Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA.

At the receptor level, OTA (within a concentration range of 0.25–2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect.

At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1–1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117 ± 14 ng/ml) over 3 times that of the solvent control (36 ± 9 pg/ml). Western blotting confirmed an increase in aromatase protein.

Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.

Cytotoxic assessment of the regulated, co-existing mycotoxins aflatoxin B1, fumonisin B1 and ochratoxin, in single, binary and tertiary mixtures

Aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) are contaminants which have been shown to regularly co-occur in a range of foods. However, only a small number of studies have evaluated the interactive effect of binary and tertiary mycotoxins. The present study evaluated the effects of low levels of each mycotoxin in combination at their EU regulatory limits. Toxic effect with respect to cell viability was measured by MTT and neutral red assays, assessing mitochondria and lysosome integrities respectively. Individual toxicity showed that OTA (10 μg/ml) was the most cytotoxic mycotoxin in all three cell lines studied (caco-2, MDBK and raw 264.7). Binary combinations were cytotoxic to the MDBK cell line in the order [OTA/FB1] > [AFB1/FB1] > [AFB1/OTA], whilst all effects observed were classified as being additive. Tertiary combinations of AFB1, FB1 and OTA at the EU regulatory limits were tested and not found to exhibit measurable cytotoxicity in MDBK, caco-2 or raw 264.7 cells. However by increasing these concentrations above the legal limits to OTA (3 μg/ml), FB1 (8 μg/ml) and AFB1 (1.28 μg/ml), cytotoxicity was observed with up to 26% reduction in cell viability and synergistic effects were evident with regard to mitochondrial integrity. © 2014 Elsevier Ltd. All rights reserved.

Determination of ochratoxin A in bread: evaluation of microwave-assisted extraction using an orthogonal composite design coupled with response surface methodology

An analytical method using microwave-assisted extraction (MAE) and liquid chromatography (LC) with fluorescence detection (FD) for the determination of ochratoxin A (OTA) in bread samples is described. A 24 orthogonal composite design coupled with response surface methodology was used to study the influence of MAE parameters (extraction time, temperature, solvent volume, and stirring speed) in order to maximize OTA recovery. The optimized MAE conditions were the following: 25 mL of acetonitrile, 10 min of extraction, at 80 °C, and maximum stirring speed. Validation of the overall methodology was performed by spiking assays at five levels (0.1–3.00 ng/g). The quantification limit was 0.005 ng/g. The established method was then applied to 64 bread samples (wheat, maize, and wheat/maize bread) collected in Oporto region (Northern Portugal). OTAwas detected in 84 % of the samples with a maximum value of 2.87 ng/g below the European maximum limit established for OTA in cereal products of 3 ng/g.

Occupational exposure to aflatoxin B1 and ochratoxin A: co-exposure in swine production

The most common scenario in occupational settings is the co-exposure to several risk factors. This aspect has to be considered in the risk assessment process because can alter the toxicity and the health effects when dealing with a co-exposure to two or more chemical agents. A study was developed aiming to elucidate if there is occupational co-exposure to aflatoxin B1 (AFB1) and ochratoxin (OTA) in Portuguese swine production. To assess occupational exposure to both mycotoxins, a biomarker of internal dose was used. The same blood samples from workers of seven swine farms and controls were consider to measure AFB1 and OTA. Twenty one workers (75%) showed detectable levels of AFB1 with values ranging from <1 ng/ml to 8.94 ng/ml and with significantly higher concentration when compared with controls. In the case of OTA, there wasn't found a statistical difference between workers and controls and the values for workers group ranged from 0.34 ng/ml to 3.12 ng/ml and 1.76 ng/ml to 3.42 ng/ml for control group. The results suggest that occupational exposure to AFB1 occurs. However, in the case of OTA results, seems that food consumption plays an important role in both groups exposure. The results claim attention for the possible implications on health of this co-exposure.

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.

The effects of ochratoxin/aluminosilicate interaction on the tissues and humoral immune response of broilers

This study aimed to evaluate the effect of dietary ochratoxin, in the presence or absence of aluminosilicate, on the histology of the bursa of Fabricius, liver and kidneys, and on the humoral immune response of broilers vaccinated against Newcastle disease virus. The exposure of birds to 2 p. p. m. ochratoxin, in the presence or absence of aluminosilicate, reduced their humoral immune response and the number of mitotic cells in the bursa. The relative weight of the livers of the birds exposed to this toxin was increased and, microscopically, there was hepatocyte vacuolation and megalocytosis with accompanying hyperplasia of the biliary epithelium. The kidneys showed hypertrophy of the renal proximal tubular epithelium, with thickening of the glomerular basement membrane. Aluminosilicate did not ameliorate the deleterious effects of the ochratoxin.

Synthesis of the stationary phase IS-Anionic (internal surface-anionic) for extraction of ochratoxin A and B from samples of beers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of hydrated sodium calcium aluminosilicate on the prevention of the toxic effects of ochratoxin

We studied the efficacy of hydrated sodium calcium aluminosilicate (HSCAS) as an inhibitor of the toxic effects of ochratoxin in broilers from 1 to 42 d of age. A total of 288 broilers was distributed into four treatments with four replicates of 18 birds each: T1, control; T2, 0.25% HSCAS; T3, 2 ppm of ochratoxin; T4, 0.25% HSCAS + 2 ppm ochratoxin. The parameters evaluated were feed intake; weight gain; feed conversion; relative weights of the liver, kidneys, and bursa; and serum levels of Ca, P, total protein (TP), aspartate aminotransferase (AST) and γ-glutamiltransferase (GGT). Ochratoxin in the diet negatively affected (P < 0.05) all performance parameters evaluated when the birds were 21 and 42 d of age. However, HSCAS did not affect performance, and there was no interaction between HSCAS and dietary ochratoxin. The liver and the kidneys of birds fed ochratoxin with or without HSCAS were relatively heavier (P < 0.05) than those of the control birds, demonstrating the influence of ochratoxin, but not of HSCAS, on the relative weight of these organs. Although the bursa of birds exposed to ochratoxin with or without HSCAS had a lower relative weight as compared to control birds, the difference was not significant. Ca, P, and TP serum levels were lower (P < 0.05) in birds exposed to ochratoxin, whereas AST and GGT levels were higher (P < 0.05) in these birds. These results reflect that ochratoxin in the diet impaired the productivity indexes and that HSCAS did not improve these parameters.

Ochratoxin A in wines from 2002 to 2008 harvest marketed in Rio de Janeiro, Brazil

The occurrence of ochratoxin A (OTA) in wine from 2002 to 2008 harvest, traded in Rio de Janeiro State, was evaluated by analysing 43 national and 37 imported wines from Argentina (32) and Chile (5), adding up to 80 samples in total. OTA determination was performed using immunoaffinity columns and high-performance liquid chromatography. In 80 wine samples analysed, 25 (31.3%) were positive, presenting levels greater than 0.020 ng OTA mL(-1). It was not detected in imported wines. Within national wines, 58.1% of the samples were contaminated, with levels ranging from 0.020 to 0.050 ng mL(-1). The toxin was detected in 18 (69.2%) of 26 samples analysed of red table wine. Wines from 2008 harvest presented 84.6% of samples contaminated in 13 samples analysed. Despite the levels found in this study, they are below Brazilian tolerance limits. Nevertheless, the presence of OTA as found contributes to the human exposure to this toxin.

Effect of commercial starter cultures and native yeasts on ochratoxin a production by aspergillus westerdijkiae and penicillium nordicum in meat products

Processed meat products are of worldwide importance and, because of their intrinsic factors as well as the processing methods, they are highly prone to fungal and mycotoxin contamination. Ochratoxin A (OTA) is the most significant mycotoxin in processed meat products. Penicillium nordicum is considered to be responsible for OTA contamination of meat products, as it is highly adapted to salt and protein-rich matrices and is moderately psycrotrophic. However, another OTA-producing fungus, Aspergillus westerdijkiae, adapted to carbon-rich matrices such as cereals and coffee beans, has been recently associated with high levels of OTA in meat products. Several Lactic Acid Bacteria (LAB) and yeasts have been tested as biocontrol agents against P. nordicum growth and OTA production in meat products, with promising results, but none of the studies have considered A. westerdijkiae. The aim of this work was to evaluate in vitro the effect of a commercial starter culture used in sausage fermentation and four yeasts isolated from dry-cured sausage on these two OTA-producing fungi, both in terms of fungal growth and of OTA production, using different meat-based culture media as model systems. The mechanisms underlying the observed effect were also studied. For this purpose, C. krusei, C. zeylanoides, R. mucilaginosa, R. glutinis, a mix of these yeasts and the starter culture were co-inoculated with P. nordicum and A. westerdijkiae in industrial sausage, traditional sausage, and ham-based media, under conditions of water activity, salt concentration and temperature that mimic real conditions at beginning and end of sausage curing process. Fungal growth was determined by measuring colony diameter, and OTA production was quantified by HPLC-FLD after extraction with methanol. Yeasts where found to inhibit significantly the growth of both fungi. P. nordicum was unable to produce detectable OTA in both sausage-based media under any condition. In ham, yeasts reduced OTA production, while the starter culture significantly increased it. Unexpectedly, OTA production by A. westerdijkiae was significantly stimulated in all media tested by all microorganisms. Matrix has a significant effect on OTA production by P. nordicum, but not by A. westerdijkiae, for which only temperature showed to have effect. By testing the mechanisms of action by which starter culture and C. zeylanoides influenced fungal responses, we were able to determine that direct contact and simultaneous growth of test organisms were the mechanisms more significantly involved in the responses. In conclusion, ochratoxigenic fungi do not all respond to antagonistic microorganisms in the same way. The use of biocontrol agents with the intent of reducing fungal growth and mycotoxin production by one fungus can have unexpected effects on others, thus leading to unforeseen safety problems. Further experiments are recommended to properly understand the reasons behind the different effects of microorganisms, to ensure their safe as biocontrol agents.

Detection of ochratoxin A in tropical wine and grape juice from Brazil.

Ochratoxin A (OTA) is the main mycotoxin found in grapes, wines and grape juices and is considered one of the most harmful contaminants to human health. In this study, samples of tropical wines and grape juices from different grape varieties grown in Brazil were analysed for their OTA content by high-performance liquid chromatography. The detection and quantification limits for OTA were 0.01 and 0.03 ?g L?1 respectively. OTA was detected in 13 (38.24%) of the samples analysed, with concentrations ranging from <0.03 to 0.62 micron g L-1. OTA was not detected in any of the grape juice samples. Most of the red wine samples proved to be contaminated with OTA (75%), while only one white wine sample was contaminated. However, the OTA levels detected in all samples were well below the maximum tolerable limit (2 micron g L-1) in wine and grape juice established by the European Community and Brazilian legislature. The results of this study indicate a low risk of exposure to OTA by consumption of tropical wines and grape juices from Brazil.

The mycotoxins 4-deoxynivalenol, zearalenone and aflatoxin in weather-damaged wheat harvested 1983-1985 in south-east Queensland

A survey for various mycotoxins was carried out on samples of all wheat delivered to nine storage and marketing depots in south-eastern Queensland, selected as most likely to receive mycotoxin-contaminated grain. All wheat was surveyed during 1983, when the degree of weather damage was high. Samples of the poorest grade of wheat from these depots were also surveyed in 1984 and 1985. The surveys included all regions where head scab of wheat caused by Fusariurn graminearurn Schwabe Group 2 had been reported to occur at significant levels. 4-Deoxynivalenol was detected in nearly all pooled samples representing bulk wheat at concentrations ranging from traces of <0.01 up to 1.7 mg kg-1. The highest concentration of zearlenone detected in a pooled wheat sample was 0.04 mg kg-1. In a few samples representing individual wheat deliveries and with up to 2.8% by weight of pink grains, 4-deoxynivalenol concentrations ranged up to 11.7 mg kg-' and zearalenone up to 0.43 mg kg-l. Aflatoxins B,, B2, G1 and G2 were detected in only one pooled sample of wheat, at a total aflatoxin concentration of 0.003 mg kg-'. Ochratoxin A, sterigmatocystin and T-2 toxin were not detected. Higher concentrations of mycotoxins were found in the poorer grades of wheat.

Mycotoxins and Fungal Damage in Maize Harvested during 1982 in Far North Queensland

A survey for mycotoxins and fungal damage in maize (Zea mays L.) grown during 1982 in Far North Queensland is reported. This season had a rainfall distribution which was typical for the reglon. The 293 samples examined came from 11 1 farms in eight maize-growing districts. The samples were first subjected to rapid screening tests for fungal damage. Aflatoxins B1, B2, G1, G2 ochratoxin A, T-2 toxin, and sterigmatocystin were not detected, but zearalenone was found in 85% of the samples. The concentrations of zearalenone were correlated with the extent of Gibberella zeae cob rot as indicated by the proportion (up to 2%) of kernels in each sample having a reddish-purple discoloration. In four samples the zearalenone concentration exceeded 1 mg kg-1, but the mean ¦ s.d. (n = 293) concentration in all samples was 0.17 ¦ 0.225 mg kg-1. Concentrations were highest in districts with the highest rainfall during the period of maize growth.

Mycotoxins and toxigenic fungi in sago starch from Papua New Guinea

Aims: To assay sago starch from Papua New Guinea (PNG) for important mycotoxins and to test fungal isolates from sago for mycotoxin production in culture. Methods and Results: Sago starch collected from Western and East Sepik Provinces was assayed for aflatoxins, ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin and zearalenone and all 51 samples were negative. Frequently isolated species of Penicillium (13), Aspergillus (five) and Fusarium (one) were cultured on wheat grain, and tested for the production of ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin, patulin and penicillic acid. All 12 isolates of P. citrinin and one of two A. flavipes isolates produced citrinin. A single isolate of A. versicolor produced sterigmatocystin. No other mycotoxins were detected in these cultures. Conclusions: No evidence was found of systemic mycotoxin contamination of sago starch. However, the isolation of several mycotoxigenic fungi shows the potential for citrinin and other mycotoxins to be produced in sago stored under special conditions. Significance and Impact of the study: Sago starch is the staple carbohydrate in lowland PNG and the absence of mycotoxins in freshly prepared sago starch is a positive finding. However, the frequent isolation of citrinin-producing fungi indicates a potential health risk for sago consumers, and food safety is dependant on promoting good storage practices.