Single and mixture effects of bisphenol A and benzophenone-3 on in vitro T helper cell differentiation.

Immune homeostasis is key to guarantee that the immune system can elicit effector functions against pathogens and at the same time raise tolerance towards other antigens. A disturbance of this delicate balance may underlie or at least trigger pathologies. Endocrine disrupting chemicals (EDCs) are increasingly recognized as risk factors for immune dysregulation. However, the immunotoxic potential of specific EDCs and their mixtures is still poorly understood. Thus, we aimed to investigate the effect of bisphenol A (BPA) and benzophenone-3 (BP-3), alone and in combination, on in vitro differentiation of T helper (TH)17 cells and regulatory T (Treg) cells. Naïve T cells were isolated from mouse lymphoid tissues and differentiated into the respective TH population in the presence of 0.001-10 µM BP-3 and/or 0.01-100 µM BPA. Cell viability, proliferation and the expression of TH lineage specific transcription factors and cytokines was measured by flow cytometry and CBA/ELISA. Moreover, the transcription of hormone receptors as direct targets of EDCs was quantified by RT-PCR. We found that the highest BPA concentration adversely affected TH cell viability and proliferation. Moreover, the general differentiation potential of both TH populations was not altered in the presence of both EDCs. However, EDC exposure modulated the emergence of TH17 and Treg cell intermediate states. While BPA and BP-3 promoted the development of TH1-like TH17 cells under TH17-differentiating conditions, TH2-like Treg cells occurred under Treg polarization. Interestingly, differential effects could be observed in mixtures of the two tested compounds compared with the individual compounds. Notably, estrogen receptor ß expression was decreased under TH17-differentiating conditions in the presence of BPA and BP-3 as mixture. In conclusion, our study provides solid evidence for both, the immune disruptive potential and the existence of cumulative effects of real nature EDC mixtures on T cell in vitro differentiation.

Single and combined exposures to bisphenol A and benzophenone-3 during early mouse pregnancy have differential effects on fetal and placental development.

Endocrine disrupting chemicals (EDCs) possess the capability to interfere with the endocrine system by binding to hormone receptors, for example on immune cells. Specific effects have already been described for individual substances, but the impact of exposure to chemical mixtures during pregnancy on maternal immune regulation, placentation and fetal development is not known. In this study, we aimed to investigate the combined effects of two widespread EDCs, bisphenol A (BPA) and benzophenone-3 (BP-3), at allowed concentrations on crucial pregnancy processes such as implantation, placentation, uterine immune cell populations and fetal growth. From gestation day (gd) 0 to gd10, female mice were exposed to 4 µg/kg/d BPA, 50 mg/kg/d BP-3 or a BPA/BP-3 mixture. High frequency ultrasound and Doppler measurements were used to determine intrauterine fetal development and hemodynamic parameters. Furthermore, uterine spiral artery remodeling and placental mRNA expression were studied via histology and CHIP-RT-PCR, respectively. Effects of EDC exposure on multiple uterine immune cell populations were investigated using flow cytometry. We found that exposure to BP-3 caused intrauterine growth restriction in offspring at gd14, while BPA and BPA/BP-3 mixture caused varying effects. Moreover, placental morphology at gd12 and placental efficiency at gd14 were altered upon BP-3 exposure. Placental gene transcription was altered particularly in female offspring after in utero exposure to BP-3. Flow cytometry analyses revealed an increase in uterine T cells and NK cells in BPA and BPA/BP-3-treated dams at gd14. Doppler measurements revealed no effect on uterine hemodynamic parameters and spiral artery remodeling was not affected following EDC exposure. Our results provide evidence that exposure to BPA and BP-3 during early gestation affects fetal development in a sex-dependent manner, placental function and immune cell frequencies at the feto-maternal interface. These results call for inclusion of studies addressing pregnancy in the risk assessment of environmental chemicals.

Absence of Heme Oxygenase-1 Affects Trophoblastic Spheroid Implantation and Provokes Dysregulation of Stress and Angiogenesis Gene Expression in the Uterus.

The enzyme heme oxygenase-1 (HO-1) is pivotal in reproductive processes, particularly in placental and vascular development. This study investigated the role of HO-1 and its byproduct, carbon monoxide (CO), in trophoblastic spheroid implantation. In order to deepen our understanding of the role of HO-1 during implantation, we conducted in vivo experiments on virgin and pregnant mice, aiming to unravel the cellular and molecular mechanisms. Using siRNA, HO-1 was knocked down in JEG-3 and BeWo cells and trophoblastic spheroids were generated with or without CO treatment. Adhesion assays were performed after transferring the spheroids to RL-95 endometrial epithelial cell layers. Additionally, angiogenesis, stress, and toxicity RT2-Profiler™ PCR SuperArray and PCR analyses were performed in uterine murine samples. HO-1 knockdown by siRNA impeded implantation in the 3D culture model, but this effect could be reversed by CO. Uteruses from virgin Hmox1-/- females exhibited altered expression of angiogenesis and stress markers. Furthermore, there was a distinct expression pattern of cytokines and chemokines in uteruses from gestation day 14 in Hmox1-/- females compared to Hmox1+/+ females. This study strongly supports the essential role of HO-1 during implantation. Moreover, CO appears to have the potential to compensate for the lack of HO-1 during the spheroid attachment process. The absence of HO-1 results in dysregulation of angiogenesis and stress-related genes in the uterus, possibly contributing to implantation failure.

Mast Cells Retard Tumor Growth in Ovarian Cancer: Insights from a Mouse Model.

Ovarian cancer has the highest mortality rate among female reproductive tract malignancies. A complex network, including the interaction between tumor and immune cells, regulates the tumor microenvironment, survival, and growth. The role of mast cells (MCs) in ovarian tumor pathophysiology is poorly understood. We aimed to understand the effect of MCs on tumor cell migration and growth using in vitro and in vivo approaches. Wound healing assays using human tumor cell lines (SK-OV-3, OVCAR-3) and human MCs (HMC-1) were conducted. Murine ID8 tumor cells were injected into C57BL6/J wildtype (WT) and MC-deficient C57BL/6-KitW-sh/W-sh (KitW-sh) mice. Reconstitution of KitW-sh was performed by the transfer of WT bone marrow-derived MCs (BMMCs). Tumor development was recorded by high-frequency ultrasonography. In vitro, we observed a diminished migration of human ovarian tumor cells upon direct or indirect MC contact. In vivo, application of ID8 cells into KitW-sh mice resulted in significantly increased tumor growth compared to C57BL6/J mice. Injection of BMMCs into KitW-sh mice reconstituted MCs and restored tumor growth. Our data show that MCs have a suppressive effect on ovarian tumor growth and may serve as a new therapeutic target.

Maternal exposure of mice to glyphosate induces depression- and anxiety-like behavior in the offspring via alterations of the gut-brain axis.

The past decade has been characterized by increased awareness and de-stigmatization of mental health issues, in particular the most common neuropsychiatric disorders depression and anxiety. Further, with growing understanding of neurodevelopmental disorders such as attention deficit and hyperactivity disorder and autism spectrum disorder, the number of diagnosed patients has increased. The pathogenesis of these behavioral disorders is multifactorial and early-life exposure to environmental chemicals has been proposed to be a relevant risk factor that might mediate these effects by disturbances on the gut-brain-axis. However, for glyphosate, the most widely used pesticide worldwide, there are only limited and inconsistent findings that link chronic low-dose exposure in particular during early life to neurobehavioral disorders. Here, we explored the impact of maternal oral glyphosate exposure (0.5 and 50 mg/kg body weight/day) during pregnancy and the lactational period on offspring's behavior, brain gene expression and gut microbiota using a cross-generational mouse model. Behavioral analyses revealed a depression- and anxiety-like behavior as well as social deficits most notably in adult female offspring of glyphosate-exposed dams. Furthermore, the expression of tryptophan hydroxylase 2, an enzyme discussed to be linked to behavioral problems, was reduced in the hippocampus of female offspring and correlated to a glyphosate-induced DNA hypermethylation of the gene. Moreover, maternal glyphosate exposure significantly altered the gut microbiota in the female offspring including a decreased abundance of Akkermansia and increased abundance of Alistipes and Blautia, bacteria involved in tryptophan metabolism and associated with depression- and anxiety-like disorders. Our results suggest that glyphosate might influence the gut-brain axis crosstalk following in-utero and lactational exposure. This study underlines the importance of understanding the impact of exposure to pesticides on the gut-brain axis and further emphasizes the need for microbiome analyses to be compulsorily included in health risk assessments of pesticides.

Undiagnosed Pediatric Elevated Blood Pressure Is Characterized by Induction of Proinflammatory and Cytotoxic Mediators.

BACKGROUND: Inflammatory processes have been suggested as a culprit of vascular damage in pediatric hypertension. We aimed to investigate transcriptional changes of immune modulators and determine their association with office blood pressure in adolescents who were not diagnosed with hypertension at the time of the study visit. METHODS: Office blood pressure measurements and blood samples were taken from adolescents of 2 German birth cohorts, GINIplus (The German Infant Study on the Influence of Nutrition Intervention Plus Air Pollution and Genetics on Allergy Development; discovery cohort, n=1219) and LISA (Influences of Lifestyle-related factors on the Immune System and the Development of Allergies in Childhood; validation cohort, n=809), during the 15-year follow-up visit and categorized based on the European Society of Hypertension Guideline. Hs-CRP (high-sensitivity C-reactive protein) and expression of 51 genes encoding cytokines/receptors and transcription factors were analyzed. RESULTS: The prevalence of elevated systolic blood pressure (overweight/obese) was 14.0% (5.1%) and 16.4% (5.2%) in the discovery and validation cohorts, respectively. An enhanced cytotoxic (GZMB, PRF1, IL2RB) and proinflammatory (FOS, IL1B, hs-CRP) immune profile was observed in association with the hypertension class in both cohorts. Expression of hs-CRP and IL1B was driven by overweight with IL1B being identified as a mediator between body mass index and elevated systolic blood pressure (adj.ß/95% CI, 0.01/0.0002-0.02). The association of GZMB (adjusted odds ratio/95% CI, 1.67/1.26-2.21; P=0.0004) and PRF1 (adjusted odds ratio/95% CI, 1.70/1.26-2.29; P=0.0005) in the hypertension class remained significant in normal-weight individuals without parental predisposition. These effects were confirmed in LISA. CONCLUSIONS: Adolescent hypertension is not limited to known risk groups. As adolescents in the hypertension class show an inflammatory profile similar to that of established hypertension in adults, blood pressure monitoring at a young age is critical to ensure early intervention and prevention of adverse sequelae.

Appearance of ghost bands in gel electrophoresis depending on agarose concentration.

Standard agarose gel electrophoresis is a widely used method to analyse diversity of nucleic acids. Certain conditions, however, may give rise to artefactual bands. We report on artefactual bands frequently occurring, especially when partially homologous nucleic acids, such as splicing variants of DNA transcripts, are analysed simultaneously. Interestingly, to some extent agarose concentration may influence the occurrence of artefactual bands.

Mixtures of per- and poly-fluoroalkyl substances (PFAS) reduce the in vitro activation of human T cells and basophils.

In the last decades, per- and poly-fluoroalkyl substances (PFAS), widely used industrial chemicals, have been in the center of attention because of their omnipotent presence in water and soils worldwide. Although efforts have been made to substitute long-chain PFAS towards safer alternatives, their persistence in humans still leads to exposure to these compounds. PFAS immunotoxicity is poorly understood as no comprehensive analyses on certain immune cell subtypes exist. Furthermore, mainly single entities and not PFAS mixtures have been assessed. In the present study we aimed to investigate the effect of PFAS (short-chain, long-chain and a mixture of both) on the in vitro activation of primary human immune cells. Our results show the ability of PFAS to reduce T cells activation. In particular, exposure to PFAS affected T helper cells, cytotoxic T cells, Natural Killer T cells, and Mucosal associated invariant T (MAIT) cells, as assessed by multi-parameter flow cytometry. Furthermore, the exposure to PFAS reduced the expression of several genes involved in MAIT cells activation, including chemokine receptors, and typical proteins of MAIT cells, such as GZMB, IFNG and TNFSF15 and transcription factors. These changes were mainly induced by the mixture of both short- and long-chain PFAS. In addition, PFAS were able to reduce basophil activation induced by anti-FcεR1α, as assessed by the decreased expression of CD63. Our data clearly show that the exposure of immune cells to a mixture of PFAS at concentrations mimicking real-life human exposure resulted in reduced cell activation and functional changes of primary innate and adaptive human immune cells.

Global hypomethylation in childhood asthma identified by genome-wide DNA-methylation sequencing preferentially affects enhancer regions.

BACKGROUND: Childhood asthma is a result of a complex interaction of genetic and environmental components causing epigenetic and immune dysregulation, airway inflammation and impaired lung function. Although different microarray based EWAS studies have been conducted, the impact of epigenetic regulation in asthma development is still widely unknown. We have therefore applied unbiased whole genome bisulfite sequencing (WGBS) to characterize global DNA-methylation profiles of asthmatic children compared to healthy controls. METHODS: Peripheral blood samples of 40 asthmatic and 42 control children aged 5-15 years from three birth cohorts were sequenced together with paired cord blood samples. Identified differentially methylated regions (DMRs) were categorized in genotype-associated, cell-type-dependent, or prenatally primed. Network analysis and subsequent natural language processing of DMR-associated genes was complemented by targeted analysis of functional translation of epigenetic regulation on the transcriptional and protein level. RESULTS: In total, 158 DMRs were identified in asthmatic children compared to controls of which 37% were related to the eosinophil content. A global hypomethylation was identified affecting predominantly enhancer regions and regulating key immune genes such as IL4, IL5RA, and EPX. These DMRs were confirmed in n = 267 samples and could be linked to aberrant gene expression. Out of the 158 DMRs identified in the established phenotype, 56 were perturbed already at birth and linked, at least in part, to prenatal influences such as tobacco smoke exposure or phthalate exposure. CONCLUSION: This is the first epigenetic study based on whole genome sequencing to identify marked dysregulation of enhancer regions as a hallmark of childhood asthma.

An old friend with a new face: YB-1 and its role in healthy pregnancy and pregnancy-associated complications.

By promoting tissue invasion, cell growth and angiogenesis, the Y-box binding protein (YB-1) became famous as multifunctional oncoprotein. However, this designation is telling only part of the story. There is one particular time in life when actual tumorigenic-like processes become undoubtedly welcome, namely pregnancy. It seems therefore reasonable that YB-1 plays also a crucial role in reproduction, and yet this biological aspect of the cold-shock protein has been overlooked for many years. To overcome this limitation, we would like to propose a new perspective on YB-1 and emphasize its pivotal functions in healthy pregnancy and pregnancy-related complications. Moreover, we will discuss findings obtained from cancer research in the light of reproductive events to elucidate the importance of YB-1 at the feto-maternal interface.

Plant Age and Soil Texture Rather Than the Presence of Root Hairs Cause Differences in Maize Resource Allocation and Root Gene Expression in the Field.

Understanding the biological roles of root hairs is key to projecting their contributions to plant growth and to assess their relevance for plant breeding. The objective of this study was to assess the importance of root hairs for maize nutrition, carbon allocation and root gene expression in a field experiment. Applying wild type and root hairless rth3 maize grown on loam and sand, we examined the period of growth including 4-leaf, 9-leaf and tassel emergence stages, accompanied with a low precipitation rate. rth3 maize had lower shoot growth and lower total amounts of mineral nutrients than wild type, but the concentrations of mineral elements, root gene expression, or carbon allocation were largely unchanged. For these parameters, growth stage accounted for the main differences, followed by substrate. Substrate-related changes were pronounced during tassel emergence, where the concentrations of several elements in leaves as well as cell wall formation-related root gene expression and C allocation decreased. In conclusion, the presence of root hairs stimulated maize shoot growth and total nutrient uptake, but other parameters were more impacted by growth stage and soil texture. Further research should relate root hair functioning to the observed losses in maize productivity and growth efficiency.

IL17F Expression as an Early Sign of Oxidative Stress-Induced Cytotoxicity/Apoptosis.

Interleukin 17F (IL17F) has been found to be involved in various inflammatory pathologies and has recently become a target for therapeutic purposes. In contrast to IL17F secreted by immune cells, the focus of this study is to describe the triggers of IL17F release in non-immune cells with a particular focus on IL17F-induced fibrosis. IL17F induction was examined in human lung epithelial (BEAS-2B) and myeloid cell lines as well as in peripheral blood mononuclear cells after in vitro exposure to aqueous cigarette smoke extract (CSE), inorganic mercury, cadmium or the apoptosis inducer brefeldin A. Fibrosis was examined in vitro, evaluating the transition of human primary dermal fibroblasts to myofibroblasts. We observed that all stressors were able to induce IL17F gene expression regardless of cell type. Interestingly, its induction was associated with cytotoxic/apoptotic signs. Inhibiting oxidative stress by N-acetylcysteine abrogated CSE-induced cytotoxic and IL17F-inducing effects. The induction of IL17F was accompanied by IL17F protein expression. The transition of fibroblasts into myofibroblasts was not influenced by either recombinant IL17F or supernatants of CSE-exposed BEAS-2B. In addition to IL17F secretion by specialized or activated immune cells, we underscored the cell type-independent induction of IL17F by mechanisms of inhibitable oxidative stress-induced cytotoxicity. However, IL17F was not involved in dermal fibrosis under the conditions used in this study.

Characterization of Three-Dimensional Trophoblast Spheroids: An Alternative Model to Study the Physiological Properties of the Placental Unit.

It was postulated that 3D cell culture models more accurately reflect the complex tissue physiology and morphology in comparison to 2D cell monolayers. Currently, there is a shortage of well-characterized and easily maintainable high-throughput experimental models of the human placenta. Here, we characterized three different 3D cultures (e.g., spheroids) derived from trophoblast cell lines and studied their functionality in comparison to primary fetal trophoblasts and placental tissue. The spheroid growth rates of JEG3, BeWo and HTR8/SVneo cell lines were similar among each other and were significantly larger in comparison to primary trophoblast spheroids. All spheroids exhibited migratory properties and shortest distances were registered for JEG3 spheroids. Even though all spheroids displayed invasive capabilities, only the invasive features of HTR8/SVneo spheroids resulted in specific branching. This was in agreement with the invasive properties of the spheroids obtained from primary trophoblasts. Human chorionic gonadotropin production was highest in JEG3 spheroids and only increased when stimulated with cAMP and forskolin in BeWo, but not HTR8/SVneo spheroids. The gene expression analysis confirmed that 3D trophoblast cell cultures and especially HTR8/SVneo spheroids showed considerable similarities with the gene expression profile of primary placental tissue. This study offers a broad characterization of 3D trophoblast spheroids that, in turn, can help in selecting the best model depending on the scientific question that needs to be answered.

EpiVisR: exploratory data analysis and visualization in epigenome-wide association analyses.

BACKGROUND: With the widespread availability of microarray technology for epigenetic research, methods for calling differentially methylated probes or differentially methylated regions have become effective tools to analyze this type of data. Furthermore, visualization is usually employed for quality check of results and for further insights. Expert knowledge is required to leverage capabilities of these methods. To overcome this limitation and make visualization in epigenetic research available to the public, we designed EpiVisR. RESULTS: The EpiVisR tool allows to select and visualize combinations of traits (i.e., concentrations of chemical compounds) and differentially methylated probes/regions. It supports various modes of enriched presentation to get the most knowledge out of existing data: (1) enriched Manhattan plot and enriched volcano plot for selection of probes, (2) trait-methylation plot for visualization of selected trait values against methylation values, (3) methylation profile plot for visualization of a selected range of probes against selected trait values as well as, (4) correlation profile plot for selection and visualization of further probes that are correlated to the selected probe. EpiVisR additionally allows exporting selected data to external tools for tasks such as network analysis. CONCLUSION: The key advantage of EpiVisR is the annotation of data in the enriched plots (and tied tables) as well as linking to external data sources for further integrated data analysis. Using the EpiVisR approach will allow users to integrate data from traits with epigenetic analyses that are connected by belonging to the same individuals. Merging data from various data sources among the same cohort and visualizing them will enable users to gain more insights from existing data.

Insights into Early-Pregnancy Mechanisms: Mast Cells and Chymase CMA1 Shape the Phenotype and Modulate the Functionality of Human Trophoblast Cells, Vascular Smooth-Muscle Cells and Endothelial Cells.

Spiral-artery (SA) remodeling is a fundamental process during pregnancy that involves the action of cells of the initial vessel, such as vascular smooth-muscle cells (VSMCs) and endothelial cells, but also maternal immune cells and fetal extravillous trophoblast cells (EVTs). Mast cells (MCs), and specifically chymase-expressing cells, have been identified as key to a sufficient SA-remodeling process in vivo. However, the mechanisms are still unclear. The purpose of this study is to evaluate the effects of the MC line HMC-1 and recombinant human chymase (rhuCMA1) on human primary uterine vascular smooth-muscle cells (HUtSMCs), a human trophoblast cell line (HTR8/SV-neo), and human umbilical-vein endothelial cells (HUVEC) in vitro. Both HMC-1 and rhuCMA1 stimulated migration, proliferation, and changed protein expression in HUtSMCs. HMC-1 increased proliferation, migration, and changed gene expression of HTR8/SVneo cells, while rhuCMA treatment led to increased migration and decreased expression of tissue inhibitors of matrix metalloproteinases. Additionally, rhuCMA1 enhanced endothelial-cell-tube formation. Collectively, we identified possible mechanisms by which MCs/rhuCMA1 promote SA remodeling. Our findings are relevant to the understanding of this crucial step in pregnancy and thus of the dysregulated pathways that can lead to pregnancy complications such as fetal growth restriction and preeclampsia.

The Role of GPR15 Function in Blood and Vasculature.

Since the first prominent description of the orphan G protein-coupled receptor 15 (GPR15) on lymphocytes as a co-receptor for the human immunodeficiency virus (HIV) type 1 and 2 and the first report about the GPR15-triggered cytoprotective effect on vascular endothelial cells by recombinant human thrombomodulin, several decades passed before the GPR15 has been recently deorphanized. Because of new findings on GPR15, this review will summarize the consequences of GPR15 signaling considering the variety of GPR15-expressing cell types and of GPR15 ligands, with a focus on blood and vasculature.

YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits.

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

Alternatives for the worse: Molecular insights into adverse effects of bisphenol a and substitutes during human adipocyte differentiation.

Bisphenol A (BPA), which is used in a variety of consumer-related plastic products, was reported to cause adverse effects, including disruption of adipocyte differentiation, interference with obesity mechanisms, and impairment of insulin- and glucose homeostasis. Substitute compounds are increasingly emerging but are not sufficiently investigated.We aimed to investigate the mode of action of BPA and four of its substitutes during the differentiation of human preadipocytes to adipocytes and their molecular interaction with peroxisome proliferator-activated receptor γ (PPARγ), a pivotal regulator of adipogenesis.Binding and effective biological activation of PPARγ were investigated by surface plasmon resonance and reporter gene assay, respectively. Human preadipocytes were continuously exposed to BPA, BPS, BPB, BPF, BPAF, and the PPARγ-antagonist GW9662. After 12 days of differentiation, lipid production was quantified via Oil Red O staining, and global protein profiles were assessed using LC-MS/MS-based proteomics. All tested bisphenols bound to human PPARγ with similar efficacy as the natural ligand 15d-PGJ2in vitroand provoked an antagonistic effect on PPARγ in the reporter gene assay at non-cytotoxic concentrations. During the differentiation of human preadipocytes, all bisphenols decreased lipid production. Global proteomics displayed a down-regulation of adipogenesis and metabolic pathways, similar to GW9662. Interestingly, pro-inflammatory pathways were up-regulated, MCP1 release was increased, and adiponectin decreased. pAKT/AKT ratios revealed significantly reduced insulin sensitivity by BPA, BPB, and BPS upon insulin stimulation.Thus, our results show that not only BPA but also its substitutes disrupt crucial metabolic functions and insulin signaling in adipocytes under low, environmentally relevant concentrations. This effect, mediated through inhibition of PPARγ, may promote hypertrophy of adipose tissue and increase the risk of developing metabolic syndrome, including insulin resistance.