Periodic Reporting for period 1 - HAZARDOmics (Liquid Chromatography-Ion Mobility-Mass Spectrometry and Capillary Electrophoresis-Mass Spectrometry as ground-breaking approaches to expand the boundaries of metabolomics in chemical risk assessment)
Reporting period: 2018-10-01 to 2020-09-30
During our life, we are exposed to various chemical agents such as food contaminants, environmental pollutants, drugs, allergens, etc., some of which are endocrine disrupting-compounds (EDCs) such as bisphenol A or polychlorinated biphenyls (PCBs). Therefore, these chemicals can cause adverse effects on our health and cause chronic diseases. From a public health perspective, it is of great interest to understand the risks associated with exposure to chemicals and the underlying modes of action (MoA) of these chemicals for the effective implementation of health prevention strategies. In this sense, chemical risk assessment is a great challenge for today’s society considering the large number of chemical substances available on the European Union (EU) market. In addition, to implement effective prevention strategies towards chemical exposure, chemical risk assessment is required to address health effects related to current exposure scenarios beyond the unrealistic and high exposure doses traditionally used in toxicological studies.
Within this framework, metabolomics has recently emerged as an innovative approach to address the current challenges of chemical risk assessment, including exposure to chemical mixtures and low dose chemical exposures. The HAZARDOmics project applies metabolomics as a novel strategy in risk assessment to generate new knowledge about the effects of chemicals related to exposure to low levels of BPA and PCBs. Furthermore, HAZARDOmics also involves the application of less frequent analytical tools in metabolomics studies, specifically ion mobility spectrometry (IMS) and capillary electrophoresis-mass spectrometry (CE-MS), for risk assessment of BPA and PCBs. Specifically, HAZARDOmics pursues analytical objectives: 1) expanding the current technical boundaries of metabolomics in risk assessment by integrating IMS into traditional liquid-chromatography-high resolution mass spectrometry (LC-HRMS) workflows, 2) implementing CE-HRMS as a complementary analytical technique to support metabolomics in risk assessment; as well as specific risk assessment objectives: 3) the identification of effect biomarkers related to exposure to a PCB ‘cocktail’ within a risk assessment framework, 4) the discovery of effect biomarkers associated with exposure to low doses of BPA.
Within this framework, metabolomics has recently emerged as an innovative approach to address the current challenges of chemical risk assessment, including exposure to chemical mixtures and low dose chemical exposures. The HAZARDOmics project applies metabolomics as a novel strategy in risk assessment to generate new knowledge about the effects of chemicals related to exposure to low levels of BPA and PCBs. Furthermore, HAZARDOmics also involves the application of less frequent analytical tools in metabolomics studies, specifically ion mobility spectrometry (IMS) and capillary electrophoresis-mass spectrometry (CE-MS), for risk assessment of BPA and PCBs. Specifically, HAZARDOmics pursues analytical objectives: 1) expanding the current technical boundaries of metabolomics in risk assessment by integrating IMS into traditional liquid-chromatography-high resolution mass spectrometry (LC-HRMS) workflows, 2) implementing CE-HRMS as a complementary analytical technique to support metabolomics in risk assessment; as well as specific risk assessment objectives: 3) the identification of effect biomarkers related to exposure to a PCB ‘cocktail’ within a risk assessment framework, 4) the discovery of effect biomarkers associated with exposure to low doses of BPA.
HAZARDomics addresses the metabolic perturbations in different groups of pigs (4-month old), including control groups, caused by exposure to PCBs (20 ng/kg bw/day of Arochlor 1260) and BPA (0.85-4 µg/kg bw/day of BPA) during an exposure period of 22 days. Serum samples have been investigated as the toxicological matrix and a Bligh and Dyer procedure has been applied for sample preparation. As a result, two different phases (hydrophilic and lipophilic phases) have been obtained for each sample, which were intended for metabolomics and lipidomics investigation, respectively, applying LC-HRMS as analytical tool. After data processing involving multivariate analysis [i.e. principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA)], a wide range of variables were identified as possible effect biomarkers related to PCBs exposure. Subsequent annotation of metabolites led to the identification of the tryptophan pathway as one of the metabolic pathways altered by exposure to PCBs at low concentration levels. Furthermore, the lipidomics study showed a relevant disturbance of glycerophosphocholines levels in serum caused by exposure to PCBs. On the other hand, it was also observed that lipid levels in serum were affected by exposure to low doses of BPA.
In an attempt to implement IMS technology into LC-HRMS workflows for metabolomics studies, the reproducibility of collision cross section (CCS) measurements between different IMS modes was also investigated. CCS can be considered as a novel molecular characteristic in metabolomics studies that provides additional information to retention indexes and mass spectra to support metabolite annotation. However, the complete implementation of this parameter for annotation purposes still requires fundamental research to give confidence in the reported CCS databases. Steroids (n ≈ 112 ions) were selected as metabolites of interest, since exposure to EDCs such as PCBs and BPA can alter steroid metabolism. CCS measurements were carried out with three different IMS modes, including drift tube ion mobility spectrometry (DTIMS), traveling wave ion mobility spectrometry (TWIMS) and trapped ion mobility spectrometry (TIMS). In general, deviations of less than 2% were obtained for all measurements and IMS modes compared to reference values, and which is the currently accepted threshold for CCS measurements compared to CCS databases generated with the same IMS mode.
Finally, as HAZARDOmics involved the implementation of different analytical tools in metabolomics approaches used for risk assessment purposes, gas chromatography (GC)-HRMS was applied for the detection of effect biomarkers related to PCBs exposure. In this sense, cholesterol was unequivocally identified as effect biomarker of PBCS exposure, although it was not previously detected in the metabolomics study applying the LC-HRMS workflow. This highlights the fact that metabolomics studies in risk assessment require multiplatforms that involve various analytical technologies to obtain as much information as possible on the metabolites and their concentration levels impacted by chemical exposure.
In an attempt to implement IMS technology into LC-HRMS workflows for metabolomics studies, the reproducibility of collision cross section (CCS) measurements between different IMS modes was also investigated. CCS can be considered as a novel molecular characteristic in metabolomics studies that provides additional information to retention indexes and mass spectra to support metabolite annotation. However, the complete implementation of this parameter for annotation purposes still requires fundamental research to give confidence in the reported CCS databases. Steroids (n ≈ 112 ions) were selected as metabolites of interest, since exposure to EDCs such as PCBs and BPA can alter steroid metabolism. CCS measurements were carried out with three different IMS modes, including drift tube ion mobility spectrometry (DTIMS), traveling wave ion mobility spectrometry (TWIMS) and trapped ion mobility spectrometry (TIMS). In general, deviations of less than 2% were obtained for all measurements and IMS modes compared to reference values, and which is the currently accepted threshold for CCS measurements compared to CCS databases generated with the same IMS mode.
Finally, as HAZARDOmics involved the implementation of different analytical tools in metabolomics approaches used for risk assessment purposes, gas chromatography (GC)-HRMS was applied for the detection of effect biomarkers related to PCBs exposure. In this sense, cholesterol was unequivocally identified as effect biomarker of PBCS exposure, although it was not previously detected in the metabolomics study applying the LC-HRMS workflow. This highlights the fact that metabolomics studies in risk assessment require multiplatforms that involve various analytical technologies to obtain as much information as possible on the metabolites and their concentration levels impacted by chemical exposure.
HAZARDOmics highlights the potential of metabolomics to address current risk assessment challenges, specifically to carry out the hazard identification of chemicals at low doses. In particular, HAZARDOmics provides new evidence for risk assessment of PCBs and BPA at low doses. This project has addressed exposure to both types of chemicals at environmental exposure doses and, in both cases, metabolism disturbances have been observed. Further research is needed to conclude whether such metabolic perturbations represent a mere adaptation of the organism to chemical exposure or, on the contrary, are indicators of adverse health effects that can lead to disease development. Within this framework, the effect biomarkers identified for PBCs exposure help to decipher the MoA of such chemicals, thus establishing the link between PCBs exposure and disease development. The identification of effect biomarkers is supported by the implementation of IMS in our analytical workflow, providing information on the geometric conformation of ions in the gas phase (i.e. collision cross section). This project contributes to the implementation of this molecular characteristic to support metabolite annotation.
The health consequences of exposure to chemicals are a growing concern among the European population. The institutions of the European Union are aware of such concern, which is why the European Commission has set the objective of achieving a toxic-free environment within the current green and digital transition. To achieve this purpose, it is necessary to understand the effects of current chemical exposure scenarios involving exposure to low-dose chemical mixtures, beyond the high exposure concentrations commonly assessed in traditional chemical risk assessments. Therefore, HAZARDOmics contributes to the implementation of novel approaches (i.e. metabolomics) and analytical tools (i.e. IMS) to adequately address actual chemical exposure scenarios from a risk assessment perspective.
The health consequences of exposure to chemicals are a growing concern among the European population. The institutions of the European Union are aware of such concern, which is why the European Commission has set the objective of achieving a toxic-free environment within the current green and digital transition. To achieve this purpose, it is necessary to understand the effects of current chemical exposure scenarios involving exposure to low-dose chemical mixtures, beyond the high exposure concentrations commonly assessed in traditional chemical risk assessments. Therefore, HAZARDOmics contributes to the implementation of novel approaches (i.e. metabolomics) and analytical tools (i.e. IMS) to adequately address actual chemical exposure scenarios from a risk assessment perspective.