Structure-activity relationships among mycotoxins

Relationships between structural features and biological effects of mycotoxins are reviewed. Structure-activity relationships are characterized at the molecular, subcellular, cellular, or supracellular level. Major chemical and physicochemical factors responsible for bioactivity of mycotoxins are stressed. A variety of chemical families of mycotoxins are then discussed from the point of view of structure-activity relationships. The structurally related families comprise small lactones, macrocyclic lactones, isocoumarin derivatives, aflatoxins and related compounds trichothecenes, anthraquinones, indole-derived tremorgens and selected amino acid-derived mycotoxins such as sporidesmins and cyclosporines. Biological effects of mycotoxins include acute and chronic toxicity, antimicrobial activity, mutagenicity and genotoxicity, carcinogenicity and biochemical modes of action.     

Mycotoxins as harmful indoor air contaminants

Fungal metabolites (mycotoxins) that pose a health hazard to humans and animals have long been known to be associated with mold-contaminated food and feed. In recent times, concerns have been raised about exposures to mycotoxin-producing fungi in indoor environments, e.g., damp homes and buildings. The principal mycotoxins that contaminate food and feed (alfatoxins, fumonisins, ochratoxin A, deoxynivalenol, zearalenone) are rarely if ever found in indoor environments, but their toxicological properties provide an insight into the difficulties of assessing the health effects of related mycotoxins produced by indoor molds. Although the Penicillium and Aspergillus genera of fungi are major contaminants of both food and feed products and damp buildings, the particular species and hence the array of mycotoxins are quite different in these environments. The mycotoxins of these indoor species and less common mycotoxins from Stachybotrys and Chaetomium fungi are discussed in terms of their health effects and the need for relevant biomarkers and long-term chronic exposure studies.     

日粮中霉菌毒素引发小熊猫中毒性肝炎的临床调查

本文报道了小熊猫霉菌毒素中毒性肝炎的群发病案。35 只小熊猫中,21 只发病,5 只死亡。通过日粮成分调查,饲料中霉菌毒素检测,病原微生物检查,血液参数测定,病理学检查和动物试验,确诊为呕吐毒素(DON)和玉米赤霉烯酮(F-2)毒素中毒。依据调查结果对小熊猫进行治疗,小熊猫预后良好。结论:(1)小熊猫对霉菌毒素(DON 和F-2 毒素)极其敏感,应予以高度重视;(2)DON 和F-2 毒素存在相互增强作用,对小熊猫的毒性作用主要为肝毒性和肾毒性,并可导致小熊猫机体免疫功能降低;(3)小熊猫对霉菌毒素的耐受性与环境的适应性有关,应激因素能提高小熊猫对霉菌毒素的敏感性。      

Sample preparation and high-resolution separation of mycotoxins possessing carboxyl groups

The chromatographic analysis of carboxyl-containing mycotoxins, such as fumonisin B₁, ochratoxin A, and citrinin, presents a continual challenge. Toxins must first be extracted from foods or tissues and then cleaned up before chromatographic separation and detection. Liquid–liquid extraction efficiencies for some carboxylic mycotoxins are marginal for spiked samples and uncertain for incurred residues. Immunoaffinity columns may be useful for concentrating mycotoxins from samples before chromatography. In almost every case, more than one analytical method must be used to confirm the identification of the mycotoxin. The fumonisins are especially troublesome to analyze because they are relatively insoluble in organic solvents, they are not separated easily by gas chromatography, and they do not respond to the usual absorbance or fluorescence detectors used in liquid chromatography. Fluorescence derivatization and electrospray liquid chromatography–mass spectrometry have now made it possible to detect trace levels of mycotoxins. The purity of mycotoxin standards for toxicological studies can be determined by liquid chromatography with either an evaporative light scattering detector or electrospray mass spectrometer. New developments in capillary electrophoresis, nonporous microsphere liquid chromatography, and detection methods for low-volatility compounds show promise for improving the analysis of mycotoxins in the future.     

Biological detoxification of mycotoxins: a review

Mycotoxins are secondary fungal metabolites and are reported to be carcinogenic, genotoxic, teratogenic, dermato-, nephro- and hepatotoxic. Several studies have shown that economic losses due to mycotoxins occur at all levels of food and feed production, including crop and animal production, processing and distribution. Therefore, there is a great demand for a novel approach to prevent both the formation of mycotoxins in food and feed and the impact of existing mycotoxin contamination. Recently, investigators have reported that many microorganisms including bacteria, yeast, moulds, actinomycetes and algae are able to remove or degrade mycotoxins in food and feed. We have reviewed various strategies for the detoxification of mycotoxins using microorganisms such as bacteria, yeast and fungi.     

Proposal of a comprehensive definition of modified and other forms of mycotoxins including “masked” mycotoxins

As the term “masked mycotoxins” encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, “modified mycotoxins” into “biologically modified” and “chemically modified” with all variations of metabolites of the former and dividing the latter into “thermally formed” and “non-thermally formed” ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term “modified mycotoxins” should be used in the future and the term “masked mycotoxins” to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.     

Fungi, quality and safety issues in fresh fruits and vegetables

The role of moulds in the spoilage of fresh fruits and vegetables is discussed. Although the major problems are economic with a significant loss of useful food materials, there are a few examples implicating a role for mycotoxins in the safety of fresh fruits. The significance of the mycotoxins patulin, ochratoxin and tenuazonic acid will be reviewed.     

Impact of food processing and detoxification treatments on mycotoxin contamination

Mycotoxins are fungal metabolites commonly occurring in food, which pose a health risk to the consumer. Maximum levels for major mycotoxins allowed in food have been established worldwide. Good agricultural practices, plant disease management, and adequate storage conditions limit mycotoxin levels in the food chain yet do not eliminate mycotoxins completely. Food processing can further reduce mycotoxin levels by physical removal and decontamination by chemical or enzymatic transformation of mycotoxins into less toxic products. Physical removal of mycotoxins is very efficient: manual sorting of grains, nuts, and fruits by farmers as well as automatic sorting by the industry significantly lowers the mean mycotoxin content. Further processing such as milling, steeping, and extrusion can also reduce mycotoxin content. Mycotoxins can be detoxified chemically by reacting with food components and technical aids; these reactions are facilitated by high temperature and alkaline or acidic conditions. Detoxification of mycotoxins can also be achieved enzymatically. Some enzymes able to transform mycotoxins naturally occur in food commodities or are produced during fermentation but more efficient detoxification can be achieved by deliberate introduction of purified enzymes. We recommend integrating evaluation of processing technologies for their impact on mycotoxins into risk management. Processing steps proven to mitigate mycotoxin contamination should be used whenever necessary. Development of detoxification technologies for high-risk commodities should be a priority for research. While physical techniques currently offer the most efficient post-harvest reduction of mycotoxin content in food, biotechnology possesses the largest potential for future developments.     

Mycotoxin producing fungi

Mycotoxins are relatively small molecules characterized by a diversity of chemical structure and a diversity of biological activity. They are often genotypically specific for a group of species, but the same compound can also be formed by fungi belonging to different genera. Most of the mycotoxins known have been recognized as metabolic products of Aspergillus, Penicillium and Fusarium species. This review will be focused on aflatoxins, ochratoxins and fumonisins because of their hazard to animal and human health. The production of these mycotoxins have been usually associated with a small number of species but some recent studies have reported the production of these mycotoxins by some other species. These results show that mycotoxin production is broader than is normally thought, so the possibility can not be ruled out that new species may be a new source of unexpected mycotoxins in their natural substrates.     

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.     

Modulation of innate and antigen-specific immune functions directed against Listeria monocytogenes by fungal toxins in vitro

Mycotoxins, a large group of secondary fungal metabolites, are ubiquitously present in the environment and are potentially harmful to exposed humans and animals. Despite increasing interest in this group of fungal metabolites it is still difficult to estimate the relative toxic potential of one individual mycotoxin compared with others. We therefore compared the effects of some of the most important mycotoxins on effector cells of the innate and adaptive immune system in an in vitro model. Our data show clear differences of various mycotoxins in regard of their immunotoxic potential on mouse macrophages and T cells. Our results also indicate differences in the susceptibility of specific immune effector functions of macrophages and T cells exposed to mycotoxins. Thus, our results enhance the understanding of role of mycotoxins in the pathogenesis of human and animal diseases.     

The mycotoxin research in foodstuffs in the Czech Republic in the 90th Years

Mycotoxins are naturally occurring secondary metabolites produced by several toxigenic microscopic fungi on a variety of crops, especially cereal grains and further foodstuffs. Series of experimental research projects on the determination of mycotoxins (aflatoxins, cyclopiazonic acid, ochratoxin A, patulin, deoxynivalenol, fumonisin B₁, T-2 toxin, zearalenone, sterigmatocystin, alternaria toxins) in several foods were realized in the National Reference Centre for Microscopic Fungi and Mycotoxins in the 90^th years. The aim of our work was an estimation of dietary exposure to mycotoxins and risk assessment. The method of a solid phase extraction (SPE), liquid — liquid extraction and immunoaffinity chromatography (f. e. R-Biopharm, VICAM) were used to elaborate for sample analyses of mycotoxins in our projects. The mycotoxins were detected most frequently by chromatographic methods (HPTLC, HPLC, GC) and immunochemical methods (ELISA). Average dietary exposure has been calculated by multiplying of concentration data for specific foods with their consumption rates per 1 kg of b. w. per day. The estimation of the dietary exposure dose of mycotoxins for the Czech population is presented.     

Fusarium species and fusarium mycotoxins in cereals from West Romania: preliminary survey

Fungal contamination of plant products is an important risk factor for health, because of the high mycotoxin potential deriving from these contaminations with multiple effects: hepatic toxicity, teratogenic, mutagenic and carcinogenic. The contamination of cereals with mycotoxins has been a serious problem in Balkan communities. Several studies implicated mycotoxins, in endemic kidney disease geographically limited to Balkan region (Balkan endemic nephropathy). The trichothecenes are of particular concern because they are ubiquitous found in wheat, corn and barley throughout the world. Fumonisins have been isolated from certain Fusarium species of which FB1, FB2 and FB3 are the major ones produced in naturally contaminated foods.These mycotoxins are produced on cereal grains infected by Fusarium while being grown in-the-field. The aim of this study is to evaluate the presence of the Fusarium species in cereals from West side of Romania and to determinate the concentrations of deoxynivalenol (DON) and fumonisine (F1+F2). Identification of Fusarium species was done using the total number of fungal species determination method. The level of mycotoxins was determined with the immune-enzymatic method ELISA. 27 cereal samples from rural households in three counties in West Romania were analysed.     

Protein Translation Inhibition by Stachybotrys chartarum Conidia with and without the Mycotoxin Containing Polysaccharide Matrix

Recent studies have correlated the presence of Stachybotrys chartarum in structures with SBS. S. chartarum produces mycotoxins that are thought to produce some of the symptoms reported in sick-building syndrome (SBS). The conidia (spores) produced by Stachybotrys species are not commonly found in the air of buildings that have been found to contain significant interior growth of this organism. This could be due in part to the large size of the Stachybotrys spores, or the organism growing in hidden areas such as wall cavities. However, individuals in buildings with significant Stachybotrys growth frequently display symptoms that may be attributed to exposure to the organism's mycotoxins. In addition, Stachybotrys colonies produce a "slime" or polysaccharide (carbohydrate) matrix that coats the hyphae and the spores. The intent of this project was to determine whether the carbohydrate matrix and the mycotoxins embedded in it could be removed from the spores by repeated washings with either aqueous or organic solvents. The results demonstrated that the process of spore washing removed compounds that were toxic in a protein translation assay as compared to spores that were washed with an organic solution, however a correlation between carbohydrate removal during the washing process and the removal of mycotoxins from the spore surface was not observed. These data demonstrated that mycotoxins are not likely to be found exclusively in the carbohydrate matrix of the spores. Therefore, mycotoxin removal from the spore surface can occur without significant loss of polysaccharide. We also showed that toxic substances may be removed from the spore surface with an aqueous solution. These results suggest that satratoxins are soluble in aqueous solutions without being bound to water-soluble moieties, such as the carbohydrate slime matrix.     

The Fate of Mycotoxins during the Distillation Process of Barley Shochu,a Distilled Alcoholic Beverage

Mycotoxins are frequent contaminants of grains and critical risk substances for brewers. Fermented barley mash contaminated artificially with 13 representative mycotoxins was distilled with small-scale apparatuses to elucidate the possibility of mycotoxin transfer from mash to distillates. None of these were detected in the distillates. The distillation process can effectively reduce the contamination risk posed by mycotoxins in distilled alcoholic beverages.     

Enniatin B and ochratoxin A in the blood serum of workers from the waste management setting

The waste management occupational environment is recognized by the simultaneous presence of several substances and biologic agents. Therefore, workers are exposed simultaneously to multiple contaminants. Occupational exposure to aflatoxin B₁ in one Portuguese waste sorting plant was already reported. However, besides this mycotoxin, data regarding fungal contamination showed that exposure to other mycotoxins could be expected. A study was developed to analyze if exposure to other mycotoxins besides aflatoxin B₁ was occurring in the workers from the waste sorting plant previously assessed and to discuss how these findings need to be considered in the risk assessment process. In addition to aflatoxin B₁ detected previously by ELISA, two additional mycotoxins and one mycotoxin degradation product were detected and quantified by a multi-mycotoxin HPLC-MS/MS approach: Enniatin B and ochratoxin A as well as 2’R-ochratoxin A. Besides the confirmation of co-exposure to several mycotoxins, results probably indicate different exposure routes for the mycotoxins reported.     

Mycotoxins in laboratory rodent feed

Twenty-one batches of fixed-formula rodent diets from three feed manufacturers were tested for the presence of five mycotoxins: deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin, T-2 toxin and ochratoxin A (OTA). Five batches were also tested for the presence of zearalenone (ZEN) and six batches for aflatoxins. Detectable levels of DON (up to 298 microg/kg), NIV (up to 118 microg/kg), OTA (up to 3.1 microg/kg) or ZEN (up to 26.7 microg/kg) were found in samples from all manufacturers. Three batches contained two (DON or NIV and OTA or ZEN) and one batch contained three (DON, OTA and ZEN) different mycotoxins. Aflatoxins, T-2 and HT-2 were not detected in any of the batches. The concentrations of mycotoxins detected in the feed were low, but indicated that feed ingredients, probably the cereal ingredients, were contaminated by mycotoxins. Since mycotoxins are known to have toxic and/or immunosuppressive effects, non-contaminated ingredients should be used for production of laboratory animal feed. The results imply that an improved quality control of ingredients used for laboratory rodent feed should be implemented.     

The fate of mycotoxins during the distillation process of barley shochu, a distilled alcoholic beverage

Mycotoxins are frequent contaminants of grains and critical risk substances for brewers. Fermented barley mash contaminated artificially with 13 representative mycotoxins was distilled with small-scale apparatuses to elucidate the possibility of mycotoxin transfer from mash to distillates. None of these were detected in the distillates. The distillation process can effectively reduce the contamination risk posed by mycotoxins in distilled alcoholic beverages.     

Mycotoxicoses of domestic animals and their diagnosis

Mycotoxins have been named "agents in search of a disease," and the considerable progress in analytical methodology over the last 10 years has not changed this very much. The following are factors that contribute to the difficulty of making a diagnosis: (1) nonspecificity of lesions; (2) masking of mycotoxic effects by secondary effects, e.g., through immunosuppression; (3) late appearance of a lesion, e.g., bone marrow damage or neoplasia; (4) interaction of several mycotoxins or presence of other toxicants or deficiency states; (5) species variation in the response to the mycotoxin(s); (6) difficulty of linking a late appearing effect with a demonstrable cause; (7) low doses of mycotoxins may cause stimulating effects; and (8) not being aware of the potential of a mycotoxin as a causative factor in disease. The mycotoxins of major importance in Canada are trichothecenes, ochratoxin, zearalenone, and ergot. It is concluded that the significance of mycotoxins for animals in Canada is likely generally underestimated.     

Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay

The application of a modified colorimetric bioassay for the evaluation of the biological effects of mycotoxins is reported. Using three different monolayer cell lines (swine kidney, Madin Darby canine kidney, HeLa) the influence of nine different mycotoxins on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated. The yellow tetrazolium salt MTT is converted by mitochondrial dehydrogenases of metabolically active cells to an insoluble purple formazan product, which was then solubilized with dimethylsulfoxide. The optical density of this homogeneous solution was suitable for a precise spectrophotometric measurement by a plate reader at a wavelength of 510 nm. Nine mycotoxins were simultaneously tested in all three cell lines, from which the swine kidney cell line proved to be the most sensitive. The effects of additional 35 mycotoxins were therefore tested using swine kidney monolayers as target cells. A total of 28 toxins of the 44 mycotoxins tested proved to be cytotoxic in the MTT-bioassay. Most of them belong to the group of trichothecene mycotoxins. Concentrations ranged between 0.01 µg and 100 µg/ml of cell culture medium. The MTT cleavage assay was found to be a quick (24 hours) and easy to perform system for the evaluation of the biological activity of many different mycotoxins and may also provide a useful tool for the testing of a large variety of sample materials.