Periodic Reporting for period 4 - HUMYCO (Investigating the Human Mycobolome through Uniting Large-scale Epidemiological and Mechanistic Poly-omic Designs)
Periodo di rendicontazione: 2025-06-01 al 2025-11-30
Chronic low-dose intake of multiple mycotoxins are hypothesized to be associated with an increased risk of developing human renal, colorectal and hepatocellular carcinomas. HUMYCO refers to a unique, holistic & multi(cross)-disciplinary research field, aiming at comprehensively investigating the human mycobolome through uniting large-scale epidemiological & mechanistic designs using a poly-omic approach. Our aim is to disseminate and publicize the research project’s results to the general population and to other important (economic) stakeholders by maximizing their impact. Accessible knowledge to all stakeholders is imperative for sustainability of fundamental human health research.
HuMyco is compiled of 4 strategic objectives as detailed: 1. To describe the human toxicokinetic profile of mycotoxins; 2. To elucidate the metabolomic profile of mycotoxins using UHPLC-HRMS; 3. To investigate the nature and extent of associations between estimated external & internal dietary mycotoxin exposures (considering single and multi-mycotoxin exposures) and developing renal cancer (RC), hepatocellular cancer (HCC) & colorectal cancer (CRC); and 4. To provide new insights into the role of mycotoxins in the aetiology and development of human RC, HCC and CRC. In conclusion, the HuMyco action has successfully advanced a holistic and interdisciplinary framework to investigate the human mycobolome and its potential role in carcinogenesis. By integrating large-scale epidemiological data with mechanistic and poly-omic approaches, the project has generated novel insights into chronic low-dose, multi-mycotoxin exposure and its relevance to human health. The action achieved its strategic objectives by (i) characterizing human toxicokinetic profiles of key mycotoxins, thereby improving understanding of internal exposure dynamics; (ii) elucidating comprehensive metabolomic signatures of mycotoxins using state-of-the-art UHPLC-HRMS methodologies and machine learning; and (iii) establishing robust associations between estimated external and internal dietary mycotoxin exposures, considering both single and combined exposures, and the risk of renal, hepatocellular, and colorectal cancers. Collectively, these findings contribute new evidence supporting the role of mycotoxins in the aetiology and development of RC, HCC, and CRC and other diseases. Beyond its scientific achievements, HuMyco has emphasized accessibility, sustainability, and societal impact by actively disseminating its results to the general public, policymakers, and economic stakeholders. This inclusive knowledge-transfer strategy enhances awareness of food safety risks, supports evidence-based decision-making, and reinforces the importance of preventive strategies in public health. Overall, HuMyco has laid a strong foundation for future research, regulatory considerations, and translational initiatives aimed at reducing mycotoxin-related health risks and promoting long-term human health protection.
HuMyco is compiled of 4 strategic objectives as detailed: 1. To describe the human toxicokinetic profile of mycotoxins; 2. To elucidate the metabolomic profile of mycotoxins using UHPLC-HRMS; 3. To investigate the nature and extent of associations between estimated external & internal dietary mycotoxin exposures (considering single and multi-mycotoxin exposures) and developing renal cancer (RC), hepatocellular cancer (HCC) & colorectal cancer (CRC); and 4. To provide new insights into the role of mycotoxins in the aetiology and development of human RC, HCC and CRC. In conclusion, the HuMyco action has successfully advanced a holistic and interdisciplinary framework to investigate the human mycobolome and its potential role in carcinogenesis. By integrating large-scale epidemiological data with mechanistic and poly-omic approaches, the project has generated novel insights into chronic low-dose, multi-mycotoxin exposure and its relevance to human health. The action achieved its strategic objectives by (i) characterizing human toxicokinetic profiles of key mycotoxins, thereby improving understanding of internal exposure dynamics; (ii) elucidating comprehensive metabolomic signatures of mycotoxins using state-of-the-art UHPLC-HRMS methodologies and machine learning; and (iii) establishing robust associations between estimated external and internal dietary mycotoxin exposures, considering both single and combined exposures, and the risk of renal, hepatocellular, and colorectal cancers. Collectively, these findings contribute new evidence supporting the role of mycotoxins in the aetiology and development of RC, HCC, and CRC and other diseases. Beyond its scientific achievements, HuMyco has emphasized accessibility, sustainability, and societal impact by actively disseminating its results to the general public, policymakers, and economic stakeholders. This inclusive knowledge-transfer strategy enhances awareness of food safety risks, supports evidence-based decision-making, and reinforces the importance of preventive strategies in public health. Overall, HuMyco has laid a strong foundation for future research, regulatory considerations, and translational initiatives aimed at reducing mycotoxin-related health risks and promoting long-term human health protection.
Over the course of the project, extensive progress has been made in characterising human exposure to mycotoxins, their metabolism, and their potential health impacts through a combination of analytical, experimental, and epidemiological approaches. Biological samples, including urine, blood, and faeces, were successfully collected from volunteers and control subjects. These samples were analysed using UHPLC-MS/MS, followed by comprehensive data processing. This work enabled the development of detailed toxicokinetic profiles and models for each investigated mycotoxin, providing key insights into their absorption, distribution, metabolism, and excretion in humans.
In parallel, a wide range of in vitro incubation studies was conducted for the targeted mycotoxins. Both in vitro-derived samples and in vivo samples from the human studies were analysed using UHPLC-HRMS. The integration of these datasets allowed for the systematic identification and structural characterisation of a broad spectrum of mycotoxin metabolites. This resulted in a comprehensive and well-curated metabolite dataset that significantly advances current knowledge on mycotoxin biotransformation.
To support exposure assessment at the population level, an EPIC end-user database was established for dietary mycotoxin exposure calculations. This resource enabled large-scale epidemiological analyses within the full EPIC cohort, leading to the investigation of associations between mycotoxin exposure and the risk of colorectal, kidney, and hepatocellular cancers. These analyses provide important evidence linking exposure biomarkers to disease outcomes.
All necessary ethical approvals were obtained to conduct three complementary sub-studies, and biological samples were successfully transferred and processed. Multi-mycotoxin biomarker profiles were measured in case-control settings across EPIC, Malawian serum, and Groningen plasma cohorts using UHPLC-MS/MS. Subsequent data processing and statistical analyses enabled the identification of exposure patterns and their associations with disease risk across geographically and demographically diverse populations.
Mechanistic investigations further strengthened the project’s findings. In vivo analyses of ochratoxin A (OTA) were completed, including the identification of a putative mutational signature. Additionally, in vitro studies for OTA, fumonisin B1 (FB1), deoxynivalenol (DON), patulin (PAT), and citrinin (CIT) were finalised, providing mechanistic insight into their toxicological effects and supporting the interpretation of epidemiological associations.
The project has generated a unique and comprehensive body of knowledge on mycotoxin exposure, metabolism, and health effects. The analytical methodologies, toxicokinetic models, and curated metabolite datasets developed during the project represent valuable tools for the scientific community and for regulatory risk assessment.
In parallel, a wide range of in vitro incubation studies was conducted for the targeted mycotoxins. Both in vitro-derived samples and in vivo samples from the human studies were analysed using UHPLC-HRMS. The integration of these datasets allowed for the systematic identification and structural characterisation of a broad spectrum of mycotoxin metabolites. This resulted in a comprehensive and well-curated metabolite dataset that significantly advances current knowledge on mycotoxin biotransformation.
To support exposure assessment at the population level, an EPIC end-user database was established for dietary mycotoxin exposure calculations. This resource enabled large-scale epidemiological analyses within the full EPIC cohort, leading to the investigation of associations between mycotoxin exposure and the risk of colorectal, kidney, and hepatocellular cancers. These analyses provide important evidence linking exposure biomarkers to disease outcomes.
All necessary ethical approvals were obtained to conduct three complementary sub-studies, and biological samples were successfully transferred and processed. Multi-mycotoxin biomarker profiles were measured in case-control settings across EPIC, Malawian serum, and Groningen plasma cohorts using UHPLC-MS/MS. Subsequent data processing and statistical analyses enabled the identification of exposure patterns and their associations with disease risk across geographically and demographically diverse populations.
Mechanistic investigations further strengthened the project’s findings. In vivo analyses of ochratoxin A (OTA) were completed, including the identification of a putative mutational signature. Additionally, in vitro studies for OTA, fumonisin B1 (FB1), deoxynivalenol (DON), patulin (PAT), and citrinin (CIT) were finalised, providing mechanistic insight into their toxicological effects and supporting the interpretation of epidemiological associations.
The project has generated a unique and comprehensive body of knowledge on mycotoxin exposure, metabolism, and health effects. The analytical methodologies, toxicokinetic models, and curated metabolite datasets developed during the project represent valuable tools for the scientific community and for regulatory risk assessment.
During the final period of the action, major progress was achieved across methodological, epidemiological, analytical, and mechanistic research lines, substantially strengthening the scientific foundation of HuMyco.
A protocol for a comprehensive systematic review was accepted for publication, consolidating and critically appraising evidence on the burden of dietary mycotoxins on human health, with explicit attention to co-exposure risks. In parallel, the World Health Organization (WHO) commissioned the HuMyco team to conduct a systematic review on the burden of aflatoxins in relation to hepatocellular carcinoma and growth impairment. Led by the ERC Principal Investigator and an international expert panel, both review protocols were finalized following extensive literature screening, and the reviews are currently being completed.
Significant advances were made in early-life exposure research. A pilot study within the Portuguese mother–child “EarlyMyco” cohort assessed mycotoxin occurrence in breast milk and urine during early infancy. In addition, the “EarlyLife Myco-cohort” was established in Bari in 2021, recruiting twenty mother–child pairs at childbirth to investigate linked exposure profiles during critical developmental windows.
On the analytical level, standardized templates for multi-mycotoxin validation and identification were developed in line with EU and international guidelines and are now fully implemented as a laboratory gold standard. A new collaboration with Agriculture and Agri-Food Canada enabled participation in a multi-laboratory study evaluating N-alkylpyridinium-3-sulfonates (NAPS) for retention time normalization in LC–MS-based mycotoxin analysis.
To address challenges in multi-exposure assessment, HuMyco advanced statistical methodology through an opinion paper published in the World Mycotoxin Journal, proposing robust tools to manage multicollinearity and high-dimensional data. In parallel, machine learning and deep learning models were established to integrate dietary, metabolomic, genomic, and metagenomic data in the context of hepatocellular carcinoma.
Mechanistic research expanded into toxico-epigenetics through collaboration with ProGenTomics (UGent), applying advanced mass spectrometry to investigate histone post-translational modifications following single and combined mycotoxin exposures. Exposure concentrations were defined based on prior toxicity studies, and analyses are ongoing with the aim of a high-impact publication.
Finally, the establishment of the genome–exposome cohort GLORIA – Health Monitor (www.gezondheidsmonitor.gent) represents a key long-term outcome, providing a sustainable platform for future integrative exposure–health research.
Overall, HuMyco successfully consolidated its interdisciplinary framework and laid a robust foundation for continued investigation into the health impacts of chronic, low-dose, multi-mycotoxin exposure.
A protocol for a comprehensive systematic review was accepted for publication, consolidating and critically appraising evidence on the burden of dietary mycotoxins on human health, with explicit attention to co-exposure risks. In parallel, the World Health Organization (WHO) commissioned the HuMyco team to conduct a systematic review on the burden of aflatoxins in relation to hepatocellular carcinoma and growth impairment. Led by the ERC Principal Investigator and an international expert panel, both review protocols were finalized following extensive literature screening, and the reviews are currently being completed.
Significant advances were made in early-life exposure research. A pilot study within the Portuguese mother–child “EarlyMyco” cohort assessed mycotoxin occurrence in breast milk and urine during early infancy. In addition, the “EarlyLife Myco-cohort” was established in Bari in 2021, recruiting twenty mother–child pairs at childbirth to investigate linked exposure profiles during critical developmental windows.
On the analytical level, standardized templates for multi-mycotoxin validation and identification were developed in line with EU and international guidelines and are now fully implemented as a laboratory gold standard. A new collaboration with Agriculture and Agri-Food Canada enabled participation in a multi-laboratory study evaluating N-alkylpyridinium-3-sulfonates (NAPS) for retention time normalization in LC–MS-based mycotoxin analysis.
To address challenges in multi-exposure assessment, HuMyco advanced statistical methodology through an opinion paper published in the World Mycotoxin Journal, proposing robust tools to manage multicollinearity and high-dimensional data. In parallel, machine learning and deep learning models were established to integrate dietary, metabolomic, genomic, and metagenomic data in the context of hepatocellular carcinoma.
Mechanistic research expanded into toxico-epigenetics through collaboration with ProGenTomics (UGent), applying advanced mass spectrometry to investigate histone post-translational modifications following single and combined mycotoxin exposures. Exposure concentrations were defined based on prior toxicity studies, and analyses are ongoing with the aim of a high-impact publication.
Finally, the establishment of the genome–exposome cohort GLORIA – Health Monitor (www.gezondheidsmonitor.gent) represents a key long-term outcome, providing a sustainable platform for future integrative exposure–health research.
Overall, HuMyco successfully consolidated its interdisciplinary framework and laid a robust foundation for continued investigation into the health impacts of chronic, low-dose, multi-mycotoxin exposure.