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Paracetamol/acetaminophen (N-acetyl-p-aminophenol, APAP) overdose is one of the most important causes of drug-induced liver injury worldwide. Hepatotoxicity induced by APAP is mainly caused by the production of N-acetyl-p-benzoquinone imine (NAPQI), a highly reactive intermediate formed predominantly via the cytochrome P450 2E1. Here, we used human studies andin vitromodels to demonstrate that NAPQI-derived thiomethyl metabolites identified using high-resolution mass spectrometry could serve to monitor NAPQI detoxification and elimination in patients (after intake at recommended dose or after intoxication), and to study inter-individual variability in NAPQI production. Usingin vitrohuman models, we showed that these thiomethyl metabolites are directly linked to NAPQI detoxification since they are mainly formed after exposure to glutathione-derived conjugates via an overlooked pathway called the thiomethyl shunt. These long-term thiomethyl metabolites have great potential in future clinical studies in order to provide a more reliable history of APAP ingestion in case of acute intoxication or to study underlying causes involved in APAP-induced hepatotoxicity.One Sentence SummaryThiomethyl metabolites are new markers to monitor the elimination of the toxic N-acetyl-p-benzoquinone imine after therapeutic use or intoxication.

International audience; Sample preparation of biological samples can have a substantial impact on the coverage of small molecules detectable using liquid chromatography–high-resolution mass spectrometry (LC-HRMS). This initial step is particularly critical for the detection of externally derived chemicals and their metabolites (internal chemical exposome) generally present at trace levels. Hence, our objective was to investigate how blood sample preparation methods affect the detection of low-abundant chemicals and to propose alternative methods to improve the coverage of the internal chemical exposome. We performed a comprehensive evaluation of 12 sample preparation methods (SPM) using phospholipid and protein removal plates (PLR), solid phase extraction plates (SPE), supported liquid extraction cartridge (SLE), and conventionally used protein precipitation (PPT). We implemented new quantitative and qualitative criteria for nontargeted analyses (detection frequency, recoveries, repeatability, matrix effect, low-level spiking significance, method detection limits, throughput, and ease of use) to amply characterize these SPM in a step-by-step-type approach. As a final step, PPT and one PLR plate were applied to cohort plasma and serum samples injected in triplicate to monitor batch repeatability, and annotation was performed on the related data sets to compare the respective impacts of these SPM. We demonstrate that sample preparation significantly affects both the range of observable compounds and the level at which they can be observed (only 43%–54% of total features are overlapping between the two SPM). We propose to use PPT and PLR on the same samples by implementing a simple analytical workflow as their complementarity would allow the broadening of the visible chemical space.

Les avancées techniques en spectrométrie de masse à haute résolution (SMHR), concomitantes au développement d’outils bio-informatiques, permettent aujourd’hui la détection simultanée de plusieurs dizaines de milliers de signaux chimiques dans des matrices biologiques, correspondant à des molécules d’origine exogène (dont les xénobiotiques) et à des molécules endogènes. Ces nouvelles approches reposant sur la SMHR, dites « non ciblées » car sans a priori, représentent une opportunité unique pour caractériser à grande échelle l’exposition de populations humaines aux composés chimiques (ce que l’on appelle exposome chimique interne), et ainsi mieux appréhender leur rôle dans la survenue de maladies chroniques.

International audience; The recent advances of novel methodologies such as non-targeted and suspect screening based on high-resolution mass spectrometry (HRMS) have paved the way to a new paradigm for exposure assessment. These methodologies allow to profile simultaneously thousands of small unknown molecules present in environmental and biological samples, and therefore hold great promises in order to identify more efficiently hazardous contaminants potentially associated with increased risks of developing adverse health outcomes. In order to further explore the potential of these methodologies and push the transition from research applications towards regulatory purposes, robust harmonized quality standards have to be implemented. Here, we discuss the feasibility of using ISO/IEC 17025: 2017 as a guideline to implement non-targeted and suspect screening methodologies in laboratories, whether it is for accreditation purposes or not. More specifically, we identified and then discussed how specificities of non-targeted HRMS methodology can be accounted for in order to comply with the specific items of ISO/IEC 17025: 2017. We also discussed other specificities of HRMS methodologies (e.g., need for digital storage capacity) that are so far not included in the ISO/IEC 17025 requirements but should be considered. This works aims to fuel and expand the discussion in order to subsidize new opportunities of harmonization for non-targeted and suspect screening.

The technological advances in high-resolution mass spectrometry (HRMS), associated with the development of bioinformatics tools, allows the simultaneous detection of tens of thousands of chemical signals in biological matrices, including exogenous (i.e. xenobiotics) and endogenous molecules. These novel approaches based on HRMS, called "non-targeted" approaches, provide a unique opportunity to capture exposures to a wide range of chemicals (i.e. the internal chemical exposome) in populations, and to better understand the links between chemical exposures and the occurrence of chronic diseases.Exposome chimique et approches « non ciblées » - Un changement de paradigme pour évaluer l’exposition des populations aux contaminants chimiques.Les avancées techniques en spectrométrie de masse à haute résolution (SMHR), concomitantes au développement d’outils bio-informatiques, permettent aujourd’hui la détection simultanée de plusieurs dizaines de milliers de signaux chimiques dans des matrices biologiques, correspondant à des molécules d’origine exogène (dont les xénobiotiques) et à des molécules endogènes. Ces nouvelles approches reposant sur la SMHR, dites « non ciblées » car sans a priori, représentent une opportunité unique pour caractériser à grande échelle l’exposition de populations humaines aux composés chimiques (ce que l’on appelle exposome chimique interne), et ainsi mieux appréhender leur rôle dans la survenue de maladies chroniques.

Background Knowledge of exact signalling events during migraine attacks is lacking. Various substances are known to trigger migraine attacks in patients and calcitonin gene-related peptide antagonising drugs are effective against migraine pain. Here, we investigated the signalling pathways involved in three different mouse models of provoked migraine and relate them to calcitonin gene-related peptide and other migraine-relevant targets. Methods In vivo mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim-induced migraine were applied utilising tactile sensitivity to von Frey filaments as measuring readout. Signalling pathways involved in the three models were dissected by use of specific knockout mice and chemical inhibitors. In vivo results were supported by ex vivo wire myograph experiments measuring arterial dilatory responses and ex vivo calcitonin gene-related peptide release from trigeminal ganglion and trigeminal nucleus caudalis from mice. Results Glyceryl trinitrate-induced hypersensitivity was dependent on both prostaglandins and transient receptor potential cation channel, subfamily A, member 1, whereas cilostazol- and levcromakalim-induced hypersensitivity were independent of both. All three migraine triggers activated calcitonin gene-related peptide signalling, as both receptor antagonism and antibody neutralisation of calcitonin gene-related peptide were effective inhibitors of hypersensitivity in all three models. Stimulation of trigeminal ganglia and brain stem tissue samples with cilostazol and levcromakalim did not result in release of calcitonin gene-related peptide, and vasodilation following levcromakalim stimulation was independent of CGRP receptor antagonism. Conclusion The mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim- induced migraine all involve calcitonin gene-related peptide signalling in a complex interplay between different cell/tissue types. These models are useful in the study of migraine mechanisms.

International audience; The technological advances of cutting-edge high-resolution mass spectrometry (HRMS) have set the stage for a new paradigm for exposure assessment. However, some adjustments of the metabolomics workflow are needed before HRMS-based methods can detect the low-abundant exogenous chemicals in human matrixes. It is also essential to provide tools to speed up marker identifications. Here, we first show that metabolomics software packages developed for automated optimization of XCMS parameters can lead to a false negative rate of up to 80% for chemicals spiked at low levels in blood. We then demonstrate that manual selection criteria in open-source (XCMS, MZmine2) and vendor software (MarkerView, Progenesis QI) allow to decrease the rate of false negative up to 4% (MZmine2). We next report an MS1 automatized suspect screening workflow that allows for a rapid preannotation of HRMS data sets. The novelty of this suspect screening workflow is to combine several predictors based on m/z, retention time (R(t)) prediction models, and isotope ratio to generate intermediate and global scorings. Several R(t) prediction models were tested and hierarchized (PredRet, Retip, retention time indices, and a log P model), and a nonlinear scoring was developed to account for R(t) variations observed within individual runs. We then tested the efficiency of this suspect screening tool to detect spiked and nonspiked chemicals in human blood. Compared to other existing annotation tools, its main advantages include the use of R(t) predictors using different models, its speed, and the use of efficient scoring algorithms to prioritize preannotated markers and reduce false positives.

International audience; The holistic characterisation of the human internal chemical exposome using high-resolution mass spectrometry (HRMS) would be a step forward to investigate the environmental ætiology of chronic diseases with an unprecedented precision. HRMS-based methods are currently operational to reproducibly profile thousands of endogenous metabolites as well as externally-derived chemicals and their biotransformation products in a large number of biological samples from human cohorts. These approaches provide a solid ground for the discovery of unrecognised biomarkers of exposure and metabolic effects associated with many chronic diseases. Nevertheless, some limitations remain and have to be overcome so that chemical exposomics can provide unbiased detection of chemical exposures affecting disease susceptibility in epidemiological studies. Some of these limitations include (i) the lack of versatility of analytical techniques to capture the wide diversity of chemicals; (ii) the lack of analytical sensitivity that prevents the detection of exogenous (and endogenous) chemicals occurring at (ultra) trace levels from restricted sample amounts, and (iii) the lack of automation of the annotation/identification process. In this article, we discuss a number of technological and methodological limitations hindering applications of HRMS-based methods and propose initial steps to push towards a more comprehensive characterisation of the internal chemical exposome. We also discuss other challenges including the need for harmonisation and the difficulty inherent in assessing the dynamic nature of the internal chemical exposome, as well as the need for establishing a strong international collaboration, high level networking, and sustainable research infrastructure. A great amount of research, technological development and innovative bio-informatics tools are still needed to profile and characterise the "invisible" (not profiled), "hidden" (not detected) and "dark" (not annotated) components of the internal chemical exposome and concerted efforts across numerous research fields are paramount.