BACKGROUND:Persistent Organic Pollutants (POP), including polychlorinated biphenyls (PCBs) and organochlorine pesticides, are established neurotoxicants in experimental models; yet it remains uncertain whether exposures in the general population increase the risk to develop brain aging pathologies. We assessed the prospective associations of plasma POP concentrations with three dementia-related outcomes in a population-based cohort of older adults. METHODS:Analyses included 515 participants from the Three-City Study, free of dementia at baseline at the time of blood measurements (1999-2000, mean age 72.5), who underwent up to 8 repeated assessments of cognitive function and dementia over 17 years and up to 3 neuroimaging examinations over 10 years. Plasma concentrations of 15 PCBs, 12 organochlorine pesticides and 1 brominated flame retardant were measured by gas chromatography coupled with tandem mass spectrometry (detection rate ≥5 %), and a POP score summarizing overall exposure was derived via factorial analysis. Associations with dementia risk, longitudinal changes in cognitive function (composite measure of four tests) and in brain volume (medial temporal lobe) were analyzed using Cox and linear mixed models, adjusted for baseline age, sex, education, apolipoprotein E (APOE) genotype, body mass index and total lipid concentrations. RESULTS:In multivariable-adjusted models, neither individual POPs nor the total POP score were significantly and consistently associated with dementia-related outcomes. Significant interactions were observed between APOE genotype and highly-chlorinated PCBs (congeners 180, 194, and 196-203) across all outcomes (p for interaction ≤0.05), whereby adverse associations were seen in carriers of the APOE-ɛ4 allele, whereas opposing trends were observed in non-carriers. CONCLUSION:Overall, this prospective study does not provide robust evidence to support an adverse association between exposure to POPs in the general population and the risk of all-cause dementia, cognitive decline, or brain atrophy in older adults.

Acetaminophen (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. Although, the medical management of APAP intoxication is well known, research is still ongoing to identify markers that could help to predict adverse issues after APAP intoxication. In this study, we aimed to study APAP biotransformation pathways in a cohort of patients with proven acute APAP intoxication to identify new biomarkers using state-of-the-art high-resolution mass spectrometry (HRMS) methodologies that could help improve the diagnosis of intoxication as well as patient follow-up. We used a cohort of 37 patients whom blood plasma samples were stratified according to the collection time after APAP intoxication. Our results showed that direct phase II metabolites from glucuronidation and sulfation pathways remain the main markers of APAP consumption. Our study also revealed that several oxidative pathways produce significant metabolites (including catechol ones) that could also help to monitor the intoxication and the elimination of the hepatotoxic NAPQI. In particular, significant levels of thiomethyl metabolites derived from the glutathione-NAPQI conjugates could be detected with a delay in their kinetics of appearance.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) enables profiling of hundreds of externally derived chemicals and their metabolites in human biofluids to capture the internal chemical exposome. Nevertheless, developing sensitive and robust high-throughput methodologies to scale up exposomics remains challenging. We compared two validated sample preparation methods (SPMs) based on protein precipitation (PPT) and PPT combined with phospholipid removal (PLR) in two cohorts using blood plasma (n = 109) and serum (n = 75) to assess their impact on LC-HRMS repeatability and chemical coverage. Additional filtration on PLR plates allowed the injection of extracts twice concentrated without affecting LC-HRMS repeatability. The comparison of chemical coverage between SPMs was then assessed using 184 compounds (including a large variety of exogenous chemicals) annotated with a suspect screening strategy. Most chemicals (78-86%) were detected with both SPMs, but a substantial fraction showed preferential (>2-fold, 37-50%) or exclusive (14-22%) detection with one SPM, reflecting differences in matrix effects and/or recoveries. Low-abundance chemicals were more sensitive to the choice of SPM, though no predictive trends related to physicochemical properties emerged, likely due to higher analytical uncertainty. Overall, both SPMs provided acceptable chemical coverage for large-scale exposomics. Nonetheless, the compound-dependent nature of detection highlights the value of applying complementary SPMs when feasible.

Persistent Organic Pollutants (POP), including polychlorinated biphenyls (PCB) and organochlorine pesticides (OCP), are established neurotoxicants, especially during developmental periods. Mechanisms of actions include disruption in neurotransmitter systems, calcium dyshomeostasis, oxidative stress and certain OCPs also promote amyloidogenesis in experimental models. Yet, it remains uncertain whether low‐grade environmental exposures in the general population increase the risk to develop brain aging pathologies. We assessed the prospective associations of plasma POP concentrations with three dementia‐related outcomes in a population‐based cohort of older adults. We included 515 participants from the French Three‐City Study, free of dementia at baseline at the time of blood measurements (1999‐2000, mean age 72.5; 19% APOE‐ɛ4 carriers), who underwent up to 8 repeated assessments of cognitive function and dementia over 17 years and 3 neuroimaging examinations over 10 years. Plasma concentrations of 12 OCPs and 15 PCBs were measured by GC/MS/MS, and a POP score summarizing overall exposure was derived via factorial analysis. Associations with dementia risk, and longitudinal changes in cognitive function (composite measure of four tests) and volume of the medial temporal lobe (MTL) were analyzed using Cox and linear mixed models, adjusted for baseline age, sex, education, APOE genotype, body mass index and total lipid concentrations. Median plasma concentrations were in accordance with expected values in the population age range. In multivariable‐adjusted models, neither individual POPs nor the POP factorial score were significantly associated with any dementia‐related outcome. Significant interactions were observed between APOE genotype and highly‐chlorinated PCBs (congeners 180, 194, and 196‐203) across all outcomes ( p for interaction ≤0.05): adverse associations were seen in carriers of the APOE‐ɛ4 allele (e.g. for each SD increase in PCB 194, HR [95% CI] for dementia = 1.51 [1.03‐2.21]; β coefficient [95% CI] for cognitive decline and MTL atrophy = ‐0.15 [‐0.34; 0.04] and ‐0.42 [‐0.74; ‐0.10] SD per decade, respectively), whereas opposing trends were observed in non‐carriers. Overall, this prospective study does not provide robust evidence to support an adverse association between low‐grade environmental exposure to POPs in the general population and the risk of all‐cause dementia, cognitive decline, or brain atrophy in older adults.

Non-targeted and suspect screening analysis using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) holds great promise to comprehensively characterize complex chemical mixtures. Data preprocessing is a crucial part of the process, however, some limitations are observed: (i) peak-picking and feature extraction might be incomplete, especially for low abundant compounds, and (ii) limited reproducibility has been observed between laboratories and software for detected features and their relative quantification. We first conducted a critical review of existing solutions that could improve the reproducibility of preprocessing for LC-HRMS. Solutions include providing repositories and reporting guidelines, open and modular processing workflows, public benchmark datasets, tools to optimize the data preprocessing and to filter out false positive detections. We then propose harmonized quality assurance/quality control guidelines that would allow to assess the sensitivity of feature detection, reproducibility, integration accuracy, precision, accuracy, and consistency of data preprocessing for human biomonitoring, food and environmental communities.

The chemical burden on the environment and human population is increasing. Consequently, regulatory risk assessment must keep pace to manage, reduce, and prevent adverse impacts on human and environmental health associated with hazardous chemicals. Surveillance of chemicals of known, emerging, or potential future concern, entering the environment-food-human continuum is needed to document the reality of risks posed by chemicals on ecosystem and human health from a one health perspective, feed into early warning systems and support public policies for exposure mitigation provisions and safe and sustainable by design strategies. The use of less-conventional sampling strategies and integration of full-scan, high-resolution mass spectrometry and effect-directed analysis in environmental and human monitoring programmes have the potential to enhance the screening and identification of a wider range of chemicals of known, emerging or potential future concern. Here, we outline the key needs and recommendations identified within the European Partnership for Assessment of Risks from Chemicals (PARC) project for leveraging these innovative methodologies to support the development of next-generation chemical risk assessment.

ObjectifsLe paracétamol (APAP) est un des médicaments antalgiques les plus utilisés et impliqué dans de très nombreux cas d’intoxication aiguë volontaire. Le métabolisme de l’APAP est décrit depuis 1980 et est présenté en deux voies : un métabolisme majoritaire de phase II direct par glucuroconjugaison et sulfoconjugaison de l’APAP pour respectivement donner l’APAP-glucuronide (APAP-GLU) et l’APAP-sulfate (APAP-SUL) et un métabolisme oxydatif minoritaire mais toxéfiant médié principalement par le CYP2E1 et donnant son métabolite hépatotoxique, le NAPQI. Récemment, ce métabolisme a été revisité grâce à des approches non ciblées, en identifiant des métabolites issus d’une voie de biotransformation peu connue (la voie du thiomethyl shunt). Ces métabolites sont issus du NAPQI conjugué au glutathion, et sont détectés plus tardivement par rapport aux métabolites de phase II, probablement en raison de la circulation entéro-hépatique. L’objectif de cette étude est d’étudier la cinétique de métabolisation de l’APAP dont ceux issus de la voie du thiomethyl shunt dans une population de sujets intoxiqués par de l’APAP, et de déterminer si ces nouveaux biomarqueurs pourraient aider à améliorer le diagnostic ainsi que le suivi des patients intoxiqués.MéthodeQuarante-deux échantillons plasmatiques et 4 urines de 31 patients (25 femmes, 6 hommes) admis au CHU de Rennes pour suspicion d’intoxication à l’APAP ont été recueillis à différents temps après l’ingestion supposée. Les prélèvements ont eu lieu majoritairement 4heures après la prise supposée afin d’évaluer la pertinence du traitement par l’antidote (N-Acetylcysteine). Certains patients ont également eu d’autres prélèvements jusqu’à 5 jours après l’ingestion. Ces échantillons ont été extraits par précipitation des protéines par du méthanol froid (ratio 4 :1). Après évaporation, 2μL des extraits sont séparés sur une colonne Acquity UHPLC HSS-T3 (1,0mm×150mm×1,8μm) maintenue à 40°C à un débit de 0,10ml/min sur un système Exon UHPLC (AB SCIEX, USA) couplé à un spectromètre de masse haute résolution AB SCIEX X500R Q-TOF-MS (SCIEX, Canada) en mode électrospray négatif et analysé en mode Full Scan (50–1100Da). Les spectres de fragmentations permettant l’élucidation structurale ont été générés à l’aide d’un mode IDA. Les résultats ont été traités avec le logiciel ScieX OS.RésultatsLa cohorte de patients intoxiqués a un âge médian de 20 ans et une concentration plasmatique moyenne en paracétamol de 67mg/L. Parmi les métabolites identifiés, nous retrouvons principalement les métabolites traditionnels : APAP-GLU et APAP-SUL dans le plasma, notamment à des temps précoces (4h). En stratifiant les prélèvements en fonction du temps par rapport à la prise, nous observons une décroissance de l’intensité du paracétamol et de ses métabolites traditionnels à partir de 4h. Comme décrit précédemment, les signaux correspondant aux dérivés thiométhylés (notamment S-CH3-APAP-S et SO-CH3-APAP-S) présentent une intensité maximale retardée par rapport aux autres métabolites ; ils sont faiblement détectables avant 4h et augmentent en fonction du temps pour être maximum après 24h. Nous observons également qu’une prise en charge par la NAC ne semble pas modifier le métabolisme du paracétamol. L’analyse des urines montre une présence importante d’APAP-GLU et APAP-SUL, mais des métabolites jusqu’ici sous-estimés comme ceux provenant d’une autre voie oxydative, i.e. la voie des catéchols (ex : 3-OCH3-APAP-GLU) ou du dérivé au glutathion issus de la voie mercapturique et du thiométhyl shunt (ex : NAC-APAP, S-CH3-APAP-S et SO-CH3-APAP-S) sont intensément détectés.ConclusionLe métabolisme du paracétamol est décrit depuis longtemps, mais celui-ci peut être rediscuté grâce aux outils analytiques de plus en plus sensibles. Les métabolites de la voie du thiomethylshunt possèdent une cinétique retardée des métabolites standards ce qui fait d’eux d’excellents marqueurs tardifs d’intoxication au paracétamol.

Over the past few decades, numerous environmental chemicals from solvents to pesticides have been suggested to be involved in the development and progression of neurodegenerative diseases. Most of the evidence has accumulated from occupational or cohort studies in humans or laboratory research in animal models, with a range of chemicals being implicated. What has been missing is a systematic approach analogous to genome-wide association studies, which have identified dozens of genes involved in Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases. Fortunately, it is now possible to study hundreds to thousands of chemical features under the exposome framework. This Perspective explores how advances in mass spectrometry make it possible to generate exposomic data to complement genomic data and thereby better understand neurodegenerative diseases. Nongenetic factors contribute to the onset, progression and severity of neurodegenerative diseases. Here, the authors describe how exposomics, the systematic analysis of environmental factors, can help neuroscientists understand these diseases.

A collaborative trial involving 16 participants from nine European countries was conducted within the NORMAN network in efforts to harmonise suspect and non-target screening of environmental contaminants in whole fish samples of bream (Abramis brama). Participants were provided with freeze-dried, homogenised fish samples from a contaminated and a reference site, extracts (spiked and non-spiked) and reference sample preparation protocols for liquid chromatography (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS). Participants extracted fish samples using their in-house sample preparation method and/or the protocol provided. Participants correctly identified 9–69 % of spiked compounds using LC-HRMS and 20–60 % of spiked compounds using GC-HRMS. From the contaminated site, suspect screening with participants’ own suspect lists led to putative identification of on average ∼145 and ∼20 unique features per participant using LC-HRMS and GC-HRMS, respectively, while non-target screening identified on average ∼42 and ∼56 unique features per participant using LC-HRMS and GC-HRMS, respectively. Within the same sub-group of sample preparation method, only a few features were identified by at least two participants in suspect screening (16 features using LC-HRMS, 0 features using GC-HRMS) and non-target screening (0 features using LC-HRMS, 2 features using GC-HRMS). The compounds identified had log octanol/water partition coefficient (KOW) values from −9.9 to 16 and mass-to-charge ratios (m/z) of 68 to 761 (LC-HRMS and GC-HRMS). A significant linear trend was found between log KOW and m/z for the GC-HRMS data. Overall, these findings indicate that differences in screening results are mainly due to the data analysis workflows used by different participants. Further work is needed to harmonise the results obtained when applying suspect and non-target screening approaches to environmental biota samples.

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 and in vitro models 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. Using in vitro human 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 Summary Thiomethyl metabolites are new markers to monitor the elimination of the toxic N-acetyl-p-benzoquinone imine after therapeutic use or intoxication.

In an increasingly chemically polluted environment, rapidly characterizing the human chemical exposome (i.e., chemical mixtures accumulating in humans) at the population scale is critical to understand its impact on health. High-resolution mass spectrometry (HRMS) profiling of complex biological matrices can theoretically provide a comprehensive picture of chemical exposures. However, annotating the detected chemical features, particularly low-abundant ones, remains a significant obstacle to implementing such approaches at a large scale. We present Scannotation (https://github.com/scannotation/Scannotation_software), an automated and user-friendly suspect screening tool for the rapid pre-annotation of HRMS preprocessed data sets. This software tool combines several MS1 chemical predictors, i.e., m/z, experimental and predicted retention times, isotopic patterns, and neutral loss patterns, to score the proximity between features and suspects, thus efficiently prioritizing tentative annotations to verify. Scannotation and MS-DIAL4 were used to annotate blood serum samples of 75 Breton adolescents. Scannotation's combination of MS1-based chemical predictors allowed us to annotate 89 chemically diverse environmental compounds with high confidence (confirmed by MS2 when available). These compounds included 62% of emerging molecules, for which no toxicological or human biomonitoring data are reported in the literature. The complementarity observed with MS-DIAL4 results demonstrates the relevance of Scannotation for the efficient pre-annotation of large-scale exposomics data sets.

Paracetamol/acetaminophen (N-acetyl-p-aminophenol, APAP) is a top selling analgesic used in more than 600 prescription and non-prescription pharmaceuticals. To study efficiently some of the potential undesirable effects associated with increasing APAP consumption (e.g., developmental disorders, drug-induced liver injury), there is a need to improve current APAP biomonitoring methods that are limited by APAP short half-life. Here, we demonstrate using high-resolution mass spectrometry (HRMS) in several human studies that APAP thiomethyl metabolite conjugates (S-methyl-3-thioacetaminophen sulfate and S-methyl-3-thioacetaminophen sulphoxide sulfate) are stable biomarkers with delayed excretion rates compared to conventional APAP metabolites, that could provide a more reliable history of APAP ingestion in epidemiological studies. We also show that these biomarkers could serve as relevant clinical markers to diagnose APAP acute intoxication in overdosed patients, when free APAP have nearly disappeared from blood. Using in vitro liver models (HepaRG cells and primary human hepatocytes), we then confirm that these thiomethyl metabolites are directly linked to the toxic N-acetyl-p-benzoquinone imine (NAPQI) elimination, and produced via an overlooked pathway called the thiomethyl shunt pathway. Further studies will be needed to determine whether the production of the reactive hepatotoxic NAPQI metabolites is currently underestimated in human. Nevertheless, these biomarkers could already serve to improve APAP human biomonitoring, and investigate, for instance, inter-individual variability in NAPQI production to study underlying causes involved in APAP-induced hepatotoxicity. Overall, our findings demonstrate the potential of exposomics-based HRMS approach to advance towards a better precision for human biomonitoring.

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.

STUDY QUESTION Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function? SUMMARY ANSWER While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action. WHAT IS KNOWN ALREADY Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy. STUDY DESIGN, SIZE, DURATION This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404. PARTICIPANTS/MATERIALS, SETTING, METHODS Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments. MAIN RESULTS AND THE ROLE OF CHANCE Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells). LIMITATIONS, REASONS FOR CAUTION The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404. WIDER IMPLICATIONS OF THE FINDINGS We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy. STUDY FUNDING/COMPETING INTEREST(S) D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE—Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

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.

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.

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.

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.