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A simple method with liquid chromatography/tandem mass spectrometry for the determination of the six trichothecene mycotoxins in rice medium.

A selective and speedy LC-MS/MS method was developed to determine six trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, and T-2 toxin) in rice medium where Fusarium graminearum were cultivated for in vitro tests. The analytes were extracted from the rice medium with acetonitrile/water (85/15, v/v), and diluted with acetonitrile/water (5/95, v/v) in order to minimize the effects of matrices. Diluted solutions were analyzed by LC-MS/MS with electrospray ionization (ESI) interface in negative or positive ion mode and the multiple reaction monitoring mode. Recovery rates were 76-106% with a spiked level at 1-6 microg/kg of mycotoxins that corresponded to the limit of quantitation. The method was applied to study the time courses of trichothecene production and the biomass of fungi by three Fusarium graminearum strains. Three strains have different mycotoxin biosynthesis pathways, wFg14 and 03E-1 were DON producer, and 03N-1 was NIV producer.

Calibration↗

Fluorescence polarization (FP) assays for the determination of grain mycotoxins (fumonisins, DON vomitoxin and aflatoxins).

Successful use of fluorescence polarization assays (FPAs) in human clinical, infectious disease, and drug discovery fields has prompted us to extend its use to the grain mycotoxin field. An antibody specific to a mycotoxin and a mycotoxin-fluorophore conjugate are developed. Free toxin (extracted from the grains with a suitable solvent) competes with the toxin-fluorophore conjugate for the antibody and a change in FP relative to the quantity of free toxin occurs. This change is compared to a standard curve obtained by using known quantities of toxin. The use of FP and toxin-fluorophore conjugates for the quantification of fumonisins, deoxynivalenol and aflatoxins is described. These assays are field portable, simple to perform, rapid and require no washing steps.

Aflatoxins↗

Effects of feeding blends of grains naturally contaminated with Fusarium mycotoxins on brain regional neurochemistry of starter pigs and broiler chickens.

Two experiments were conducted to compare the effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on brain regional neurochemistry of starter pigs and broiler chickens. A polymeric glucomannan mycotoxin adsorbent (GM polymer) was also tested for its efficacy in preventing Fusarium mycotoxicoses. In Exp. 1, a total of 150 starter pigs (initial weight = 9.3+/-1.1 kg) were fed five diets (six pens of five pigs per diet) for 21 d. Diets (as-fed basis) included control, 17% contaminated grains, 24.5% contaminated grains, 24.5% contaminated grains + 0.2% GM polymer, and a pair-fed control for comparison with pigs receiving 24.5% contaminated grains. In Exp. 2,360 1-d-old male broiler chicks were fed for 56 d one of four diets containing the same source of contaminated grains as was fed to pigs. The diets included control, 37% contaminated grains, 58% contaminated grains, and 58% contaminated grains + 0.2% GM polymer (as fed). Neurotransmitter concentrations in the cortex, hypothalamus, and pons were analyzed by HPLC. The following brain neurotransmitter alterations (P < or = 0.05) were observed. In pigs, inclusion of contaminated grains in the diet 1) linearly increased cortex 5-hydroxytryptamine (5HT, serotonin) concentrations, while linearly decreasing hypothalamic tryptophan concentrations; 2) quadratically increased hypothalamic and pons 5-hydroxyindoleacetic acid (5HIAA):5HT ratios, whereas the ratio decreased linearly in the cortex; and 3) linearly increased the ratio of hypothalamic 3,4-dihydroxyphenylacetic acid:dopamine (DA) concentrations, whereas hypothalamic norepinephrine (NRE) and pons DA and homovanillic acid (HVA) concentrations linearly decreased. In broiler chickens, inclusion of contaminated grains in the diet 1) linearly increased concentrations of 5HT and 5HIAA in the pons and 5HT concentrations in the cortex; 2) linearly decreased 5HIAA:5HT ratio; and 3) linearly increased pons NRE, 3-methoxy-4-hydroxyphenylethylene glycol, DA, and HVA concentrations. Supplementation of GM polymer to the contaminated diet decreased (P < 0.05) 5HT and 5HIAA concentrations in the cortex of pigs. It was concluded that the differences in alterations of brain neurochemistry might explain the species differences in the severity of Fusarium mycotoxin-induced feed refusal.

Adsorption↗

Immunotoxicity of mycotoxins.

The immune system is primarily responsible for defense against invading organisms. The effects of several mycotoxins on the immune responses have been investigated; however, most data concern laboratory animals. In some instances, farm animals and cells derived from livestock species have been employed to evaluate the immunotoxicity of mycotoxins. Immune responses are highly variable, and cost considerations usually preclude the use of dairy cattle as experimental models. Immunosuppression caused by aflatoxin B1 has been demonstrated in various livestock species (e.g., turkeys, chickens, and pigs) and also in laboratory animals (mice, guinea pigs, and rabbits). The response of bovine lymphocytes to aflatoxin in vitro is similar to that of other laboratory animals. Trichothecenes are potent immunosuppressive agents that directly affect immune cells and also modify immune responses as a consequence of tissue damage elsewhere. Sheep and calves treated with fusarium T-2 toxin develop leukopenia and decreased functioning of peripheral lymphocytes. Immunosuppressive effects of ochratoxin A, rubratoxin B, and patulin have been reported. Citrinin produced lymphopenia but stimulated responses against antigens. Antibodies against mycotoxins conjugated to proteins have been produced and are useful for analytical purposes.

Animals↗

Lipoic acid as a potential first agent for protection from mycotoxins and treatment of mycotoxicosis.

Mycotoxins--toxic substances produced by fungi or molds--are ubiquitous in the environment and are capable of damaging multiple biochemical mechanisms, resulting in a variety of human symptoms referred to collectively as "mycotoxicosis." In fact, mycotoxins mimic multiple xenobiotics, not only with respect to their ultimate damage, but also in their routes of detoxification. This suggests potential therapeutic options for the challenging treatment of mycotoxicosis. In this brief review, the author examines the use of lipoic acid as an example of an inexpensive and available nutrient that has been shown to protect against, or reverse, the adverse health effects of mycotoxins.

Air Pollution, Indoor↗

Aflatoxin B1 and clinoptilolite in feed for laying hens: effects on egg quality, mycotoxin residues in livers, and hepatic mixed-function oxygenase activities.

Ninety-six laying hens were allocated to four groups administered different diets (group 0-0 received a complete diet, group 0-AF received a diet supplemented with 2.5 ppm of aflatoxin B1 [AFB1], group 2-0 received a diet supplemented with 2% clinoptilolite [CPL], and group 2-AF received a diet supplemented with 2% CPL and 2.5 ppm of AFB1) for 4 weeks to evaluate the effect of AFBI and/or CPL on egg quality and the ability of CPL to interact with the oral administration of AFB1. The possible effects of AFB1 on cytochrome P450-dependent hepatic mixed-function oxygenase (MFO) activities were also evaluated. Mycotoxin reduced yolk weight, while CPL influenced albumen percentage relative to that of eggs laid by chickens in group 0-AF Eggs laid by chickens in groups 0-AF and 2-AF had stronger shells and weighed less than the eggs of other groups. The eggs of treated groups were lighter in color than those of the control group (P < 0.01), and the tendency to yellowness in eggs was increased by CPL, probably through the affinity of red pigments for adsorbents and a consequent prevalence of yellow tonality. Color parameters might be connected with AFB1's interference with lipid metabolism and pigment deposition. The livers of hens in groups 0-AF and 2-AF showed very low mycotoxin concentrations that were significantly different (P < 0.01). The highest levels observed were those in the livers of the hens receiving the diet supplemented with the mycotoxin alone. AFB1 did not exert any significant effects on the hepatic MFO activities examined.

Adsorption↗

Multiplexed detection of mycotoxins in foods with a regenerable array.

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

Biosensing Techniques↗

Mycotoxins and toxigenic fungi.

Mycotoxins and the fungi that produce them are of increasing importance as causes of human illness, but the diseases produced remain poorly understood at the clinical level. This paper explores four aspects: the increase of interest in ochratoxin A, factors affecting mycotoxin production, toxicology of the major mycotoxins, and the identification of Penicillium species which cause food spoilage and are important in indoor air.

Animals↗

[Mycotoxins in spices].

Since antiquity, spices have been used for flavoring foods and beverages and for medication. They play an important role in the national economy of several of the producing, exporting and importing countries. Tropical climatic conditions under which spices are grown offer a favorable environment for the fungal and mycotoxin contamination. This review summarized information from 44 reports on the occurrence of mycotoxins (aflatoxins, ochratoxins zearalenone, fumonisins, trichothecenes) and their levels in different spices. Aflatoxins which are highly carcinogenic compounds, produced by A. flavus and A. parasiticus, are the leading toxins present in red peppers (paprika, chilli, cayenne) nutmeg, mustard, ginger, black and white peppers, coriander. Ochratoxins have been reported also as a natural contaminants in many spices. Although the levels of mycotoxins are generally low when compared with other food products, the testing of spices (imported and domestic) by regulatory agencies seems to be in need of expansion.

Aflatoxins↗

Evaluation of certain mycotoxins in food. Fifty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives.

This report presents the conclusions of a Joint FAO/WHO Expert Committee convened to assess the risks associated with the consumption of food contaminated with certain mycotoxins. The first part of the report contains a general discussion of the principles for evaluating mycotoxins in food, including those concerning analytical methods, sampling, data on food consumption and dietary intake, and prevention and control. The second part provides a summary of the Committee's evaluations of toxicological data on specific mycotoxins, including aflatoxin M1, fumonisins B1, B2 and B3, ochratoxin A, deoxynivalenol, and T-2 and HT-2 toxins.

Food Contamination↗

Comparison of phytotoxicity and mammalian cytotoxicity of nontrichothecene mycotoxins.

The phytotoxicity and mammalian cytotoxicity of four nontrichothecene mycotoxins (apicidin, sambutoxin, wortmannin, HC-toxin) were compared. Phytotoxicity was evaluated in terms of electrolyte leakage, growth inhibition, and reduction in chlorophyll content. Based on the parameters evaluated, the relative order of phytotoxicity to duckweed (Lemna pausicostata L.) was wortmannin > HC-toxin > apicidin >> sambutoxin. A 48-hr exposure to 10 microM wortmannin, HC-toxin or apicidin caused electrolyte leakage from duckweed. The IC50 values for growth inhibition and chlorophyll reduction for wortmannin, HC-toxin, and apicidin were 0.2 and 2.6 microM, 15.4 and 12.6 microM, and 27.7 and 45.3 microM, respectively. Based on the parameters measured, a 72-hr exposure to 100 microM sambutoxin was not toxic to duckweed. Kudzu (Pueraria lobata L.) leaf disc assays revealed a similar trend in relative toxicities, but higher mycotoxin concentrations were required to elicit phytotoxic effects compared to duckweed. All four mycotoxins were cytotoxic to four mammalian cell cultures. However, in contrast to plants, wortmannin was the least toxic (IC50 = 10 to 20 microM) and sambutoxin exhibited a high level of toxicity (IC50 = 0.5 to 1 microM).

Animals↗

Occurrence of mycotoxins in animal feeds.

A total of 1120 grain samples of oats, wheat, rye, barley, and maize, delivered for processing of mixed feeds for animals, were collected during the years 1975 to 1979 from commercial feed mills located throughout Poland. In addition, 625 samples of the commercially mixed feeds and protein concentrates were collected during 1976. For the mycotoxin survey, 751 laboratory samples were chosen at random and analyzed. When applying confirmatory tests neither aflatoxins B1, B2, G1, G2 nor sterigmatocystin, zearalenone, or ochratoxin B were found to be present in any of the samples of barley, wheat, rye, or oats. Aflatoxins were detected in about 4% of the maize samples. The presence of ochratoxin A in the range of 2 to 200 micrograms/kg was evident in 9% of the grain samples. The commercially mixed feeds were found to be more contaminated with mycotoxins than were the grains. The aflatoxins were confirmed in about 13% of the samples of mixed feeds. After the preliminary multimycotoxin analysis, out of 42 feed samples that could be suspected of containing ochratoxins, 32 failed to prove their presence. Similarly, out of 27 suspected feed samples, zearalenone was found only in one (0.5%) sample. The lowest percentage of samples contaminated with mycotoxins was found in poultry mixed feeds (4%). The highest contamination occurred among the samples of swine rations, where 17% of the samples contained aflatoxins and 13% ochratoxins. The protein concentrates contained only aflatoxins. Out of 31 analyzed samples, aflatoxins were detected in 19 (61%) in concentrations ranging from 5 micrograms/kg to 500 micrograms/kg. In one sample, aflatoxin concentration (B1 + B2) reached 1140 micrograms/kg. Practical implications of the results are discussed in relation to animal and human safety.

Aflatoxins↗

[Methods of detecting and determining nitrogen-containing mycotoxins].

The influence of various nitrogen functional groups used for extraction cleanup and determination of N-containing mycotoxins (NM) in feeds and foodstuffs have been considered. TLC and LC are the most common techniques for detection and determination of nitrogen-containing mycotoxins. Gas chromatography has been used for determination (with or without derivatization) of several nitrogen-containing mycotoxins and/or their degradation products. Immunochemical techniques, in particular ELISA are available for only a very limited number of NM (e.g. ochratoxin A). Numerous methods for determination of ochratoxin A in feeds, grains, animal products and other foodstuffs have been developed. Methods for which recoveries have been carried out on spiked samples are also available for several other NM.

Animal Feed↗

[Monitoring the important mycotoxin biomarkers (ochratoxin A, aflatoxin M1) in the Czech population].

BACKGROUND: In among mycotoxins, secondary metabolites of toxinogenic moulds, ochratoxin A and aflatoxins occupy a prominent place. These mycotoxins have been--as etiologic agents-- associated with a wide numbers of acute and chronic human diseases including mycotoxicoses like Balkan endemic nephropathy or liver cancer. While the risk of acute toxic effects of ochratoxin A and aflatoxin B1 is usually considered to be minimal in the Czech Republic, the situation is different as far as the risk of the late toxic effects (particularly carcinogenic), which result from a single or repeated intake of low doses of these mycotoxins from foodstuffs is concerned. METHODS AND RESULTS: The presence of ochratoxin A and aflatoxins in human environment has been monitored within the program "Monitoring the health state of the population". As for ochratoxin A, 2206 samples of blood serum were investigated, 2077 (94 %) of them turned out to be positive (with levels > or = 0.1 microg x l(-1)), the average was 0.28 microg x l(-1), the median was 0.2 microg x (l(-1), and the percentile (90%) was 0.5 microg x l(-1). The ochratoxin A levels ranged from 0.1 to 13.7 microg x l(-1) of sera. The presence of ochratoxin A was also analyzed in 30 samples of human kidneys; 12 samples were positive (with levels > or = 0.1 microg l(-2), the average was 0.07 microg x kg(-1), the median was 0,05 microg x kg(-1). As for aflatoxins, in 1997-1998 the presence of aflatoxin M1 was investigated in 205 samples of human urine; 118 samples (58%) were positive (with levels >125 pg x l(-1) of urine). CONCLUSIONS: When calculated to a concentration of creatinine in urine, the average was 391 pg x g(-1), the median was 127 pg x g(-1), and the percentile (90%) was 585 pg x g(-1). The aflatoxin M1 levels ranged from 19 to 19 219 pg x g(-1) of creatinine.

Aflatoxin M1↗

Reliable identification and quantification of trichothecenes and other mycotoxins by electron impact and chemical ionization-gas chromatography-mass spectrometry, using an ion-trap system in the multiple mass spectrometry mode. Candidate reference method for complex matrices.

Highly toxic mycotoxins like the trichothecenes can be found as contaminants from the metabolism of fungi in food and food preparations. They can be identified and quantified with great accuracy by GC/MS-measurements. Reliable analytical methods are urgently needed because such mycotoxins are not only toxic substances occurring in nature but also are in the list of biological weapons (e.g. T2-toxin, HT-2-toxin) and have some potential for terroristic attacks. By using GC/MS in the EI- and NCI- or PCI-mode and MS(n)-measurements with a 30 m Rtx 5MS fused-silica capillary column it is possible to identify and quantify all relevant mycotoxins either as underivatized substances or as their TMS-derivatives in extracts from food, food preparations or beverages with very complex matrix-derived background. This method can also be used to determine free ricinine as a biological marker for ricine in terroristic attacks. So laborious and time-consuming steps of sample-preparation can often be diminished. The LOD is in the range of 10-50 pg and the LOQ with linear calibration curves is in the range of 50-5000 pg. The high specificity of these methods helps not only to detect the existence of intentional terroristic or natural food contamination but also to avoid faulty alarm with unnecessary panic in the public. Furthermore, these methods have a high potential in ameliorating the safety of basic food and food products.

Calibration↗

[Safety of rice grains and mycotoxins - a historical review of yellow rice mycotoxicoses].

Aflatoxins, the most powerful mycotoxins, were brought to the attention fo the people in the early 1960s with the outbreak of the turkey "X" disease in England. However, the history of mycotoxin research in Japan began 100 years ago. In 1891, Sakaki demonstrated that moldy, unpolished rice was fatal to experimental animals, with symptoms indicating paralysis of the central nervous system (Shoshin-kakke). In 1920, Prof. I. Miyake and Dr. Takada first reported that Penicillium commune, which was known as a causal agent of "Mossy diseased rice" was found to be toxic to experimental animals by feeding the moldy rice to rabbits and rats.With such a historical background, taking the idea of "rice, fungus and toxin" as a working hypothesis, Miyake and his co-workers discovered the first sample of yellow rice grains from Taiwanese and domestic rice, from which was isolated a species of Penicillium and later identified it with P. citreonigrum (=P. toxicarium). The fungus produced a highly toxic metabolite, citreoviridin. Unfortunately because this study was published during wartime, it failed to alert the world to the potential or actual dangers of the toxicity of common molds. After World War II, Japanese people suffered for some years from a shortage in domestic rice production and depended on foreign countries to supply rice, which led to the toxicological screening on fungal isolates from polluted rice grains by Dr. Tsunoda and his co-workers. AMong the isolates from imported rice, there were two species of Penicillium which were particularly associated with high toxicity; P. islandicum responsible for brownish discolored rice, and P. citrinum responsible for yellowish rice. P. islandicum produces two hepatotoxic metabolites: luteoskyrin and cyclochlorotine, while a nephrotoxic of P. citrinum is citrinin. These toxicological characters, including the induction of cancer and chemical structures, were studied by Profs. uraguchi, Saito, Shibata, Tatsuno and their co-workers. In this way, toxic disturbances associated with the consumption of rice contaminated with these Peniciliium species occurred and became known under the collective name of the "Yellow rice syndrome". Although no human cases of poisoning were recorded, scientists in the field of mycotoxin research recommended to the Japanese Government that administrative action should be taken to protect the people against any possible hazards from "yellow rice". Consequently, mycological inspections of imported rice were started by the government. The safety control system for domestic rice during post-harvest handling and long-term storage has been progressively established after learning of these problems.

Animals↗

Lack of relationship between sorghum consumption, mycotoxin contamination and pellagra in a traditionally sorghum eating population.

Traditionally, the disease pellagra has been associated with corn consumption and niacin deficiency, and has presently been recognised as a multiple factor nutritional syndrome. In the recent past, it has been suggested that consumption of mycotoxin contaminated sorghum/corn may be involved in the development of pellagra in a sorghum/corn eating population. The present study relates to the assessment of levels of mycotoxins (aflatoxin B1 and T-2 toxin) in sorghum collected from a traditionally sorghum eating population. Despite 25% fungal contamination in the sorghum samples collected, the levels of mycotoxins were minimal (1.4%) and there was no incidence of pellagra in the survey area. The absence of pellagra in the survey areas may be attributed to the changing dietary pattern.

Edible Grain↗

The public health significance of mycotoxins.

Diseases of man and animals associated with foodstuffs contaminated by molds have been known for centuries, but only recently have mycotoxicoses been recognized as a significant hazard to man. A detailed review of the literature involving the major mycotoxin-producing fungi is presented. Because of their hazards to both human and animal health, increasing concern has developed regarding the carcinogenic activity of aflatoxins and other mycotoxins. The symptoms of mycotoxicoses in man and experimental animals is reviewed and certain specific case histories reported. The industry is urged to implement all available techniques in order to minimize the contamination of foods by mycotoxins.

Aflatoxins↗