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.

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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.

The analgesic paracetamol (N-acetyl-4-aminophenol, APAP) is commonly used to relieve pain, fever and malaise. While sales have increased worldwide, a growing body of experimental and epidemiological evidence has suggested APAP as a possible risk factor for various health disorders. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is criticical. However, retrospectively assessing pharmaceutical use of APAP in humans is challenging because of its short half-life. The objective of this study was to address the key biomonitoring issues with APAP using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. Using non-targeted analyses based on high-resolution mass spectrometry, we identified in a controlled longitudinal exposure study with male volunteers, unrecognized APAP metabolites with delayed formation and excretion rates. We postulate that these metabolites are formed via the thiomethyl shunt after the enterohepatic circulation as already observed in rodents. Importantly, the conjugated thiomethyl metabolites were (i) of comparable diagnostic sensitivity as the free APAP and its phase II conjugates detected by current methods; (ii) had delayed peak levels in blood and urine compared to other APAP metabolites and therefore potentially extend the window of exposure assessment; and (iii) provide relevant information regarding metabolic pathways of interest from a toxicological point of view. Including these metabolites in future APAP biomonitoring methods provide an option to decrease potential underestimation of APAP use and challenges the notion that the standard methods in biomonitoring based exclusively on the parent compound and its phase II metabolites are adequate for human biomonitoring of non-persistant chemical such as APAP. <br>

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.

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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.