Periodic Reporting for period 3 - HUMYCO (Investigating the Human Mycobolome through Uniting Large-scale Epidemiological and Mechanistic Poly-omic Designs)
Reporting period: 2023-12-01 to 2025-05-31
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 below:
O1 To describe the human toxicokinetic profile of mycotoxins; O2 To elucidate the metabolomic profile of mycotoxins using UHPLC-HRMS; O3 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); O4 To provide new insights into the role of mycotoxins in the aetiology and development of human RC, HCC and CRC.
HuMyco is compiled of 4 strategic objectives as detailed below:
O1 To describe the human toxicokinetic profile of mycotoxins; O2 To elucidate the metabolomic profile of mycotoxins using UHPLC-HRMS; O3 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); O4 To provide new insights into the role of mycotoxins in the aetiology and development of human RC, HCC and CRC.
WP1
M1. Achieve the urine, blood and faecal samples of the volunteers and control subjects: 100%
M2. Analysis and data-processing of the samples using UHPLC-MS/MS: 100%
M3. Establish a toxicokinetic profile and model for each investigated mycotoxin: 100%
WP2
M1. In vitro incubations for each mycotoxin:100%
M2. In vitro samples analysed by UHPLC-HRMS: 100%
M3. In vivo samples (from WP1) analysed by UHPLC-HRMS: 100%
M4. Gathering all the mycotoxin metabolites data, mostly gained via WP1/2: 100%
M5. ID-description of each metabolite: 100%
WP3
M1. Establishment of an EPIC end-user database for dietary mycotoxin exposure calculations: 100%
M2. Associative analyses with colorectal, kidney and hepatocellular cancer risk using the full EPIC cohort: 100%
WP4
M1. Acquiring ethical approvals for the three sub-studies for performing the analysis at Ghent University: 100%
M2. Shipping of biological samples to Ghent University: 100%
M3. Multi-mycotoxin biomarker profiles analysed for cases and controls in the three sub-studies using UHPLC-MS/MS: EPIC samples755%, Malawian serum samples 100%, Groningen plasma samples 100%.
M4. Data processing and associative analyses: 75%
WP5
M1. Finalization of OTA in vivo analysis with putative OTA mutational signature identified: 75%
M2. Finalization of OTA, FB1, DON, PAT and CIT in vitro analysis: 75%
M1. Achieve the urine, blood and faecal samples of the volunteers and control subjects: 100%
M2. Analysis and data-processing of the samples using UHPLC-MS/MS: 100%
M3. Establish a toxicokinetic profile and model for each investigated mycotoxin: 100%
WP2
M1. In vitro incubations for each mycotoxin:100%
M2. In vitro samples analysed by UHPLC-HRMS: 100%
M3. In vivo samples (from WP1) analysed by UHPLC-HRMS: 100%
M4. Gathering all the mycotoxin metabolites data, mostly gained via WP1/2: 100%
M5. ID-description of each metabolite: 100%
WP3
M1. Establishment of an EPIC end-user database for dietary mycotoxin exposure calculations: 100%
M2. Associative analyses with colorectal, kidney and hepatocellular cancer risk using the full EPIC cohort: 100%
WP4
M1. Acquiring ethical approvals for the three sub-studies for performing the analysis at Ghent University: 100%
M2. Shipping of biological samples to Ghent University: 100%
M3. Multi-mycotoxin biomarker profiles analysed for cases and controls in the three sub-studies using UHPLC-MS/MS: EPIC samples755%, Malawian serum samples 100%, Groningen plasma samples 100%.
M4. Data processing and associative analyses: 75%
WP5
M1. Finalization of OTA in vivo analysis with putative OTA mutational signature identified: 75%
M2. Finalization of OTA, FB1, DON, PAT and CIT in vitro analysis: 75%
A protocol for a systematic review was accepted for publication, aiming to cover all existing scientific information relevant to the overarching burden of dietary mycotoxins on human health, and addressing co-exposure risks, as well as providing evidence appraisal, in a systematic manner. In addition, a task was granted by the World Health Organization (WHO) to construct a systematic review on the burden of aflatoxins on hepatocellular cancer, and growth impairment. With a dedicated team of international experts (ERC PI is head of this team) we are currently finalized the two protocols for systematic reviews, upon screening the publications and compiling the two reviews.
A pilot study has been performed on the Portuguese mother-child “EarlyMyco” cohort to determine the occurrence of mycotoxins in breast milk and urine during the first months of life of the newborn. A second birth cohort was established in Bari during 2021 in collaboration with Prof. Nicola Laforgia and Prof. Antonio Moretti: “EarlyLife Myco-cohort”. Twenty mothers hospitalised during childbirth were recruited to test the hypothesis of an exposure profile, linking mother-child pairs in terms of mycotoxin occurrence. Finally, during the past year, new templates for validation and identification of multi-mycotoxin analysis were standardized in alignment with documents of 2002/657/EC, SANTE/12089/2016 and ICH guidelines M10 (2019). These templates are now utilised in the lab as a useful tool and golden standard to confirm the presence of mycotoxins after experimental analysis. In the last months, a new collaboration has been established with Dr. Mark Sumarah (Agriculture and Agri-food Canada) to test the feasibility of N-alkylpyridinium-3-sulfonates (NAPS) as references for the normalisation of retention times. The ERC-research group is participating in a multi-laboratory study using the NAPS retention index system to normalise mycotoxin analysis by LC-MS.
To guide multi-exposure assessment, careful considerations of the statistical approaches available are required. In addition, the issue of multicollinearity in high-dimensional settings of multiple exposure analysis underlies the controversy surrounding the reliability and consistency of statistical conclusions about the exposure-health outcome associations. For that reason, an opinion paper was manuscripted and published in World Mycotoxin Journal in order to propose for the first time convenient and robust tools for data analysis in the context of multi-mycotoxin exposure. Additionally, hepatocellular carcinoma which is the most common type of liver cancer, comprises plenty of aetiological factors such as hepatitis B, C viral infection, chronic alcohol consumption, and mycotoxins, which even complicates the problem of high dimensional data. To deepen the understanding of that disease, novel models with machine learning and deep learning are currently developed to integrate the complexity of dietary information, metabolome, genome and metagenome.
Toxico-epigenetics is a growing field of interest that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms with histone post-translational modifications (hPTMs), amongst others. Integration of hPTM analysis in the field of toxicology has shown to be promising, giving the opportunity to gain advanced and innovative insights into toxicological events. Therefore, a collaboration was established with a research group of highly praised histone experts (ProGenTomics, UGent) that developed a data-dependent acquisition (DDA) mass spectrometry-based method, capable of simultaneously detecting multiple hPTM-changes in human embryonic stem, HepG2 and Caco-2 cells, a.o. to assess not only developmental toxicity, but also hepatotoxicity, nephrotoxicity and other toxicities associated with the exposure to mycotoxins. Consequently, the effect of single as well as multiple-mycotoxins exposure on the hPTM-dynamics is set up to be examined, to get an insight in the epigenetic role mycotoxins play in the human body. Before analysing the hPTMs from exposed HepG2 and Caco-2 cells, the appropriate mycotoxin concentrations were determined based on the cyto- and genotoxicity studies described under WP5. This research strives to obtain a high-impact publication on the impact of multiple-mycotoxins on the hPTM-dynamics.
Establishment of a new genome-exposome population cohort GLORIA - HEALTH MONITOR (www.gezondheidsmonitor.gent)
A pilot study has been performed on the Portuguese mother-child “EarlyMyco” cohort to determine the occurrence of mycotoxins in breast milk and urine during the first months of life of the newborn. A second birth cohort was established in Bari during 2021 in collaboration with Prof. Nicola Laforgia and Prof. Antonio Moretti: “EarlyLife Myco-cohort”. Twenty mothers hospitalised during childbirth were recruited to test the hypothesis of an exposure profile, linking mother-child pairs in terms of mycotoxin occurrence. Finally, during the past year, new templates for validation and identification of multi-mycotoxin analysis were standardized in alignment with documents of 2002/657/EC, SANTE/12089/2016 and ICH guidelines M10 (2019). These templates are now utilised in the lab as a useful tool and golden standard to confirm the presence of mycotoxins after experimental analysis. In the last months, a new collaboration has been established with Dr. Mark Sumarah (Agriculture and Agri-food Canada) to test the feasibility of N-alkylpyridinium-3-sulfonates (NAPS) as references for the normalisation of retention times. The ERC-research group is participating in a multi-laboratory study using the NAPS retention index system to normalise mycotoxin analysis by LC-MS.
To guide multi-exposure assessment, careful considerations of the statistical approaches available are required. In addition, the issue of multicollinearity in high-dimensional settings of multiple exposure analysis underlies the controversy surrounding the reliability and consistency of statistical conclusions about the exposure-health outcome associations. For that reason, an opinion paper was manuscripted and published in World Mycotoxin Journal in order to propose for the first time convenient and robust tools for data analysis in the context of multi-mycotoxin exposure. Additionally, hepatocellular carcinoma which is the most common type of liver cancer, comprises plenty of aetiological factors such as hepatitis B, C viral infection, chronic alcohol consumption, and mycotoxins, which even complicates the problem of high dimensional data. To deepen the understanding of that disease, novel models with machine learning and deep learning are currently developed to integrate the complexity of dietary information, metabolome, genome and metagenome.
Toxico-epigenetics is a growing field of interest that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms with histone post-translational modifications (hPTMs), amongst others. Integration of hPTM analysis in the field of toxicology has shown to be promising, giving the opportunity to gain advanced and innovative insights into toxicological events. Therefore, a collaboration was established with a research group of highly praised histone experts (ProGenTomics, UGent) that developed a data-dependent acquisition (DDA) mass spectrometry-based method, capable of simultaneously detecting multiple hPTM-changes in human embryonic stem, HepG2 and Caco-2 cells, a.o. to assess not only developmental toxicity, but also hepatotoxicity, nephrotoxicity and other toxicities associated with the exposure to mycotoxins. Consequently, the effect of single as well as multiple-mycotoxins exposure on the hPTM-dynamics is set up to be examined, to get an insight in the epigenetic role mycotoxins play in the human body. Before analysing the hPTMs from exposed HepG2 and Caco-2 cells, the appropriate mycotoxin concentrations were determined based on the cyto- and genotoxicity studies described under WP5. This research strives to obtain a high-impact publication on the impact of multiple-mycotoxins on the hPTM-dynamics.
Establishment of a new genome-exposome population cohort GLORIA - HEALTH MONITOR (www.gezondheidsmonitor.gent)