Periodic Reporting for period 1 - BIOTOXDoc (Safe food in a world of changing climate: The doctoral training programme to develop novel control, mitigation and risk assessment methods for biotoxins)
Reporting period: 2023-09-01 to 2025-08-31
There is a massive and urgent need to ensure security and safety of the food supply of the growing world population. However, agriculture and food industries continue to be vulnerable to problems of contamination with biotoxins produced by plants, algae and particularly by fungi. Global warming and extreme weather events make the occurrence of these toxic metabolites even less predictable. Alarmingly, the EU currently faces a lack of food safety specialists. These challenges lay the foundation for BIOTOXDoc – Safe food in a world of changing climate: The doctoral training programme to develop novel control, mitigation and risk assessment methods for biotoxins. The objective of BIOTOXDoc is to train doctoral students in a broad range of skills and complementary competencies – necessary to innovate various scientific fields and approaches so urgently needed to control and mitigate biotoxins – by taking advantage of a multidisciplinary, multi-sectoral team of world-class experts. The training and research will include development of early warning systems and on‑site testing by portable mass spectrometry. Doctoral candidates will develop novel detoxification strategies of biotoxins and will assess the combined toxicity of co‑occurring biotoxins. Moreover, doctoral candidates will develop much-needed rapid as well as confirmatory tests for biotoxins and aim to close major gaps in our current knowledge of biotoxins. The major common link between all doctoral candidates, working on a wide range of biotoxins at different points along the food and feed chain, is the influence of climate change on biotoxin occurrence and the resulting demand of revised strategies to mitigate its impact on the European population.
• An empirical model on the distribution and risk for mycotoxin producing fungal pathogens in Europe was developed. The model was used to calculate a mycotoxin risk index driven by ambient temperature thresholds relevant to A. flavus, F. graminearum, and F. verticillioides. It uses climatic conditions to quantify the microclimatic conditions suitable for mycotoxin habitats as well as maize and wheat distribution data. A northward fungal expansion was identified, especially in Southern and Central Europe.
• A model for aflatoxin prediction in maize was re-developed, incorporating high-resolution meteorological datasets. A hybrid approach combining both reanalysis and forecast data was implemented for seasonal risk prediction. Also, an experimental workflow was developed to study fungal co-occurrence involving A. flavus, F. verticillioides, and F. graminearum under controlled environmental conditions. Quantitative data were generated using spectrophotometric growth measurements and qPCR-based quantification of fungal contributions through temperature gradient.
• A method for fungal volatile component (VOC) sampling and analysis using solid-phase microextraction fibers combined with two-dimensional GC high-resolution mass spectrometry (GC×GC-hrMS) was developed. Fungal growth and changes in the volatile profiles were monitored. Also a liquid chromatography high-resolution mass spectrometry (LC-hrMS) method was developed for mycotoxin determination. Monitored VOCs, such as trichodiene and other sesquiterpenes, showed correlations with mycotoxin presence. First field tests with a portable GC-MS system were conducted.
• A large-scale screening of pyrrolizidine (PA) and tropane (TA) alkaloid as well as mycotoxins in more than 200 rooibos products from the European market was conducted, using liquid chromatography tandem mass spectrometry (LC-MS/MS). Secondly, an experimental study on the fate of PAs during the ensiling of forage contaminated with toxic weed species was perfomed. Thirdly, ergot alkaloids were determined in rye and sclerotia samples.
• In vitro cell culture studies with aflatoxin B1 (AFB1), sterigmatocystin (STC) and versicolorin A (VerA) were conducted. No significant cytotoxicity was detected under any tested condition. AFB1 and STC induced significant DNA damage. Following recovery, all 3 mycotoxins induced dose-dependent DNA strand breaks. To complement the in vitro results, porcine intestinal explants were used to evaluate the individual effects of the same mycotoxins on the expression of DNA damage response genes. Preliminary results did not reveal significant differences in gene expression.
• Microvascular endothelial cells from mice and humans and intestinal Caco-2 cells were treated with okadaic acid (OA) to compare their responses. The treated cells were tested for cell viability, cellular toxicity of OA, interleukin release, reactive oxygen species release, mitochondrial membrane potential and ATP production. The differentiated cells were also tested for viability when treated with OA, cyclosporin A and both, and intracellular and extracellular levels of cyclophilin A, B, C and D were measured. The results show that Okadaic acid triggers NFκB and STAT3 phosphorylation followed by a release of inflammatory markers in human and mouse endothelial cells. Also human cells are 5 times more sensitive to the toxins than mice cells. This supports the urgent need for an update on current TEF.
• 53 fungal strains were screened for the production of ochratoxin A (OTA). A multi-step purification protocol was developed, including liquid-liquid extraction, solid-phase extraction, and preparative HPLC, yielding several grams of high-purity OTA. The workflow was adapted for 13C-labelling, resulting in 20 mg of high-purity 13C-OTA. The labelled compound has been used for treatment of maize ears to identify soluble OTA metabolites. Treated maize samples have been measured with LC-HRMS, and metabolite identification is in progress.
• Enzymatic detoxification pathways of aflatoxin B1 were investigated in certain bacteria. Both the generation of mutants lacking certain biosynthesis genes and the heterologous expression of candidate bacterial enzyme in Escherichia coli were tested. Although the knockout was not successful, likely due to the low recombination efficiency, the expressed enzymes degraded aflatoxin B1 in vitro. LC-MS/MS analysis of the degradation products revealed the presence of multiple AFB1 metabolites. Purification of metabolites for further toxicological testing is ongoing.
• Bivalves encode a chemical defensome that participate in the metabolism of xenobiotic compounds. A component of the defensome in vertebrates are Pregnane X receptor (PXR) orthologs. The responses of those receptors, when exposed to natural agonist compounds, was characterized in Mytilus galloprovincialis and Ruditapes decussatus. 13 out of 18 tested compounds exhibited moderate to potent agonist activity on at least one paralogs of M. galloprovincialis and R. decussatus and 7 were chosen as promising agonist compounds to be evaluated in future in vivo assays.
• Our project associated partner from the UK studied rapid detection of tropane alkaloids using Surface-Enhanced Raman Spectroscopy (SERS). Spherical gold nanoparticles were synthesized, enabeling the generation of high-quality SERS spectra. Chemometric tools were employed, resulting in promising sensitivity and specificity of the developed method. Initial tests using a hand-held Raman spectrometer showed that the SERS signals for atropine were consistent with those obtained from the high-resolution microscope.
• A model for aflatoxin prediction in maize was re-developed, incorporating high-resolution meteorological datasets. A hybrid approach combining both reanalysis and forecast data was implemented for seasonal risk prediction. Also, an experimental workflow was developed to study fungal co-occurrence involving A. flavus, F. verticillioides, and F. graminearum under controlled environmental conditions. Quantitative data were generated using spectrophotometric growth measurements and qPCR-based quantification of fungal contributions through temperature gradient.
• A method for fungal volatile component (VOC) sampling and analysis using solid-phase microextraction fibers combined with two-dimensional GC high-resolution mass spectrometry (GC×GC-hrMS) was developed. Fungal growth and changes in the volatile profiles were monitored. Also a liquid chromatography high-resolution mass spectrometry (LC-hrMS) method was developed for mycotoxin determination. Monitored VOCs, such as trichodiene and other sesquiterpenes, showed correlations with mycotoxin presence. First field tests with a portable GC-MS system were conducted.
• A large-scale screening of pyrrolizidine (PA) and tropane (TA) alkaloid as well as mycotoxins in more than 200 rooibos products from the European market was conducted, using liquid chromatography tandem mass spectrometry (LC-MS/MS). Secondly, an experimental study on the fate of PAs during the ensiling of forage contaminated with toxic weed species was perfomed. Thirdly, ergot alkaloids were determined in rye and sclerotia samples.
• In vitro cell culture studies with aflatoxin B1 (AFB1), sterigmatocystin (STC) and versicolorin A (VerA) were conducted. No significant cytotoxicity was detected under any tested condition. AFB1 and STC induced significant DNA damage. Following recovery, all 3 mycotoxins induced dose-dependent DNA strand breaks. To complement the in vitro results, porcine intestinal explants were used to evaluate the individual effects of the same mycotoxins on the expression of DNA damage response genes. Preliminary results did not reveal significant differences in gene expression.
• Microvascular endothelial cells from mice and humans and intestinal Caco-2 cells were treated with okadaic acid (OA) to compare their responses. The treated cells were tested for cell viability, cellular toxicity of OA, interleukin release, reactive oxygen species release, mitochondrial membrane potential and ATP production. The differentiated cells were also tested for viability when treated with OA, cyclosporin A and both, and intracellular and extracellular levels of cyclophilin A, B, C and D were measured. The results show that Okadaic acid triggers NFκB and STAT3 phosphorylation followed by a release of inflammatory markers in human and mouse endothelial cells. Also human cells are 5 times more sensitive to the toxins than mice cells. This supports the urgent need for an update on current TEF.
• 53 fungal strains were screened for the production of ochratoxin A (OTA). A multi-step purification protocol was developed, including liquid-liquid extraction, solid-phase extraction, and preparative HPLC, yielding several grams of high-purity OTA. The workflow was adapted for 13C-labelling, resulting in 20 mg of high-purity 13C-OTA. The labelled compound has been used for treatment of maize ears to identify soluble OTA metabolites. Treated maize samples have been measured with LC-HRMS, and metabolite identification is in progress.
• Enzymatic detoxification pathways of aflatoxin B1 were investigated in certain bacteria. Both the generation of mutants lacking certain biosynthesis genes and the heterologous expression of candidate bacterial enzyme in Escherichia coli were tested. Although the knockout was not successful, likely due to the low recombination efficiency, the expressed enzymes degraded aflatoxin B1 in vitro. LC-MS/MS analysis of the degradation products revealed the presence of multiple AFB1 metabolites. Purification of metabolites for further toxicological testing is ongoing.
• Bivalves encode a chemical defensome that participate in the metabolism of xenobiotic compounds. A component of the defensome in vertebrates are Pregnane X receptor (PXR) orthologs. The responses of those receptors, when exposed to natural agonist compounds, was characterized in Mytilus galloprovincialis and Ruditapes decussatus. 13 out of 18 tested compounds exhibited moderate to potent agonist activity on at least one paralogs of M. galloprovincialis and R. decussatus and 7 were chosen as promising agonist compounds to be evaluated in future in vivo assays.
• Our project associated partner from the UK studied rapid detection of tropane alkaloids using Surface-Enhanced Raman Spectroscopy (SERS). Spherical gold nanoparticles were synthesized, enabeling the generation of high-quality SERS spectra. Chemometric tools were employed, resulting in promising sensitivity and specificity of the developed method. Initial tests using a hand-held Raman spectrometer showed that the SERS signals for atropine were consistent with those obtained from the high-resolution microscope.