Projet: EXPOSURE: EXPOsome Signatures of Ultra-processed food pollutants: outcomes on brain Regulation
Présentation
Key words: Pollutants, Phthalates, Plastics,Ultra-processed food, Exposome, Brain disorders.
| DESCRIPTION OF THE PROJECT |
| EXPOSURE: EXPOsome Signatures of Ultra-processed food pollutants: outcomes on brain Regulation Plastics present in our environment represent a worrying source of environmental pollution. Micro and nanoparticles (MP/NP) resulting from abrasion of plastics carry pollutant chemicals that diffuse in biological tissues. Ultra-processed foods not only have an imbalanced composition (high levels of salt, refined sugars and saturated fats) but also contain a substantial number of harmful chemicals, which are either added or formed during industrial processing. Chemicals present in ultra-processed foods are likely adding up to pollutants originating from MPs and NPs. The consumption of ultra-processed foods accounts for more than 60% of the calories consumed in some parts of continental Europe. In a recent study from our group where men were exposed to unprocessed and ultra-processed diet for 3 weeks, we discovered that the consumption of ultra-processed foods deteriorates mood (as measured by an impaired depression score), impairs reproductive function and alters the secretion of the follicle stimulating hormone, a hormone secreted by the brain. In the same cohort, we identified that the consumption of ultra-processed diet was associated with higher level of a phthalate involved in hormonal disruption. Given the effect of ultra-processed diet on hormonal imbalance and mood, we therefore hypothesize that the consumption of ultra-processed foods deteriorates brain function through the action of associated pollutants chemicals. |
| Context: Plastic is the third most produced material in the world, after cement and steel. Global production doubled between 2000 and 2020 to reach 460 million tons per year worldwide. More than half of plastic waste is not recycled properly and ends up in the environment. This human-made pollution is ubiquitous1 and persists for years in the environment. It reaches record levels in the seas where the quantity of plastics outreaches the biomass2,3. In the environment, plastics are subjected to abrasion and photodegradation, fragmenting into MPs and NPs. These particles are a particularly worrying source of environmental pollution, because the plastic polymers currently produced are not biodegradable and MPs/NPs can act as buffers and transporters for plastic additives and other pollutants. The toxicity induced by MPs and NPs is size and shape-dependent, as smaller particles have better adsorption capacity and larger surface-volume ration, releasing more associated chemicals. MPs/NPs and associated chemicals are present in the air, house dust, water, food, cosmetics and everyday plastic objects. People are exposed through direct contact, inhalation and ingestion. It is estimated that an adult ingests the equivalent of 5g of plastic particles per week4. Once ingested, MPs and NPs cross the body’s epithelial barriers and enter the bloodstream. NPs have been shown to cross the blood-brain barrier to accumulate in the brain just a few hours after ingestion5. Fetuses and infants are exposed during critical periods of nervous system development, since MPs have been observed in the human placenta6 and maternal milk7. In the body, plastic particles are immune stimulants, inducing the production of cytokines and chemokines8, and cause oxidative stress9. Environmental pollution by plastics has been proposed to be associated with a higher risk of neurodevelopmental disorders and endocrine dysfunction, psychiatric illnesses and debilitating neurological disorders10 and chronic pain11, however, definitive demonstration of the link between exposure and toxicity is lacking and the pathophysiological mechanisms are still unknown. Ingestion of NPs is accompanied by exposure to additives (as Di(2-ethylhexyl) phthalate (DEHP) or Mono-carboxy-isononyl phthalate (cxMINP), Bisphenol A, PFAS) used in plastic polymer synthesis. Ultra-processed foods (UPFs) are a source of contamination with phthalates which occurs during the food process and packaging. In particular, high-molecular weight phthalates are thought to be endocrine and metabolic disruptors, and reprotoxic to humans. Epidemiological surveys of exposed workers show a high incidence of central, peripheral and autonomic neuropathology. Adverse effects have been documented on the male reproductive system, adipogenesis and neurodevelopment. Exposure is linked to asthma, endometriosis, infertility in adults, and of particular interest to our project, in childhood obesity, type 2 diabetes (T2DM), attention deficit disorder with or without hyperactivity (ADHD) and neurodevelopment. In mice, exposure to phthalates is a risk factor for cerebrovascular function12. While phthalates and NPs could act synergistically, no study thus far, to the best of our knowledge, has evaluated the effect of their co-exposure on the development and function of the central nervous system. The consumption of UPFs has markedly increased globally, now accounting for over 60% of total energy intake in some European countries13. UPFs are defined as industrially processed foods, and composed of highly transformed, derived or synthesized ingredients. In addition to many macro- and micronutrients associated with poor cardiometabolic health, UPFs are suspected to contain contaminants originating from the industrial process, as consumption of UPFs is associated with increased urinary concentration of phthalates, bisphenols and nitrates14. Our team conducted a human nutritional intervention study testing the effect of UPFs on metabolic and male reproductive health (in press). Using a randomized 2×2 cross over design and outpatient setting in 50 men (Figure 1A), we found that the consumption of UPFs was associated with increased fat mass, depression score and sperm quality (Figure 1B). This was associated with striking hormonal disruption with altered levels of the hormones Follicle Stimulating Hormone (FSH) (Figure 1B), testosterone and growth/differentiation factor 15 (GDF-15) (not shown). Of interest, the metabolite cxMINP was accumulated in blood and seminal fluid from the participants after the consumption of UPFs (Figure 1C). Given the documented role of phthalates in hormone disruption, our results collectively constitute the foundation of our hypothesis that pollutants occurring in UPFs contribute to alter brain function, including the central regulation of mood and reproductive function. |
| Methods: WP1: To map the exposome associated with UPFs consumption in blood and seminal fluid from our human participants In this project, we will apply bioinformatics techniques and machine learning to high-throughput metabolomic and exposome datasets to extract molecular signatures specific to each contaminant class with collaboration with Drs A. Droit (Laval University’s Faculty of Medicine, Québec – Chaire IA, Equipe MAASAI – Inria Sophia, UniCA) and S. Dagnino (PHEN-X, UniCA). For phthalates (e.g., DEHP, cxMINP) and PFAS, we will train supervised classifiers to pinpoint metabolite panels (i.e., signatures) that distinguish exposed samples from controls. For NPs/MPs exposure, we will develop algorithms to detect shifts in metabolic pathways associated with particle uptake. By combining rigorous data preprocessing, dimensionality reduction, and model validation, we will define sets of biomarkers that reliably signal each pollutant and build predictive models for the early detection of exposure effects. WP2: To determine the effect of candidate pollutants on brain function in the mouse We will test the effect of NPs and phthalates on feeding behaviour, memory, anhedonia, anxiety and pain sensibility. Based on our preliminary results, we will start experiments with the high molecular weight phthalates cxMINP and DEHP, and we will use other phthalates identified in WP1. We have recently set up a model of exposure of mice (C57Bl/6j) to polystyrene NPs (NP-PS) and DEHP or (NP-PS, diameter 100 nm, 0.3 mg/kg body weight/d; DEHP, 0.2 mg/kg body weight/d) via food supplementation. Mice are exposed from embryonic stage E15 and during lactation after birth via the mother’s diet, then, after weaning, via individual diet. This reproduces pre and postnatal exposure, which is crucial for investigating the impact on critical periods of the nervous system development. A longitudinal study will be carried out on mice at 4, 8, 12 and 16 wks of age. Both sexes are included to explore potential sexual dimorphism in the response. While plastic is widespread, control groups cannot be entirely shielded, so the dosage for test groups is in the higher range of estimated quantities present in the environment. All experiments adhere to ethical guidelines for animal research, following the 3Rs principle (ethical approval APAFIS #45285-2023100413393163 v7; APAFIS #57659-2025102711529883 v3). WP2.1: Impact of NPs and phthalates exposure on mice feeding behaviour. NPs and phthalates (cxMINP, DEHP and phthalates identified in WP1) will be tested to analyse if these components influence mice body weight and/or food consumption using metabolic cages available at IPMC, on both genders. We will assess the impact of NPs and phthalates on organs involved in phthalate metabolism and storage (i.e. liver and adipose tissues (AT) by quantifying markers of tissue inflammation and tissue histology. Body composition will be compared (EchoMRITM available at IPMC) over 16 weeks of exposure. The intestinal barrier’s integrity will be measured by quantification of circulating lipopolysaccharides. Glucose homeostasis and insulin tolerance (GTT, ITT) will be tested to evaluate progressive onset of T2DM. These experiments evaluate the mice energy balance, which is crucial for the project as we have shown that metabolic syndrome causes hypersensitivity to pain. WP2.2: Impact of NPs and phthalates on mental and cognitive disorders (memory, anhedonia, anxiety) and pain hypersensitivity. Memory (Morris water maze for special memory test, Novel Object Recognition Test), anhedonia (Saccharose preference test) and anxiety (Elevated Plus Maze, Open Field test) will be tested. The effect on inflammatory and neuropathic pain (von Frey test for sensitivity to mechanical pain and Hargreaves test for thermal heat pain) will be measured. All these behavioral tests are available from IPMC (https://www.ipmc.cnrs.fr/fr/facility/4exploration-fonctionnelle-animex/). WP3: Infiltration of NP-PS and phthalates in the nervous system and metabolic organs. WP3.1: Mapping of NPs in the nervous system. We propose to validate a reliable and quantitative technique, compatible with biological tissues, for measuring NP-PS collection in the nervous system. SC’s team will feed mice with commercially available blue dyed polystyrene nano-particles (100 nm in diameter; DiagPoly™ Blue Colored Polystyrene Particles, # DDB-L003; CD Bioparticles®). These fluorescent particles, once fed to the mice, will be detected in the brain, lumbar spinal cord, lumbar dorsal root using brain clearing technique (Adipoclear+) and high-resolution light sheet fluorescent microscopy with 3D reconstruction for whole tissue mapping of NPs or confocal microscopy. In parallel experiments, we will map NP-PS in the nervous system with monoclonal antibodies directed at polystyrene polymers, obtained from Jun Lin’s lab of University of Tennessee (accessible to the consortium through a Material Transfer Agreement (MTA) with the UTRF (#28472 UT MTA)) and Adipoclear+ whole-mount immunolabeling. Intracellular cytotoxic accumulation of NP-PS in neurons and glia will be characterized with single and bi-photon confocal microscopy (available at IPMC, https://www.ipmc.cnrs.fr/fr/facility/2imagerie-de-cytometrie/) and electron microscopy (collaboration with the Centre Commun de Microscopie Appliquée, UniCA). WP3.2: Mapping of phthalates and its metabolites in metabolic tissues and the nervous system. Phthalates and their metabolites will be quantified in the regions of the nervous system studied in WP3.1, and in body organs involved in metabolism (AT and liver) studied in WP2, in exposed mice vs control. Samples will undergo a rigorous analysis, leveraging advanced mass spectrometry techniques, including Ultra-High-Performance Liquid Chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) housed in a state-of-the-art metabolomics platform of PHEN-X lab of Sonia Dagnino. We aim at finding associations between the dosage of phthalates and the metabolites and the effect on metabolic organs and the nervous system (WP2). This will not only confirm the presence of higher level of phthalates in exposed mice brain and metabolic tissues but also enable us to quantify specific products of phthalates biotransformation, thereby providing a deeper understanding of its metabolic fate within metabolic organs and the nervous system. WP4: Impact of NPs and phthalates on metabolic pathways, oxidative stress, neuroinflammation, cell- based mechanism and neuronal network. WP4.1. Impact of NPs and phthalates on metabolic pathways and inflammation. We intend to elucidate the complex mechanisms by which NP-PS and candidate phthalates disrupt cellular functions. Lou Ruffino, in collaboration with Sonia Dagnino, will use biological samples (AT, liver, cortex, hypothalamus, hippocampe, amygdale, spinal cord, autonomic and sensory ganglia) collected from mice exposed to NP-PS and candidate phthalates to drive a comprehensive metabolomic analysis, using advanced mass spectrometry techniques (UHPLC-MS/MS) to obtain a panoramic view of metabolic disturbances and map out the metabolic signatures that are altered as a result of exposure. We will then dissect these metabolic pathways at cellular level in glial cells and neurons. In parallel experiments, we aim to explore how these disturbances correlate with neuroinflammation. We will quantify the neuroinflammation in brain regions of interest involved in feeding behavior memory, anhedonia, anxiety and pain perception, by measuring inflammatory markers (IL1-b, IL6, TNFa, CCL2 and CCL5) with multiplex immunoassays. We will characterize in detail the populations of cells expressing inflammatory markers (astroglial and neuronal populations) by in situ hybridization (RNAScope® ISH technology) on histological brain slices to determine the location and the proportion of pro-inflammatory cytokines and chimiokines positive cells in brain regions of interest with RNA in situ hybridization experiments using the RNAScope® technology. To complement these experiments, cell types will also be identified by coupling RNAScope in situ hybridization for each inflammatory mediator with fluorescent immunohistochemical experiments with cell types specific antibodies (NeuN, Iba1 and GFAP for neurons, microglia, and astrocytes). Fluorescent images will be acquired with confocal microscopy. The activation of glia (microglia and astrocytes) will be quantified in these areas with cell morphometry, using a fully automated algorithm, NutriMorph15, developed by SC team in collaboration with Eric Debreuve (Team Morpheme, I3S-Inria-UniCA). Lou Ruffino and Arnaud Droit will process the UHPLC-MS/MS output through tailored bioinformatics workflows, perform statistical analyses and train supervised machine learning models to identify sets of metabolites that reliably distinguish NPs and phthalates exposure from controls, then integrate these metabolite panels with neuroinflammatory readouts. WP4.2. Impact of NPs and phthalates on neuron function. First, we will perform spatial transcriptomics to get better resolution on the different brain regions affected by pollutant exposure. The spatial transcriptomics analysis provides evidence about spatial differential gene expression, allowing us to conclude on pathway targeted by the candidate pollutants exposure. The 10X GENOMICS protocol will be implemented by the Single-Cell Omics Platform (SCOP) of the Center for Basics Metabolic Research (CBMR) at the University of Copenhagen. We will use the panel of 257 brain probes provided by the 10X GENOMICS supplier. These images and the ones from Xenium scanning are overlapped for cells segmentation, and help for bioinformatics analysis. Secondly, we aim to explore the functional consequences of NPs and phthalates contamination in identified cells of relevant brain areas. Population neuron activity will be monitored using injections of AAV9-CAG-GCaMP6f-WPRE and in vivo calcium imaging in freely moving mice (Miniscope). Fiber photometry will be used for recordings in the hypothalamus. The promotor may be adapted to the particular subpopulations of cells identified above. To test pain perception, calcium imaging will be analyzed in response to peripheral cutaneous stimulation to establish the network activation shift in exposed mice. We will then examine the effect of contamination on neuron excitability and synaptic activity using whole-cell patch-clamp recording in brain slices. In parallel experiments, we will measure sensory thresholds and discharge activity of peripheral sensory neurons using the nerve-skin recording technique in collaboration with Jacques Noël team (IPMC). Neurons from different regions will be cultured with NPs and phthalates or its metabolites (identified in WP3) for a full characterization of neurons functional properties in vitro. Lou Ruffino and Arnaud Droit will apply AI-based analyses to the calcium imaging and patch-clamp datasets, building classifiers to detect pollutant-induced shifts in neuronal activity patterns and linking these electrophysiological changes to behavioral measures for early exposure detection. All procedures planned in this proposal are performed routinely by our team or collaborative teams. We have developed the necessary expertise and know-how. The material is available for all experiments described. References 1 – Landrigan et al., The Minderoo-Monaco Commission on Plastics and Human Health. Ann Glob Health. 2023, 89(1):23. 2 – The Mediterranean: Mare Plasticum. ed Boucher and Billard, 2020, IUCN, Global Marine and Polar Programme. 3 – UN Environment Mediterranean Action Plan Barcelona Convention, 2017 Mediterranean Quality Status Report. 4 – Cox et al., Human Consumption of Microplastics. Environ Sci Technol. 2019, 53(12):7068-7074. 5 – Kopatz et al., Micro- and Nanoplastics Breach the Blood-Brain Barrier (BBB): Biomolecular Corona’s Role Revealed. Nanomaterials (Basel). 2023, 13(8):1404. 6 – Ragusa et al., Plasticenta: First evidence of microplastics in human placenta. Environ Int. 2021, 6:106274. 7 – Ragusa et al., Raman Microspectroscopy Detection and Characterisation of Microplastics in Human Breastmilk. Polymers. 2022. 8 – Hwang et al., Potential toxicity of polystyrene microplastic particles. Sci Rep. 2020, 10(1):7391. 9 Kadac-Czapska et al., Microplastics and Oxidative Stress-Current Problems and Prospects. Antioxidants (Basel). 2024, 13(5):579. 10 – Jin et al., Evaluation of Neurotoxicity in BALB/c Mice following Chronic Exposure to Polystyrene Microplastics. Environ Health 2022, 130(10):107002. 11 – Brockmeyer et al., How air pollution alters brain development: the role of neuroinflammation. Transl Neurosci. 2016, 7(1):24-30. 12 – Ahmadpour et al., Disruption of the blood-brain barrier and its close environment following adult exposure to low doses of di(2-ethylhexyl) phthalate alone or in an environmental phthalate mixture in male mice. Chemosphere. 2021, 282:131013. 13– Mertens et al., Ultra-processed food consumption in adults across Europe. Eur J Nutr. 2022, 61(3):1521-1539. 14 – Martínez et al., Association between dietary contribution of ultra-processed foods and urinary concentrations of phthalates and bisphenol in a nationally representative sample of the US population aged 6 years and older. PLoS One. 2020, 15(7):e0236738. 15 – Sanchezet al.,Computational detection, characterization, and clustering of microglial cells in a mouse model of fat-induced postprandial hypothalamic inflammation. Methods. 2025, 236:28-38. |
| Interdisciplinary Impact: The interdisciplinary impact of the project results from its fundamental ambition to integrate different scales of analysis and several scientific fields (neuroscience, immunology, endocrinology, metabolomics, exposome, computational biology) to understand the complex mechanisms linking food pollutants to brain health. The use of innovative computational methods to integrate diverse data is a cornerstone of this approach. |
| Originality: The originality of the project lies in its integrated and translational approach (from human to animal), the exploitation of unique data on the UPF-related exposome, the extensive application of AI and computational biology methods to analyze complex data, and the exploration of still largely unknown mechanisms by which pollutants present in UPF directly affect brain function. |
Les travaux sont soutenus par le projet PLASTIC-BRAIN financé par l’Université Côte d’Azur (UCA-IDEX, Académie 3 « Espace, environnement, risques et résilience ») sur la période 2025-2028, à hauteur de 50 000 € par an sur trois ans, dans le cadre d’une co-coordination avec le Jacques Noël.
Ils bénéficient également du soutien du programme européen Horizon Europe (HORIZON-MSCA-2021-SE-01-01, projet PsyCoMed) sur la période 2023-2026, avec un financement de 110 400 € attribué à l’équipe en tant que partenaire.
