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Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. Recently, exposure to phthalates has been associated with preterm birth, low birth weight, and pregnancy loss. There is limited information about the possible mechanisms linking maternal phthalate exposure and placental development, but one such mechanism may be mediated by peroxisome proliferator–activated receptor γ (PPARγ). PPARγ belongs to the nuclear receptor superfamily that regulates, in a ligand-dependent manner, the transcription of target genes. Studies of PPARγ-deficient mice have demonstrated its essential role in lipid metabolism and placental development. In the human placenta, PPARγ is expressed in the villous cytotrophoblast (VCT) and is activated during its differentiation into syncytiotrophoblast. The goal of this study was to investigate the action of mono(2-ethylhexyl) phthalate (MEHP) on PPARγ activity during differentiation of VCTs. We combined immunofluorescence, PPARγ activity/hCG assays, western blotting, and lipidomics analyses to characterize the impacts of physiologically relevant concentrations of MEHP (0.1, 1, and 10 μM) on cultured VCTs isolated from human term placentas. Doses of 0.1 and 1 μM MEHP showed significantly lower PPARγ activity and less VCT differentiation in comparison with controls, whereas, surprisingly, a 10 μM dose had the opposite effect. MEHP exposure inhibited hCG production and significantly altered lipid composition. In addition, MEHP had significant effects on the mitogen-activated protein kinase (MAPK) pathway. This study suggests that MEHP has a U-shaped dose–response effect on trophoblast differentiation that is mediated by the PPARγ pathway and acts as an endocrine disruptor in the human placenta. https://doi.org/10.1289/EHP3730.

In the central nervous system, the process of myelination involves oligodendrocytes that wrap myelin around axons. Myelin sheaths are mainly composed of lipids and ensure efficient conduction of action potentials. Oligodendrocyte differentiation is an essential preliminary step to myelination which, in turn, is a key event of neurodevelopment. Bisphenol A (BPA), a ubiquitous endocrine disruptor, is suspected to disrupt this developmental process and may, thus, contribute to several neurodevelopmental disorders. In this study, we assessed the effect of BPA on oligodendrocyte differentiation through a comprehensive analysis of cell lipidome by UHPLC-HRMS. For this purpose, we exposed the oligodendroglial cell line Oli-neu to several BPA concentrations for 72 h of proliferation and another 72 h of differentiation. In unexposed cells, significant changes occurred in lipid distribution during Oli-neu differentiation, including an increase in characteristic myelin lipids, sulfatides, and ethanolamine plasmalogens, and a marked remodeling of phospholipid subclasses and fatty acid contents. Moreover, BPA induced a decrease in sulfatide and phosphatidylinositol plasmalogen contents and modified monounsaturated/polyunsaturated fatty acid relative contents in phospholipids. These effects counteracted the lipid remodeling accompanying differentiation and were confirmed by gene expression changes. Altogether, our results suggest that BPA disrupts lipid remodeling accompanying early oligodendrocyte differentiation.

The in-depth knowledge of lipid biological functions needs a comprehensive structural annotation including a method to locate fatty acid unsaturations, which remains a thorny problem. For this purpose, we have associated Grubbs’ cross-metathesis reaction and liquid chromatography hyphenated to tandem mass spectrometry to locate double bond positions in lipid species. The pretreatment of lipid-containing samples by Grubbs’ catalyst and an appropriate alkene generates substituted lipids through cross-metathesis reaction under mild, chemoselective, and reproducible conditions. A systematic LC-MS/MS analysis of the reaction mixture allows locating unambiguously the double bonds in fatty acid side chains of phospholipids, glycerolipids, and sphingolipids. This method has been successfully applied at a nanomole scale to commercial standard mixtures consisting of 10 lipid subclasses as well as in lipid extracts of human corneal epithelial (HCE) cell line allowing to pinpoint double bond of more than 90 species. This method has also been useful to investigate the lipid homeostasis alteration in an in vitro model of corneal toxicity, i.e., HCE cells incubated with benzalkonium chloride. The association of cross-metathesis and tandem mass spectrometry appears suitable to locate double bond positions in lipids involved in relevant biological processes.

Identification of xenobiotics and their phase I/II metabolites in poisoned patients remains challenging. Systematic approaches using bioinformatic tools are needed to detect all compounds as exhaustively as possible. Here, we aimed to assess an analytical workflow using liquid chromatography coupled to high-resolution mass spectrometry with data processing based on a molecular network to identify tramadol metabolites in urine and plasma in poisoned patients. The generated molecular network from liquid chromatography coupled to high-resolution tandem mass spectrometry data acquired in both positive and negative ion modes allowed for the identification of 25 tramadol metabolites in urine and plasma, including four methylated metabolites that have not been previously reported in humans or in vitro models. While positive ion mode is reliable for generating a network of tramadol metabolites displaying a dimethylamino radical in their structure, negative ion mode was useful to cluster phase II metabolites. In conclusion, the combined use of molecular networks in positive and negative ion modes is a suitable and robust tool to identify a broad range of metabolites in poisoned patients, as shown in a fatal tramadol-poisoned patient.

Metabolomics in clinical toxicology aim at reliably identifying and semi-quantifying a broad array of endogenous and exogenous metabolites using dedicated analytical methods. Here, we developed a three-step-based workflow to investigate the metabolic impact of the antidepressant drug venlafaxine in a poisoned patient who developed life-threatening cardiac failure managed with extracorporeal membrane oxygenation. Both targeted quantitative and untargeted semi-quantitative metabolomic analyses using liquid chromatography hyphenated to high-resolution tandem mass spectrometry were performed to determine the plasma kinetics of venlafaxine, O-desmethyl-venlafaxine, and N-desmethyl-venlafaxine and to identify sixteen different venlafaxine-derived metabolites including one unknown (i.e., venlafaxine conjugated to a hexosyl-radical), respectively. Correlations between the quantitative metabolomic data and annotated endogenous metabolites suggested impaired amino acid and lipid metabolism, Krebs cycle, and kynurenine pathway. This preliminary study represents a first step towards a more extensive application of toxicometabolomics in clinical toxicology and a useful workflow to identify the biomarkers of toxicity.

P‐glycoprotein (P‐gp) is an efflux transporter involved in the bioavailability of many drugs currently on the market. P‐gp is responsible for several drug–drug interactions encountered in clinical practice leading to iatrogenic hospital admissions, especially in polypharmacy situations. ABCB1 genotyping only reflects an indirect estimate of P‐gp activity. Therefore, it would be useful to identify endogenous biomarkers to determine the P‐gp phenotype to predict in vivo activity prior to the initiation of treatment and to assess the effects of drugs on P‐gp activity. The objective of this study was to assess changes in plasma lipidome composition among healthy volunteers selected on the basis of their ABCB1 genotype and who received clarithromycin, a known inhibitor of P‐gp. Untargeted lipidomic analysis based on liquid chromatography–tandem mass spectrometry was performed before and after clarithromycin administration. Our results revealed changes in plasma levels of some ceramides (Cers) Cer(d18:1/22:0), Cer(d18:1/22:1), and Cer(d18:1/20:0) by ~38% ( p  < 0.0001), 13% ( p  < 0.0001), and 13% ( p  < 0.0001), respectively and phosphatidylcholines (PCs) PC(17:0/14:1), PC(16:0/18:3), and PC(14:0/18:3) by ~24% ( p  < 0.001), 10% ( p  < 0.001), and 23.6% ( p  < 0.001) associated with both ABCB1 genotype and clarithromycin intake. Through the examination of plasma lipids, our results highlight the relevance of untargeted lipidomics for studying in vivo P‐gp activity and, more generally, to safely phenotyping transporters.

Annotation of lipids in untargeted lipidomic analysis remains challenging and a systematic approach needs to be developed to organize important datasets with the help of bioinformatic tools. For this purpose, we combined tandem mass spectrometry-based molecular networking with retention time (tR) prediction to annotate phospholipid and sphingolipid species. Sixty-five standard compounds were used to establish the fragmentation rules of each lipid class studied and to define the parameters governing their chromatographic behavior. Molecular networks (MNs) were generated through the GNPS platform using a lipid standards mixture and applied to lipidomic study of an in vitro model of dry eye disease, i.e., human corneal epithelial (HCE) cells exposed to hyperosmolarity (HO). These MNs led to the annotation of more than 150 unique phospholipid and sphingolipid species in the HCE cells. This annotation was reinforced by comparing theoretical to experimental tR values. This lipidomic study highlighted changes in 54 lipids following HO exposure of corneal cells, some of them being involved in inflammatory responses. The MN approach coupled to tR prediction thus appears as a suitable and robust tool for the discovery of lipids involved in relevant biological processes.

Impairment in cholesterol metabolism is associated with many neurodegenerative disorders including Alzheimer's disease (AD). However, the lipid alterations underlying neurodegeneration and the connection between altered cholesterol levels and AD remains not fully understood. We recently showed that cholesterol accumulation in hippocampal neurons, induced by silencing gene expression, leads to neurodegeneration with a progressive neuronal loss associated with AD-like phenotype in wild-type mice. We used a targeted and non-targeted lipidomics approach by liquid chromatography coupled to high-resolution mass spectrometry to further characterize lipid modifications associated to neurodegeneration and cholesterol accumulation induced by CYP46A1 inhibition. Hippocampus lipidome of normal mice was profiled 4 weeks after cholesterol accumulation due to gene expression down-regulation at the onset of neurodegeneration. We showed that major membrane lipids, sphingolipids and specific enzymes involved in phosphatidylcholine and sphingolipid metabolism, were rapidly increased in the hippocampus of AAV-shCYP46A1 injected mice. This lipid accumulation was associated with alterations in the lysosomal cargoe, accumulation of phagolysosomes and impairment of endosome-lysosome trafficking. Altogether, we demonstrated that inhibition of cholesterol 24-hydroxylase, key enzyme of cholesterol metabolism leads to a complex dysregulation of lipid homeostasis. Our results contribute to dissect the potential role of lipids in severe neurodegenerative diseases like AD.

The Aryl hydrocarbon Receptor(AhR) is among the most important receptors which bind pollutants; however it also regulates signaling pathways independently of such exposure. We previously demonstrated that AhR is expressed during development of the central nervous system(CNS) and that its deletion leads to the occurrence of a congenital nystagmus. Objectives of the present study are to decipher the origin of these deficits, and to identify the role of the AhR in the development of the CNS. We show that the AhR-knockout phenotype develops during early infancy together with deficits in visual-information-processing which are associated with an altered optic nerve myelin sheath, which exhibits modifications in its lipid composition and in the expression of myelin-associated-glycoprotein(MAG), a cell adhesion molecule involved in myelin-maintenance and glia-axon interaction. In addition, we show that the expression of pro-inflammatory cytokines is increased in the impaired optic nerve and confirm that inflammation is causally related with an AhR-dependent decreased expression of MAG. Overall, our findings demonstrate the role of the AhR as a physiological regulator of myelination and inflammatory processes in the developing CNS. It identifies a mechanism by which environmental pollutants might influence CNS myelination and suggest AhR as a relevant drug target for demyelinating diseases.

Background: Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. Recently, exposure to phthalates has been associated with preterm birth, low birth weight, and pregnancy loss. There is limited information about the possible mechanisms linking maternal phthalate exposure and placental development, but one such mechanism may be mediated by peroxisome proliferator-activated receptor [gamma] (PPAR[gamma]). PPAR[gamma] belongs to the nuclear receptor superfamily that regulates, in a ligand- dependent manner, the transcription of target genes. Studies of PPAR[gamma]-deficient mice have demonstrated its essential role in lipid metabolism and placental development. In the human placenta, PPAR[gamma] is expressed in the villous cytotrophoblast (VCT) and is activated during its differentiation into syncytiotrophoblast. Objectives: The goal of this study was to investigate the action of mono(2-ethylhexyl) phthalate (MEHP) on PPAR[gamma] activity during in vitro differentiation of VCTs. Methods: We combined immunofluorescence, PPAR[gamma] activity/hCG assays, western blotting, and lipidomics analyses to characterize the impacts of physiologically relevant concentrations of MEHP (0.1, 1, and 10 [micro]M) on cultured VCTs isolated from human term placentas. Results: Doses of 0.1 [micro]M and 1 [micro]M MEHP showed significantly lower PPAR[gamma] activity and less VCT differentiation in comparison with controls, whereas, surprisingly, a 10 [micro]M dose had the opposite effect. MEHP exposure inhibited hCG production and significantly altered lipid composition. In addition, MEHP had significant effects on the mitogen-activated protein kinase (MAPK) pathway. Conclusions: This study suggests that MEHP has a U-shaped dose-response effect on trophoblast differentiation that is mediated by the PPAR[gamma] pathway and acts as an endocrine disruptor in the human placenta.