Metformin strongly affects transcriptome of peripheral blood cells in healthy individuals

Funding Information: The study was supported by the European Regional Development Fund under the project ?Investigation of interplay between multiple determinants influencing response to metformin: search for reliable predictors for efficacy of type 2 diabetes therapy? (Project No.: 1.1.1.1/16/A/091, https://ec.europa.eu/regional_policy/en/funding/ erdf/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors would like to thank all the volunteers for their participation and acknowledge the Genome Database of the Latvian Population for providing biological material and data. Publisher Copyright: © 2019 Ustinova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Metformin is a commonly used antihyperglycaemic agent for the treatment of type 2 diabetes mellitus. Nevertheless, the exact mechanisms of action, underlying the various therapeutic effects of metformin, remain elusive. The goal of this study was to evaluate the alterations in longitudinal whole-blood transcriptome profiles of healthy individuals after a one-week metformin intervention in order to identify the novel molecular targets and further prompt the discovery of predictive biomarkers of metformin response. Next generation sequencing-based transcriptome analysis revealed metformin-induced differential expression of genes involved in intestinal immune network for IgA production and cytokine-cytokine receptor interaction pathways. Significantly elevated faecal sIgA levels during administration of metformin, and its correlation with the expression of genes associated with immune response (CXCR4, HLA-DQA1, MAP3K14, TNFRSF21, CCL4, ACVR1B, PF4, EPOR, CXCL8) supports a novel hypothesis of strong association between metformin and intestinal immune system, and for the first time provide evidence for altered RNA expression as a contributing mechanism of metformin’s action. In addition to universal effects, 4 clusters of functionally related genes with a subject-specific differential expression were distinguished, including genes relevant to insulin production (HNF1B, HNF1A, HNF4A, GCK, INS, NEUROD1, PAX4, PDX1, ABCC8, KCNJ11) and cholesterol homeostasis (APOB, LDLR, PCSK9). This inter-individual variation of the metformin effect on the transcriptional regulation goes in line with well-known variability of the therapeutic response to the drug.publishersversionPeer reviewe

Evaluation of 309 Environmental Chemicals Using a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity Assay

The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC50) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation

Toxicity Testing in the 21st Century: Defining New Risk Assessment Approaches Based on Perturbation of Intracellular Toxicity Pathways

The approaches to quantitatively assessing the health risks of chemical exposure have not changed appreciably in the past 50 to 80 years, the focus remaining on high-dose studies that measure adverse outcomes in homogeneous animal populations. This expensive, low-throughput approach relies on conservative extrapolations to relate animal studies to much lower-dose human exposures and is of questionable relevance to predicting risks to humans at their typical low exposures. It makes little use of a mechanistic understanding of the mode of action by which chemicals perturb biological processes in human cells and tissues. An alternative vision, proposed by the U.S. National Research Council (NRC) report Toxicity Testing in the 21st Century: A Vision and a Strategy, called for moving away from traditional high-dose animal studies to an approach based on perturbation of cellular responses using well-designed in vitro assays. Central to this vision are (a) “toxicity pathways” (the innate cellular pathways that may be perturbed by chemicals) and (b) the determination of chemical concentration ranges where those perturbations are likely to be excessive, thereby leading to adverse health effects if present for a prolonged duration in an intact organism. In this paper we briefly review the original NRC report and responses to that report over the past 3 years, and discuss how the change in testing might be achieved in the U.S. and in the European Union (EU). EU initiatives in developing alternatives to animal testing of cosmetic ingredients have run very much in parallel with the NRC report. Moving from current practice to the NRC vision would require using prototype toxicity pathways to develop case studies showing the new vision in action. In this vein, we also discuss how the proposed strategy for toxicity testing might be applied to the toxicity pathways associated with DNA damage and repair

The steroid metabolome in women with premenstrual dysphoric disorder during GnRH agonist-induced ovarian suppression: effects of estradiol and progesterone addback.

Clinical evidence suggests that symptoms in premenstrual dysphoric disorder (PMDD) reflect abnormal responsivity to ovarian steroids. This differential steroid sensitivity could be underpinned by abnormal processing of the steroid signal. We used a pharmacometabolomics approach in women with prospectively confirmed PMDD (n=15) and controls without menstrual cycle-related affective symptoms (n=15). All were medication-free with normal menstrual cycle lengths. Notably, women with PMDD were required to show hormone sensitivity in an ovarian suppression protocol. Ovarian suppression was induced for 6 months with gonadotropin-releasing hormone (GnRH)-agonist (Lupron); after 3 months all were randomized to 4 weeks of estradiol (E2) or progesterone (P4). After a 2-week washout, a crossover was performed. Liquid chromatography/tandem mass spectrometry measured 49 steroid metabolites in serum. Values were excluded if >40% were below the limit of detectability (n=21). Analyses were performed with Wilcoxon rank-sum tests using false-discovery rate (q<0.2) for multiple comparisons. PMDD and controls had similar basal levels of metabolites during Lupron and P4-derived neurosteroids during Lupron or E2/P4 conditions. Both groups had significant increases in several steroid metabolites compared with the Lupron alone condition after treatment with E2 (that is, estrone-SO4 (q=0.039 and q=0.002, respectively) and estradiol-3-SO4 (q=0.166 and q=0.001, respectively)) and after treatment with P4 (that is, allopregnanolone (q=0.001 for both PMDD and controls), pregnanediol (q=0.077 and q=0.030, respectively) and cortexone (q=0.118 and q=0.157, respectively). Only sulfated steroid metabolites showed significant diagnosis-related differences. During Lupron plus E2 treatment, women with PMDD had a significantly attenuated increase in E2-3-sulfate (q=0.035) compared with control women, and during Lupron plus P4 treatment a decrease in DHEA-sulfate (q=0.07) compared with an increase in controls. Significant effects of E2 addback compared with Lupron were observed in women with PMDD who had significant decreases in DHEA-sulfate (q=0.065) and pregnenolone sulfate (q=0.076), whereas controls had nonsignificant increases (however, these differences did not meet statistical significance for a between diagnosis effect). Alterations of sulfotransferase activity could contribute to the differential steroid sensitivity in PMDD. Importantly, no differences in the formation of P4-derived neurosteroids were observed in this otherwise highly selected sample of women studied under controlled hormone exposures

Pharmacometabolomic Assessments of Atenolol and Hydrochlorothiazide Treatment Reveal Novel Drug Response Phenotypes.

Achieving hypertension (HTN) control and mitigating the adverse health effects associated with HTN continues to be a global challenge. Some individuals respond poorly to current HTN therapies, and mechanisms for response variation remain poorly understood. We used a nontargeted metabolomics approach (gas chromatography time-of-flight/mass spectrometry gas chromatography time-of-flight/mass spectrometry) measuring 489 metabolites to characterize metabolite signatures associated with treatment response to anti-HTN drugs, atenolol (ATEN), and hydrochlorothiazide (HCTZ), in white and black participants with uncomplicated HTN enrolled in the Pharmacogenomic Evaluation of Antihypertensive Responses study. Metabolite profiles were significantly different between races, and metabolite responses associated with home diastolic blood pressure (HDBP) response were identified. Metabolite pathway analyses identified gluconeogenesis, plasmalogen synthesis, and tryptophan metabolism increases in white participants treated with HCTZ (P &lt; 0.05). Furthermore, we developed predictive models from metabolite signatures of HDBP treatment response (P &lt; 1 × 10(-5)). As part of a quantitative systems pharmacology approach, the metabolites identified herein may serve as biomarkers for improving treatment decisions and elucidating mechanisms driving HTN treatment responses

Pharmacometabolomic Assessments of Atenolol and Hydrochlorothiazide Treatment Reveal Novel Drug Response Phenotypes

Achieving hypertension (HTN) control and mitigating the adverse health effects associated with HTN continues to be a global challenge. Some individuals respond poorly to current HTN therapies, and mechanisms for response variation remain poorly understood. We used a nontargeted metabolomics approach (gas chromatography time‐of‐flight/mass spectrometry gas chromatography time‐of‐flight/mass spectrometry) measuring 489 metabolites to characterize metabolite signatures associated with treatment response to anti‐HTN drugs, atenolol (ATEN), and hydrochlorothiazide (HCTZ), in white and black participants with uncomplicated HTN enrolled in the Pharmacogenomic Evaluation of Antihypertensive Responses study. Metabolite profiles were significantly different between races, and metabolite responses associated with home diastolic blood pressure (HDBP) response were identified. Metabolite pathway analyses identified gluconeogenesis, plasmalogen synthesis, and tryptophan metabolism increases in white participants treated with HCTZ (P < 0.05). Furthermore, we developed predictive models from metabolite signatures of HDBP treatment response (P < 1 × 10(−5)). As part of a quantitative systems pharmacology approach, the metabolites identified herein may serve as biomarkers for improving treatment decisions and elucidating mechanisms driving HTN treatment responses