Analytical strategies to profile the internal chemical exposome and the metabolome of human placenta.

As a non-invasive biological matrix, the placenta offers great and novel opportunities to monitor fetal exposure to exogenous chemicals and their biotransformation products (the internal chemical exposome), as well as the biological responses associated, in large-scale epidemiological studies. However, it is first crucial to ensure that analytical methods based on high-resolution mass spectrometry (HRMS) can detect the low abundant components of the internal chemical exposome present in these complex biological matrices. In this study, we aimed to develop a robust analytical method (extraction and sample preparation) sensitive enough to profile the internal chemical exposome and the metabolome of placenta using high-resolution mass spectrometry (HRMS) for future application in mother child cohorts. Several extraction solvents (methanol, methanol/H2O (50/50 v/v), methyl tert-butyl ether/methanol/H2O) were tested and their ability to extract components of the internal exposome and metabolome were compared. Then, sample preparation methods commonly used for metabolomics application (methanolic protein precipitation) were compared with solid phase extraction (SPE), protein and phospholipid removal plates (PPRP) and combination of SPE and PPRP. The methods were compared and validated using qualitative (i.e., numbers of features and chemical classes ID), quantitative parameters adopted from targeted multi-residue analysis (recovery experiments, repeatability and matrix effect) as well as the ability of these methods to be implemented for high-throughput applications. The analytical repeatability of the two most effective methods (methanolic extraction followed by either protein precipitation or PPRP) were tested at the batch level to determine the best concentration factors to be used for improving detection of components of the internal chemical exposome and metabolome without impacting on the analytical response. Finally, these two methods based on protein precipitation and PPRP were tested on 40 placenta samples from the French PELAGIE birth cohort, and annotation was performed on the related datasets to compare the respective impacts of PPT and PPRP. A wide range of exogenous (e.g., biocides, pharmaceuticals, personal care products) and endogenous chemicals (steroids, prostanoids, lipids, carnitins) could be detected and annotated (some of them for the first time in placenta). We show that both methods are complementary but that PPRP allows the injection of more concentrated extracts without impacting the LC repeatability and therefore improve the detection (presence and signal area fold change) of many exogenous and endogenous chemicals.

Further Biochemical Profiling of Hypholoma fasciculare Metabolome Reveals Its Chemogenetic Diversity.

Natural products with novel chemistry are urgently needed to battle the continued increase in microbial drug resistance. Mushroom-forming fungi are underutilized as a source of novel antibiotics in the literature due to their challenging culture preparation and genetic intractability. However, modern fungal molecular and synthetic biology tools have renewed interest in exploring mushroom fungi for novel therapeutic agents. The aims of this study were to investigate the secondary metabolites of nine basidiomycetes, screen their biological and chemical properties, and then investigate the genetic pathways associated with their production. Of the nine fungi selected, Hypholoma fasciculare was revealed to be a highly active antagonistic species, with antimicrobial activity against three different microorganisms: Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. Genomic comparisons and chromatographic studies were employed to characterize more than 15 biosynthetic gene clusters and resulted in the identification of 3,5-dichloromethoxy benzoic acid as a potential antibacterial compound. The biosynthetic gene cluster for this product is also predicted. This study reinforces the potential of mushroom-forming fungi as an underexplored reservoir of bioactive natural products. Access to genomic data, and chemical-based frameworks, will assist the development and application of novel molecules with applications in both the pharmaceutical and agrochemical industries.

Acetaminophen metabolism revisited using non-targeted analyses: Implications for human biomonitoring.

The analgesic paracetamol/acetaminophen (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 in humans. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is critical. 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 issue of potential underestimation of APAP use using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. The question we address is whether investigating additional metabolites than direct phase II conjugates could improve the monitoring of APAP. Using non-targeted analyses based on high-resolution mass spectrometry, we identified, in a controlled longitudinal exposure study with male volunteers, overlooked 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, these 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 therefore provides an option to decrease potential underestimation of APAP use. Moreover, our data challenge 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 a non-persistent chemical such as APAP.

Distinguishing between the metabolome and xenobiotic exposome in environmental field samples analysed by direct-infusion mass spectrometry based metabolomics and lipidomics.

Environmental metabolomics is increasingly used to investigate organismal responses to complex chemical mixtures, including waste water effluent (WWE). In parallel, increasingly sensitive analytical methods are being used in metabolomics studies, particularly mass spectrometry. This introduces a considerable, yet overlooked, challenge that high analytical sensitivity will not only improve the detection of endogenous metabolites in biological specimens but also exogenous chemicals. If these often unknown xenobiotic features are not removed from the "biological" dataset, they will bias the interpretation and could lead to incorrect conclusions about the biotic response. Here we illustrate and validate a novel workflow classifying the origin of peaks detected in biological samples as: endogenous, xenobiotics, or metabolised xenobiotics. The workflow is demonstrated using direct infusion mass spectrometry-based metabolomic analysis of testes from roach exposed to different concentrations of a complex WWE. We show that xenobiotics and their metabolic products can be detected in roach testes (including triclosan, chloroxylenol and chlorophene), and that these compounds have a disproportionately high level of statistical significance within the total (bio)chemical changes induced by the WWE. Overall we have demonstrated that this workflow extracts more information from an environmental metabolomics study of complex mixture exposures than was possible previously.

Global metabolite profiling reveals transformation pathways and novel metabolomic responses in Solea senegalensis after exposure to a non-ionic surfactant.

Alcohol polyethoxylate (AEO) surfactants are widely used in household and industrial products, but the health effects arising from short-term exposure to sublethal concentrations are unknown. A metabolomic approach was used to investigate the biotransformation and effects of exposure to sublethal concentrations of hexaethylene glycol monododecylether (C12EO6) in juvenile sole, Solea senegalensis. After 5 days, C12EO6 was rapidly metabolized in the sole by oxidation, glucuronidation, and ethoxylate chain shortening. C12EO6 exposure at either 146 or 553 µg L(-1) resulted in significant metabolite disruption in liver and blood samples, including an apparent fold increase of >10(6) in the circulating levels of C24 bile acids and C27 bile alcohols, disturbance of glucocorticoid and lipid metabolism, and a 470-fold decrease in levels of the fatty acid transport molecule palmitoyl carnitine. Depuration resulted in rapid elimination of the surfactant and normalization of metabolites toward pre-exposure levels. Our findings show for the first time the ability of metabolomic analyses to discern effects of this AEO on metabolite homeostasis at exposure levels below its no effect concentrations for survival and reproduction in juvenile fish. The pronounced alteration in levels of liver metabolites, phospholipids, and glucocorticoids in S. senegalensis in response to surfactant exposure may indicate that this contaminant could potentially impact a number of health end points in fish.

Methodology for profiling anti-androgen mixtures in river water using multiple passive samplers and bioassay-directed analyses.

The identification of endocrine disrupting chemicals in surface waters is challenging as they comprise a variety of structures which are often present at nanomolar concentrations and are temporally highly variable. Hence, a holistic passive sampling approach can be an efficient technique to overcome these limitations. In this study, a combination of 4 different passive samplers used for sampling polar (POCIS Apharm and POCIS Bpesticide) and apolar compounds (LDPE low density polyethylene membranes, and silicone strips) were used to profile anti-androgenic activity present in river water contaminated by a wastewater effluent. Extracts of passive samplers were analysed using HPLC fractionation in combination with an in vitro androgen receptor antagonist screen (YAS). Anti-androgenic activity was detected in extracts from silicone strips and POCIS A/B at (mean ± SD) 1.1 ± 0.1 and 0.55 ± 0.06 mg flutamide standard equivalents/sampler respectively, but was not detected in LDPE sampler extracts. POCIS samplers revealed higher selectivity for more polar anti-androgenic HPLC fractions compared with silicone strips. Over 31 contaminants were identified which showed inhibition of YAS activity and were potential anti-androgens, and these included fungicides, germicides, flame retardants and pharmaceuticals. This study reveals that passive sampling, using a combination of POCIS A and silicone samplers, is a promising tool for screening complex mixture of anti-androgenic contaminants present in surface waters, with the potential to identify new and emerging structures with endocrine disrupting activity.

The xenometabolome and novel contaminant markers in fish exposed to a wastewater treatment works effluent.

Organisms exposed to wastewater treatment works (WwTW) effluents accumulate complex mixtures of xenobiotics but there is a scarcity of information on the nature and impacts of these chemical mixtures. We applied metabolomics techniques as a novel approach to identify xenobiotics and their metabolites (the xenometabolome) that bioconcentrate in fish exposed to a WwTW effluent. Exposed juvenile rainbow trout (Oncorhynchus mykiss) accumulated surfactants, naphthols, chlorinated xylenols, and phenoxyphenols, chlorophenes, resin acids, mefenamic acid, oxybenzone, and steroidal alkaloids in the bile or plasma, and there were perturbations in the plasma concentrations of bile acids and lipids. Exposure of adult roach (Rutilus rutilus) to 50% or 100% concentrations of the same effluent resulted in dose-dependent increases in plasma concentrations of xenometabolites as well as cyprinol sulfate and taurocholic acid, lysophospholipids, and a decrease in sphingosine levels (a key component of cell membrane lipids). Our findings reveal the highly complex nature of xenobiotics accumulating in effluent-exposed fish, and the great potential of metabolomics for both identifying plasma marker (bio)chemicals for monitoring exposure to wastewater effluents, and for targeting studies on potential consequent impacts on fish health.