There is an increasing awareness of the biohazards posed to both human and animal health by the presence of mycotoxins, the toxins produced by filamentous fungi (molds) genera Aspergillus, Alternaria, Claviceps, Fusarium, Penicillium, and Stachybotrys. Mycotoxins are highly toxic for human and animal organisms, secondary metabolites of molds contaminating cereals, fruits, berries, and vegetables both pre- and post-harvest, other food and feed raw materials and products as well as the environment (soil, water and air). The most common ways of exposure to toxigenic molds and mycotoxins are contaminated food and feed raw materials and products. The deleterious effects of mycotoxins on human and animal health may include both acute food poisonings (alimentary mycotoxicoses) and various hard chronic diseases, depending on concentration and duration of exposure. Mycotoxins are highly stable compounds, and the toxigenic molds producing them are ubiquitous in the environment. These toxins greatly resist decomposition during digestion, so they remain in the food chain in edible products (e.g., meat, fish, eggs and dairy products) or under the form of metabolites of the parent toxin ingested. Temperature treatments such as heating or freezing not affect mycotoxins. Another characteristic feature of mycotoxins is that being hydrophobic (lipophilyc) these compounds have an ability to pass through cell membrane and accumulate in humans and animals tissues. Today contamination of food with mycotoxins represents a major public health problem worldwide, particularly in developing and emerging economy countries, where crop, other food and feed raw materials and products storage conditions are frequently conducive to fungal growth and mycotoxin production, much of the population relies on subsistence farming or on unregulated local markets. Another important concern with mycotoxins is that these toxic compounds were used as biological warfare, e.g. in Southwest Asia and Afghanistan (1974-1981) and during the Gulf War (1991), and thus represent potential bioterrorist weapons.
Mycotoxins have carcinogenic, mutagenic, genotoxic, teratogenic, embryotoxic, hemorrhagic, immunosuppressive, nephrotoxic, hepatotoxic, dermotoxic, neurotoxic, and gonadotoxic effects (affecting reproductive and developmental systems) on human and animal organisms. These compounds are cytotoxic, trigger formation of reactive oxygen species and oxidative stress (enhanced lipid peroxidation in tissues along with decreased concentrations of endogenous antioxidants have been demonstrated as mycotoxins-caused deleterious effects), induce both necrotic and apoptotic cell death including eryptosis. There are reports on damaging effects of mycotoxins on cell membrane function, in particular, related to the ion transport machinery. Among mycotoxins the most harmful and dangerous are aflatoxin B1, ochratoxin A, and T2-toxin produced by Aspergillus and Fusarium mold species, respectively. Development of the efficient measures on neutralization of toxic effects of these compounds and prevention of pathogenic changes they caused in human and animal organisms is currently considered an extremely important and urgent issue worldwide. Here, as potential prospective "antimycotoxic" agents Schiff base cyclic amino acid derivatives may be considered. Our previous studies (supported by ISTC A-1321 and A-1764 grants) performed by using animal and cell culture models of radiation injury have demonstrated that these compounds simultaneously possess free-radical scavenging, anti-cytotoxic, anti-genotoxic and anti-mutagenic properties and ability to elevate capacities of the antioxidant and immune systems. Among them the most efficient are picolinyl-L-phenylalanine, picolinyl-L-tryptophan and nicotinyl-L-tryptophan. Importantly, Schiff bases are used for design and development of many novel physiologically active compounds with fewer side effects. Based on these results we suggest that the above mentioned multifunctional compounds may have protective effects against mycotoxicoses. This suggestion is also based on the results obtained by other research groups indicating anti-mycotoxin activities of some Schiff base derivatives as well as phenylalanine and tryptophan amino acids.
Therefore, the overall aim of the proposed project is to evaluate a potential ability of Schiff base cyclic amino acid derivatives including picolinyl-L-phenylalanine (PLP), picolinyl-L-tryptophan (PLT), nicotinyl-L-tryptophan (NLT) to act against damaging effects of aflatoxin B1 (AFB1), ochratoxin A (OTA), and T2-toxin (T2T) mycotoxins.
To meet the objective of this study the following research tasks will be fulfilled using animal model of mycotoxicoses:
1. Assessment of the ability of PLP, PLT, and NLT to reduce genotoxic effects of AFB1, OTA, and T2T;
2. Assessment of the ability of PLP, PLT, and NLT to reduce immunosuppressive effects of AFB1, OTA, and T2T;
3. Assessment of the ability of PLP, PLT, and NLT to stabilize free radical scavenger system and intensify neutralization of reactive oxygen species during the exposure to AFB1, OTA, and T2T;
4. Assessment of the ability of PLP, PLT, and NLT to decrease accelerated apoptosis rate induced by AFB1, OTA, and T2T;
5. Assessment of the ability of PLP, PLT, and NLT to limit the progression of cytotoxic (necrosis and cell lysis) and hemorrhagic effects of AFB1, OTA, and T2T.
6. Assessment of the ability of PLP, PLT, and NLT to reduce gonadotoxic effects of AFB1, OTA, and T2T.
The works on Project will be mostly implemented in the laboratories of the Institute of Molecular Biology NAS RA, incuding "GMO and mycotoxin testing" analytical laboratory where all necessary equipment and safety conditions required for working with mycotoxins are provided. Each of three selected Schiff base cyclic amino acid derivatives (PLP, PLT, and NLT) will be tested for each of three types of mycotoxicoses (AFB1-, OTA- and T2T-caused). In each experiment three groups of animals will be included: intact animals, animals developed AFB1-, OTA- or T2T-mycotoxicosis, and animals treated with PLP, PLT or NLT after developing mycotoxicosis. Animal care and handling will be performed in the animal house of the Institute of Molecular Biology NAS RA. White pubescent rats will be used in all experiments. Animal care, handling and use in research will be performed according to international regulations adopted by the Ministry of Health of the Republic of Armenia. Methodological design will include a variety of preparative and analytical procedures including enzyme-linked immunosorbent assays, Comet assay and micronuclei test to examine DNA and chromosome damage, photochemiluminescent measurements, flow cytometry, ionometry, histochemical and cytochemical methods to examine cell damage and viability and some other.
Research team of experienced scientists and qualified young specialists will be engaged in the works on the Project, The team has skills and expertise in working with mycotoxins, studying the effects of mycotoxins, developing mycotoxicoses, synthesizing and screening of Schiff base cyclic amino acid derivatives, and in all Project related methodologies. Weapons scientists represent 55.2% of the total number of Project participants. Funding of the proposed Project will provide these scientists with the opportunity to apply the knowledge and experience gained in former defense science and technology laboratories/research groups to solve public health and social problems through implementing peaceful biomedical research. In addition, since the proposed study will be performed in collaboration with foreign scientists, the implementation of the works on Project will facilitate the integration of Armenian scientific community into the international research area.
The results obtained during the implementation of this project will largely contribute to deciphering the molecular and cellular mechanisms of action and biological activities of PLP, PLT, and NLT as well as cyclic amino acids and Schiff base derivatives, in general. Moreover the project implementation will create strong ground for the development of novel therapeutic approaches based on application of Schiff base cyclic amino acids in treatment of mycotoxin-induced health complications in humans and animals.
