoxin production in Alternaria alternata.

Alternaria alternata is a common filamentous fungus that contaminates various fruits, grains and vegetables causing important economic losses to farmers and the food industry. A. alternata is a mycotoxigenic mould, which may jeopardize human and animal health. Two of the most common A. alternata mycotoxins found in food and feed are alternariol and alternariol monomethyl ether. In this study we examined the role of LaeA and VeA, two regulatory proteins belonging to the velvet family, which have been described to be involved in several functions in many fungi including secondary metabolism. We found that deletion of laeA and veA genes, respectively, greatly reduced sporulation and strongly compromised mycotoxin production, both in vitro or during pathogenesis of tomato fruits. We have also studied how the loss of laeA and veA may affect expression of genes related to alternariol and alternariol monomethyl ether biosynthesis (pksJ and altR), and to melanin biosynthesis (cmrA, pksA).

The effect of enhanced carotenoid content of transgenic maize grain on fungal colonization and mycotoxin content.

Novel strategies that address vitamin A deficiency have been developed, such as high-carotenoid maize, a biofortified transgenic maize line rich in carotenoids generated by genetic transformation. The South African white maize inbred (M37W), which is devoid of carotenoids, was engineered to accumulate high levels of ß-carotene (provitamin A), lutein, and zeaxanthin. Maize seeds contaminated with fumonisins and other mycotoxins pose a serious threat to both humans and livestock. During three consecutive harvests, the fungal incidence and the fumonisin and aflatoxin content of maize seeds grown in an experimental field in Catalonia (Northeastern Spain) were evaluated. Fungal infection was similar in high-carotenoid maize and its isogenic line, with Fusarium verticillioides being the most prevalent fungus in all the harvests. Neither Aspergillus spp. nor aflatoxin contamination was found. Fumonisin levels were lower in high carotenoid than in its isogenic line, but this reduction was statistically significant in only 2 of the 3 years of study. Our results suggest that high carotenoid content reduces fumonisin levels in maize grains.

Effect of 1-methylcyclopropene on the development of black mold disease and its potential effect on alternariol and alternariol monomethyl ether biosynthesis on tomatoes infected with Alternaria alternata.

Ethylene is a naturally produced plant regulator involved in several plant functions, such as regulation of fruit ripening. Inhibition of ethylene perception by using 1-methylcyclopropene (1-MCP) slows down the ripening of the fruit maintaining its quality and freshness. The use of 1-MCP is a commercial strategy commonly used in the food industry to extend the postharvest life of several fruits, including tomatoes. To assess how 1-MCP affected infection by Alternaria alternata on tomatoes, three different cultivars were artificially inoculated with 5µL of an A. alternata conidial suspension (10(5)conidia/mL). Tomatoes were treated with 0.6µL/L of 1-MCP for 24h. Spiked but untreated tomatoes were considered controls. Then, fruit were stored 6days at 10°C and one more week at 20°C to simulate shelf-life. Fungal growth development and mycotoxin production (alternariol, AOH and alternariol monomethyl ether, AME) were assessed both on the first and on the second week. After the first 6days at 10°C, in just one variety the black mold disease was higher in the 1-MCP treated samples. However, after two weeks of storage, in all cases, tomatoes treated with 1-MCP showed more significant fungal growth disease. Regarding mycotoxin production, no large differences were observed among different treatments, which was corroborated with gene expression analysis of pksJ, a gene related to AOH and AME biosynthesis.

Modulation of the xenobiotic transformation system and inflammatory response by ochratoxin A exposure using a co-culture system of Caco-2 and HepG2 cells.

Cytotoxicity of ochratoxin A (OTA) was evaluated using the MTS assay, and membrane integrity was measured using transepithelial electrical resistance (TEER). A transwell system was used to investigate the effect of OTA on the expression of the CYP450 (1A1, 2A6, 2B6, 3A4 and 3A5), NAT2, COX-2, LOX-5, and MRP2 genes in Caco-2 and HepG2 cells. TEER decreased by a mean of 63.2% after 24 h in Caco-2 differentiated cells without inducing cell detachment; revealing damage to the intestinal epithelial cell tight junction proteins and an increase in cell permeability. Gene expression analysis showed that modulation of gene expression by OTA was higher in Caco-2 cells than in HepG2 cells, and generally, the duration of exposure to OTA had a more significant effect than the OTA dose. A general OTA down-regulation effect was observed in Caco-2 cells, in contrast with the down- and up-regulation observed in HepG2 cells. In Caco-2 cells, CYP1A1 was the gene with the highest regulation, followed by CYP3A4 and CYP3A5. Conversely, in HepG2 cells, CYP2B6 was highly regulated at 3 and 12 h compared to the other cytochromes; CYP1A1 was slightly modulated during the first 12 h, but an overexpression was observed at 24 h. Our data support the involvement of the COX-2 and 5-LOX genes in liver metabolism of OTA. On the basis of the gene expression analysis, the results suggest a possible impairment in OTA secretion at the intestinal and hepatic level due to MRP2 repression. In addition, we provide evidence of the effect of OTA on NAT2 gene expression, which had not been reported before.

Cytotoxicity of the mycotoxins deoxynivalenol and ochratoxin A on Caco-2 cell line in presence of resveratrol.

Exposure to mycotoxins through dietary food intake involves a highly complex scenario where co-contamination of different mycotoxins has been frequently demonstrated. On the other hand, the effect of the interaction of mycotoxins with other generally considered beneficial food components, as the antioxidants, has been scarcely studied. The main goal of the present work was to assess the cytotoxic effects on Caco-2 cells of the mycotoxins deoxynivalenol (DON) and ochratoxin A (OTA), alone or combined, and to explore potential protective effects of resveratrol (RES), an antioxidant frequently found in wine. In parallel, reactive oxygen species (ROS) production has also been studied as a first approach to understand the underlying mechanism of cytotoxicity. Results indicate a higher toxic effect of the mycotoxins when they are co-exposed. This increase in cytotoxicity was not accompanied by an increase in ROS production. The co-exposure of OTA or DON with RES did not result in a decrease in cytotoxicity; on the contrary, it resulted in increased cytotoxicity not associated with an increase in ROS production.

Targeting Fusarium graminearum control via polyamine enzyme inhibitors and polyamine analogs.

Fusarium graminearum not only reduces yield and seed quality but also constitutes a risk to public or animal health owing to its ability to contaminate grains with mycotoxins. Resistance problems are emerging and control strategies based on new targets are needed. Polyamines have a key role in growth, development and differentiation. In this work, the possibility of using polyamine metabolism as a target to control F. graminearum has been assessed. It was found that putrescine induces mycotoxin production, correlating with an over expression of TRI5 and TRI6 genes. In addition, a homolog of the Saccharomyces cerevisiae TPO4 involved in putrescine excretion was up-regulated as putrescine concentration increased while DUR3 and SAM3 homologues, involved in putrescine uptake, were down-regulated. When 2.5 mM D, l-α-difluoromethylornithine (DFMO) was added to the medium, DON production decreased from 3.2 to 0.06 ng/mm(2) of colony and growth was lowered by up to 70 per cent. However, exogenous putrescine could overcome DFMO effects. Five polyamine transport inhibitors were also tested against F. graminearum. AMXT-1505 was able to completely inhibit in vitro growth and DON production. Additionally, AMXT-1505 blocked F. graminearum growth in inoculated wheat spikes reducing DON mycotoxin contamination from 76.87 µg/g to 0.62 µg/g.

The fate of deoxynivalenol and ochratoxin A during the breadmaking process, effects of sourdough use and bran content.

Deoxynivalenol (DON) and ochratoxin A (OTA) are mycotoxins produced by fungal species which can contaminate, alone or simultaneously, cereal-based products such as bread. Due to the increasing interest in the beneficial effects of dietary bran, bran bread has attained high consumption. Usually, the higher mycotoxin concentrations in cereals are found in the external layers of the grain (bran), leading to higher concentration of DON and OTA in breads with added bran. Moreover, the use of sourdough in breadmaking is increasing, but no studies about its effect in the mycotoxins content exist. The objective of this study was to determine the variation of concentration of these mycotoxins during the breadmaking process including the following factors: two initial mycotoxin concentrations in the initial mix of ingredients, four different bran contents, and use of sourdough. OTA was confirmed to be quite stable during the breadmaking process, regardless of the assayed factors. DON concentration during breadmaking was not significantly affected by bran content of bread. However, it was significantly affected by kneading and fermentation steps in opposite way depending on sourdough use and flour contamination level: if DON reduction occurs during fermentation, this leads to a safer situation, but the possible increase in DON should be considered with care, as it can compensate the expected dilution effect by recipe. Finally, the results on deoxynivalenol-3-glucoside (DON-3-G), although preliminar, suggest an increase of this toxin during fermentation, but mainly during baking.

Ecophysiological characterization of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger isolated from grapes in Spanish vineyards.

The aim of this study was to evaluate the diversity of black aspergilli isolated from berries from different agroclimatic regions of Spain. Growth characterization (in terms of temperature and water activity requirements) of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger was carried out on synthetic grape medium. A. tubingensis and A. niger showed higher maximum temperatures for growth (>45 °C versus 40-42 °C), and lower minimum aw requirements (0.83 aw versus 0.87 aw) than A. carbonarius. No differences in growth boundaries due to their geographical origin were found within A. niger aggregate isolates. Conversely, A. carbonarius isolates from the hotter and drier region grew and produced OTA at lower aw than other isolates. However, little genetic diversity in A. carbonarius was observed for the microsatellites tested and the same sequence of ß-tubulin gene was observed; therefore intraspecific variability did not correlate with the geographical origin of the isolates or with their ability to produce OTA. Climatic change prediction points to drier and hotter climatic scenarios where A. tubingensis and A. niger could be even more prevalent over A. carbonarius, since they are better adapted to extreme high temperature and drier conditions.

VeA and LaeA transcriptional factors regulate ochratoxin A biosynthesis in Aspergillus carbonarius.

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, teratogenic and immunotoxic properties which represents a serious risk for human and animal health. Aspergillus carbonarius is considered the main OTA-producing species in grapes and products such as raisins, wine or juices, although it has also been isolated from coffee, cocoa and cereals. Till now not much information is available about regulatory mechanisms of OTA production by A. carbonarius. A better understanding of how environmental factors influence OTA production and which genes are involved in its regulation could help us design new control strategies. In this study, we have evaluated the role of VeA and LaeA transcriptional factors, which have been shown to regulate secondary metabolism in response to light in A. carbonarius. To this aim, veA and laeA genes were deleted in an ochratoxigenic A. carbonarius strain by targeted gene replacement using Agrobacterium tumefaciens-mediated transformation. Loss of veA and laeA in A. carbonarius yields to an organism with slight differences in vegetative growth but a strong reduction in conidial production. A drastic decrease of OTA production that ranged from 68.5 to 99.4% in ΔveA and ΔlaeA null mutants was also observed, which was correlated with a downregulation of a nonribosomal peptide synthetase involved in OTA biosynthesis. These findings suggest that VeA and LaeA have an important role regulating conidiation and OTA biosynthesis in response to light in A. carbonarius in a similar way to other fungi where functions of VeA and LaeA have been previously described. This is the first report of a transcriptional factor governing the production of OTA by A. carbonarius.

Mycotoxins: occurrence, toxicology, and exposure assessment.

Mycotoxins are abiotic hazards produced by certain fungi that can grow on a variety of crops. Consequently, their prevalence in plant raw materials may be relatively high. The concentration of mycotoxins in finished products is usually lower than in raw materials. In this review, occurrence and toxicology of the main mycotoxins are summarised. Furthermore, methodological approaches for exposure assessment are described. Existing exposure assessments, both through contamination and consumption data and biomarkers of exposure, for the main mycotoxins are also discussed.

Occurrence and exposure assessment of aflatoxins in Catalonia (Spain).

The main objective of this study was to assess the exposure of Catalonian (Spain) population to AFs. Thus, two sub-objectives were considered: (1) to assess the occurrence of AFs in food marketed in Catalonia, and (2) to assess the consumption of those foods susceptible to AFs contamination by Catalonian population. AFs were analysed in a total of 603 samples considering special commodities as free-gluten, ethnic or baby foods. Analytical method consisted of an extraction and clean-up of aflatoxins step using immunoaffinity columns, and determination by HPLC with post-column photochemical derivatization and fluorescence detection. Food dietary intake was assessed using a food frequency questionnaire, administered to 1387 individuals by trained interviewers. Contamination and consumption raw datasets were combined by means of a direct method and a stochastic method, building the pseudo-parametric bootstrap confidence intervals of the main outputs. Margins of exposure (MoE) and cancer incidence were estimated for the different collectives. The highest percentages of positive samples were found in red pepper, pistachios and peanuts. Considering our results, the most exposed group was the celiac sufferer collective followed by the adolescents; however health concern should not be expected in the population groups.

Modelling Aspergillus flavus growth and aflatoxins production in pistachio nuts.

Aflatoxins (AFs) are the main contaminants in pistachio nuts. AFs production in pistachio has been attributed to Aspergillus flavus. The aim of this study was to apply existing models to predict growth and AFs production by an A. flavus isolated from pistachios as a function of moisture content and storage temperature of pistachios in order to test their usefulness and complementarities. A full factorial design was used: the moisture content levels assayed were 10, 15, 20, 25 and 30% and incubation temperatures were 10, 15, 20, 25, 30, 37 and 42 °C. Both kinetic and probability models were built to predict growth of the strain under the assayed conditions. Among the assayed models, cardinal ones gave a good quality fit for radial growth rate data. Moreover, the progressive approach, which was developed based on a reduced number of experimental points led to an improved prediction in the validation step. This is quite significant as may allow for improved experimental designs, less costly than full factorial ones. Probability model proved to be concordant in 91% of the calibration set observations. Even though the validation set included conditions around the growth/no-growth interface, there was a 100% agreement in the predictions from the data set (n = 16, cut off = 0.5) after 60 days. Similarly, the probability for AF presence was rightly predicted in 89% of the cases. According to our results EC maximum aflatoxin levels would be surpassed in a period as short as 1 month if pistachio nuts reach 20 °C, unless %mc is ≤10%.

Exposure assessment to ochratoxin A in Catalonia (Spain) based on the consumption of cereals, nuts, coffee, wine, and beer.

Ochratoxin A (OTA) was analysed in composite samples of cereal-based baby foods, beer, breakfast cereals (corn- and rice and wheat-based), loaf bread, peanuts and pistachios. Foodstuffs were collected in hypermarkets and supermarkets from 12 cities in the Spanish region of Catalonia, and composite samples were prepared for analysis involving liquid-liquid extraction, followed by immunoaffinity column clean-up and HPLC with fluorescence detection. Consumption data for the selected foodstuffs were collected by means of a food-frequency questionnaire. The studied population was grouped by age in infants, children, adolescents and adults; and exposure to OTA through the specified foodstuffs, and through wine and coffee, was assessed. Exposure assessment was done through deterministic and probabilistic modelling of the contamination and consumption data. OTA occurrence and mean of positive samples (ng g(-1) or ng ml(-1), for beer) were the following: 8.7% and 0.233 in baby foods; 88.7% and 0.022 in beer; 2.8% and 0.728 in corn-based breakfast cereals; 25% and 0.293 in wheat-based breakfast cereals; 12.9% and 0.283 in loaf bread; 41.7% and 0.241 in peanuts; and 2.9% and 0.228 in pistachios. The median estimated daily intake of OTA through the foodstuffs by each age group were below the latest provisional tolerable daily intakes (PTDIs) of 17 and 14 ng kg(-1) bw day(-1) recommended by the European Food Safety Authority (EFSA) in 2006 and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2007, respectively, ranging from 1% and 2% of those values in adolescents and children, to 3% and 11% in adults and infants.

Temperature-dependent production of various PlcR-controlled virulence factors in Bacillus weihenstephanensis strain KBAB4.

The Bacillus cereus sensu lato complex has recently been divided into several phylogenetic groups with clear differences in growth temperature range. However, only a few studies have investigated the actual pathogenic potential of the psychrotolerant strains of the B. cereus group at low temperature, and little information is available concerning gene expression at low temperature. We found that vegetative cells of the psychrotolerant B. weihenstephanensis strain KBAB4 were pathogenic against the model insect Galleria mellonella at 15°C but not at 30°C. A similar temperature-dependent difference also was observed for the supernatant, which was cytotoxic to Vero epithelial cell lines and to murine macrophage J774 cells at 15°C but not at 30°C. We therefore determined the effect of low temperature on the production of various proteins putatively involved in virulence using two-dimensional protein gel electrophoresis, and we showed that the production of the Hbl enterotoxin and of two proteases, NprB and NprP2, was greater at a growth temperature of 15°C than at 30°C. The quantification of the mRNA levels for these virulence genes by real-time quantitative PCR at both temperatures showed that there was also more mRNA present at 15°C than at 30°C. We also found that at 15°C, hbl mRNA levels were maximal in the mid- to late exponential growth phase. In conclusion, we found that the higher virulence of the B. cereus KBAB4 strain at low temperature was accompanied by higher levels of the production of various known PlcR-controlled virulence factors and by a higher transcriptional activity of the corresponding genes.

Exposure assessment of T2 and HT2 toxins in Catalonia (Spain).

HT-2 toxin (HT2) and T-2 toxin (T2) are mycotoxins produced in several cereals by species of Fusarium. The aim of the present study was to assess the exposure of Catalonian population to the combined mycotoxins T2 and HT2. Three different approaches were considered to handle the left censored data: (1) a substitution method, (2) a parametric method using the maximum likelihood estimation (MLE) and (3) a non-parametric method using the Kaplan-Meier (KM) estimator. Raw contamination data was provided by a large survey conducted in this region, in addition to the raw consumption data from a nutritional study specifically designed to assess the dietary intake of the main foodstuffs related to T2 and HT2 contamination for all population age groups. Contamination and consumption data were combined by simulation using a stochastic method. Accuracy and reliability of the statistic estimates were assessed building the related confidence intervals using a pseudo-parametric bootstrap method. Considering the results from the present study, the most exposed group population to the toxin T2 and HT2 should be expected to be the children.