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Invisible danger with long-term consequences

According to official figures, mycotoxins, which are toxic secondary metabolites produced by moulds, are found in around of 20 per cent of the European crop harvest. Even the smallest concentrations of mycotoxins can have devastating toxic effects, causing cancer or posing a risk to fertility and development. Due to their remarkable resistance to heat and acids, myotoxins can survive in products made from previously contaminated crops, coffee beans, eggs, etc. Mycotoxins pose a particular danger when mouldy products are processed because the contamination cannot be detected visually. Alexandra Heussner from the University of Konstanz is investigating ochratoxins, whose chronic effect represents a huge danger to human health. She has recently successfully produced a monoclonal antibody against the toxin that has the potential to be used for the routine detection of ochratoxins in food and beverages for improved determination of ochratoxin risk.

Alexandra Heussner from the University of Konstanz is investigating the long-term consequences of ochratoxins. © Heussner

Mycotoxins are toxic secondary metabolites produced by moulds under certain conditions, including high humidity and temperature. They can have devastating long-term effects on human and animal health. The most frequent cause of mycotoxin infection is the sub-optimal crop storage method: mycotoxin production is favoured by a broad range of different factors, including nutrient and water supply, ambient air humidity, temperature, pH and the presence of other fungi. Several hundred different mycotoxins are known, which can also occur in combination with each other due to one mould species producing several different mycotoxins. Moulds can contaminate crops on the field or after harvest; they are also found in food such as coffee beans and fruit and their secondary products such as coffee and wine. "The mycotoxins expose farm animals to a very high level of risk as the animals can take up the mycotoxins with their feed and transmit them to the milk, eggs and meat they produce," said Alexandra Heussner, a technical expert in the Department of Human and Environmental Toxicology at the University of Konstanz who is currently working on mycotoxin research.

Heussner's particular focus is ochratoxins (OTA and OTB), common contaminants of human food and animal feed. The toxins have been shown to be teratogenic, immunotoxic and carcinogenic in various laboratory animals. Animals such as pigs, horses and chicken that are fed mouldy feedstuff are exposed to specific dangers and can also transmit the toxins to humans. Heussner is currently working on the characterisation of previously unknown ochratoxin mechanisms of action with the goal of improving risk assessments related to animal and human health.

Toxic combination affects nervous system

The reasons for the development of mycotoxins and the majority of secondary metabolites found in nature are not yet known. One possible explanation may be that the fungus uses them as a strategy to improve the environment for proliferation or as a means to communicate among the fungal hyphae, for example as signalling substance to promote optimal growth. "The environment plays a major role in the development of fungi and the production of toxins, which is why ochratoxin A and B are only found in moderate climatic zones," said Alexandra Heussner, admitting that it is difficult to characterise the different types of fungus because one mould species can produce several different mycotoxins and/or the same mycotoxin as another species. The resulting synergistic effects represent an additional danger. "OTA and OTB may increase the negative effects of other mycotoxins. Mycotoxin mixtures are believed to pose a greater danger to human and animal health than individual toxins," said Heussner.

Different mycotoxins may have a broad range of effects on health, both with regard to disease symptoms and disease. Certain pathogenic fungi of the genus Fusarium may lead to vomiting and gastrointestinal diseases whereas neurotoxic toxins act on the nervous system. "The trichothecenes of a mould fungus that is found in mouldy houses can lead the suppression of the immune system as well as increase an individual's susceptibility to asthma," said the expert from Konstanz.

In vitro models for the identification of the potential danger

Ochratoxins with a damaging effect on the kidneys and carcinogenic effects have been found in several different animal species. Since many mycotoxins exert their effect quite some time after exposure, it has so far not been possible to establish a causal relationship for certain human diseases. However, the ochratoxins appear to be the causal agents in mycotoxic porcine nephropathy (MPN) and are most likely involved in the aetiology of a similar human disease (Balkan endemic nephropathy; BEN). "The primary goal of our research into the development of renal fibroses and renal and urothelial tumours was to develop relevant in vitro models for investigating the mechanisms caused by ochratoxin effects," said Alexandra Heussner.

A number of primary cell cultures from different species, including humans, rats and pigs, as well renal cell lines have been used for such investigations. The cytotoxicity of the toxins was investigated under different conditions, including individual effects, effects resulting from repeated exposure and level of toxicity of mycotoxin mixtures. "We found that the two major representatives of the ochratoxin family, OTA and OTB, are produced by the same species and can occur simultaneously. Although OTA and OTB are quite similar, they nevertheless lead to different effects in the cells. In addition, we were able to show that, upon toxin removal, the cytotoxic effects of OTB were only reversible with difficulty, or not at all, whereas they were reversible with OTA," explained Alexandra Heussner. While other mycotoxins have acute toxic effects, the danger of ochratoxins lies in their chronic effect.

Cultured kidney cells exposed to ochratoxin: A, untreated control; B, 20 μM OTA (48 h); C, 20 μM OTB (48 h) © A.H. Heussner, 2001

Production of an antibody using hybridoma technology

Since mycotoxins have no immunogenic effect, the toxin needs to be modified to enable the researchers to produce antibodies using the hybridoma technology (production of hybrid cell lines). The researchers therefore modify the toxin so that it leads to an immunogenic effect in an organism, a mouse for example. This is done by fusing the toxin molecule to a much larger protein from another, distantly related organism. The mice are then immunised with this toxin-protein complex and they produce antibodies against the intruder. The antibody-producing cells are subsequently isolated from the mouse spleen and used for the production of immortal cells. "The greatest problem is to find the cell that produces the sought-after antibody since the murine cells also produce antibodies against the protein to which the toxin is attached. Further characterisation of the positive hybridoma clones leads to the selection of the most suitable candidate, which can then be cultivated in greater quantities to isolate the sought-after antibody," said Alexandra Heussner describing the technology used to produce a specific monoclonal antibody against ochratoxin B. Future investigations will be aimed at achieving a complete characterisation of the antibodies to explore potential laboratory applications, e.g. Western blot analyses and enzyme-linked immunosorption (ELISA), for the ability to efficiently identify the toxin in blood samples.

Prevention is the best countermeasure

In order to protect consumers from mycotoxin-related risks, many countries have defined regulatory limits for mycotoxins due to the fact that even small mycotoxin concentrations may lead to long-term cytotoxic effects. However, no regulations have been put in place for food products such as beer, meat products and spices that are frequently contaminated with mycotoxin. "Since only random samples are taken, it is virtually impossible to avoid exposing consumers to mycotoxins. The only sensible and effective solution to this would be to prevent the mycotoxins from growing during transport and storage," explains Alexandra Heussner.

Further information:

Alexandra H. Heussner
Human and Environmental Toxicology
University of Konstanz
Universitätsstraße 10
78457 Konstanz
Tel.: +49 (0)7531/88-4106
E-mail: alexandra.heussner(at)uni-konstanz.de

Website address: https://www.biooekonomie-bw.de/en/articles/news/invisible-danger-with-long-term-consequences