Common deregulated gene expression profiles and morphological changes in developing zebrafish larvae exposed to environmental-relevant high to low concentrations of glucocorticoids.

Synthetic glucocorticoids have been detected in environmental waters and their biological potency have raised concerns of their impact on aquatic vertebrates especially fish. In this study, developing zebrafish larvae exposed to representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) at 50 pM to 50 nM from 3 h post-fertilisation to 5 days post-fertilisation were investigated. Microarray analysis identified 1255, 1531, and 2380 gene probes, which correspondingly mapped to 660, 882 and 1238 human/rodent homologs, as deregulated by dexamethasone, prednisolone and triamcinolone, respectively. A total of 248 gene probes which mapped to 159 human/rodent homologs were commonly deregulated by the three glucocorticoids. These homologs were associated with over 20 molecular functions from cell cycle to cellular metabolisms, and were involved in the development and function of connective tissue, nervous, haematological, and digestive systems. Glucocorticoid receptor signalling, NRF2-mediated oxidative stress response and RAR signalling were among the top perturbed canonical pathways. Morphological analyses using four transgenic zebrafish lines revealed that the hepatic and endothelial-vascular systems were affected by all three glucocorticoids while nervous, pancreatic and myeloid cell systems were affected by one of them. Quantitative real-time PCR detected significant change in the expression of seven genes at 50 pM of all three glucocorticoids, a concentration comparable to total glucocorticoids reported in environmental waters. Three genes (cry2b, fbxo32, and klhl38b) responded robustly to all glucocorticoid concentrations tested. The common deregulated genes with the associated biological processes and morphological changes can be used for biological inference of glucocorticoid exposure in fish for future studies.

[1]  P. Jeffrey,et al.  Structural Basis of UV DNA-Damage Recognition by the DDB1–DDB2 Complex , 2008, Cell.

[2]  Z. Gong,et al.  Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations. , 2016, Chemosphere.

[3]  F. Jeanneteau,et al.  Molecular Biology of Glucocorticoid Signaling. , 2015, Advances in experimental medicine and biology.

[4]  J. Cidlowski,et al.  Glucocorticoid receptor signaling in health and disease. , 2013, Trends in pharmacological sciences.

[5]  Ran Zhao,et al.  Oncogenes and Tumor Suppressors DDB 2 Suppresses Tumorigenicity by Limiting the Cancer Stem Cell Population in Ovarian Cancer , 2014 .

[6]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[7]  J. Cidlowski,et al.  Glucocorticoid signaling in the heart: A cardiomyocyte perspective , 2015, The Journal of Steroid Biochemistry and Molecular Biology.

[8]  Erin M. Snyder,et al.  In vitro bioassays to evaluate complex chemical mixtures in recycled water. , 2015, Water research.

[9]  B. Cleveland,et al.  Molecular characterization of atrogin-1/F-box protein-32 (FBXO32) and F-box protein-25 (FBXO25) in rainbow trout (Oncorhynchus mykiss): Expression across tissues in response to feed deprivation. , 2010, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[10]  John Calvin Reed,et al.  Analysis of gene expression patterns during glucocorticoid-induced apoptosis using oligonucleotide arrays. , 2002, Biochemical and biophysical research communications.

[11]  P. Raychaudhuri,et al.  Damaged DNA Binding Protein 2 in Reactive Oxygen Species (ROS) Regulation and Premature Senescence , 2012, International journal of molecular sciences.

[12]  K. Cooper,et al.  Glucocorticoids alter craniofacial development and increase expression and activity of matrix metalloproteinases in developing zebrafish (Danio rerio). , 2008, Toxicological sciences : an official journal of the Society of Toxicology.

[13]  Richard J. Williams,et al.  Prediction of environmental concentrations of glucocorticoids: the River Thames, UK, as an example. , 2012, Environment international.

[14]  L. Myatt,et al.  Induction of PGF2α synthesis by cortisol through GR dependent induction of CBR1 in human amnion fibroblasts. , 2014, Endocrinology.

[15]  S. Herzig,et al.  How Do Glucocorticoids Regulate Lipid Metabolism? , 2015, Advances in experimental medicine and biology.

[16]  E. Bruford,et al.  Update on the Kelch-like (KLHL) gene family , 2013, Human Genomics.

[17]  A. Meijer,et al.  Discovery of a functional glucocorticoid receptor beta-isoform in zebrafish. , 2008, Endocrinology.

[18]  V. Sharma,et al.  Simultaneous determination of corticosteroids, androgens, and progesterone in river water by liquid chromatography-tandem mass spectrometry. , 2010, Chemosphere.

[19]  B. Necela,et al.  A Single Amino Acid Change in the First Zinc Finger of the DNA Binding Domain of the Glucocorticoid Receptor Regulates Differential Promoter Selectivity* , 2004, Journal of Biological Chemistry.

[20]  M. Weger,et al.  The circadian clock and glucocorticoids – Interactions across many time scales , 2013, Molecular and Cellular Endocrinology.

[21]  L. Colombo,et al.  A living biosensor model to dynamically trace glucocorticoid transcriptional activity during development and adult life in zebrafish , 2014, Molecular and Cellular Endocrinology.

[22]  S. Snyder,et al.  Balancing the Budget: Accounting for Glucocorticoid Bioactivity and Fate during Water Treatment. , 2016, Environmental science & technology.

[23]  V. Laudet,et al.  ZebRA: An overview of retinoic acid signaling during zebrafish development. , 2015, Biochimica et biophysica acta.

[24]  Chunaram Choudhary,et al.  Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control , 2015, Nature communications.

[25]  A. Beggs,et al.  Kelch proteins: emerging roles in skeletal muscle development and diseases , 2014, Skeletal Muscle.

[26]  Jian Wang,et al.  A functional insertion/deletion polymorphism in the promoter of PDCD6IP is associated with the susceptibility of hepatocellular carcinoma in a Chinese population. , 2013, DNA and cell biology.

[27]  Robert A. Harris,et al.  Regulation of pyruvate dehydrogenase kinase expression by peroxisome proliferator-activated receptor-alpha ligands, glucocorticoids, and insulin. , 2002, Diabetes.

[28]  M. Oppermann,et al.  Glucocorticoids induce gastroparesis in mice through depletion of l-arginine. , 2014, Endocrinology.

[29]  M. Fishman.  Genomics. Zebrafish--the canonical vertebrate. , 2001, Science.

[30]  Chen Cao,et al.  Glucocorticoid receptor repression mediated by BRCA1 inactivation in ovarian cancer , 2014, BMC Cancer.

[31]  Xiaojuan Qi,et al.  All-trans retinoic acid-induced hypothalamus–pituitary–adrenal hyperactivity involves glucocorticoid receptor dysregulation , 2013, Translational Psychiatry.

[32]  P. Forgez,et al.  Loss of glucocorticoid receptor activation is a hallmark of BRCA1-mutated breast tissue , 2013, Breast Cancer Research and Treatment.

[33]  S. Ghavami,et al.  Association between Programmed Cell Death 6 Interacting Protein Insertion/Deletion Polymorphism and the Risk of Breast Cancer in a Sample of Iranian Population , 2015, Disease markers.

[34]  J. Goldstone,et al.  10 - Perspectives on zebrafish as a model in environmental toxicology , 2010 .

[35]  D. Choi,et al.  Glucocorticoid Receptor (GR)-Associated SMRT Binding to C/EBPβ TAD and Nrf2 Neh4/5: Role of SMRT Recruited to GR in GSTA2 Gene Repression , 2005, Molecular and Cellular Biology.

[36]  A. Meijer,et al.  Transcriptional and metabolic effects of glucocorticoid receptor α and β signaling in zebrafish. , 2015, Endocrinology.

[37]  M. Vijayan,et al.  The Transcriptomics of Glucocorticoid Receptor Signaling in Developing Zebrafish , 2013, PloS one.

[38]  M. Kiniwa,et al.  Species-specific differences in the glucocorticoid receptor transactivation function upon binding with betamethasone-esters. , 2002, International immunopharmacology.

[39]  A. Papavassiliou,et al.  The glucocorticoid receptor signalling in breast cancer. , 2007, Journal of cellular and molecular medicine.

[40]  Zhimin Zhang,et al.  BTG2: a rising star of tumor suppressors (review). , 2015, International journal of oncology.

[41]  Danhong Lu,et al.  Regulation of islet beta-cell pyruvate metabolism: interactions of prolactin, glucose, and dexamethasone. , 2010, Endocrinology.

[42]  W. Yuan,et al.  Dexamethasone Enhanced Functional Recovery after Sciatic Nerve Crush Injury in Rats , 2015, BioMed research international.

[43]  L. A. White,et al.  Larval zebrafish as a model for glucose metabolism: expression of phosphoenolpyruvate carboxykinase as a marker for exposure to anti-diabetic compounds. , 2007, Journal of molecular endocrinology.

[44]  D. Franchimont.  Overview of the Actions of Glucocorticoids on the Immune Response: A Good Model to Characterize New Pathways of Immunosuppression for New Treatment Strategies , 2004, Annals of the New York Academy of Sciences.

[45]  Chang-Ju Kim,et al.  Modulation of glucocorticoid receptor induction properties by core circadian clock proteins , 2014, Molecular and Cellular Endocrinology.

[46]  H. Tamary,et al.  Inhaled corticosteroids increase blood neutrophil count by decreasing the expression of neutrophil adhesion molecules Mac-1 and L-selectin. , 2016, The American journal of emergency medicine.

[47]  John P Sumpter,et al.  Synthetic glucocorticoids in the environment: first results on their potential impacts on fish. , 2011, Environmental science & technology.

[48]  Shane A Snyder,et al.  Ultra high performance liquid chromatography tandem mass spectrometry for rapid analysis of trace organic contaminants in water , 2013, Chemistry Central Journal.

[49]  D. Buhler,et al.  Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva. , 2005, Toxicology and applied pharmacology.

[50]  R. Schleimer,et al.  An overview of glucocorticoid anti-inflammatory actions , 2005, European Journal of Clinical Pharmacology.

[51]  S. Lightman,et al.  Temporal control of glucocorticoid neurodynamics and its relevance for brain homeostasis, neuropathology and glucocorticoid-based therapeutics , 2016, Neuroscience & Biobehavioral Reviews.

[52]  R. Evans,et al.  Cryptochromes mediate rhythmic repression of the glucocorticoid receptor , 2011, Nature.

[53]  A. Meijer,et al.  Glucocorticoid-Induced Attenuation of the Inflammatory Response in Zebrafish. , 2016, Endocrinology.

[54]  A. Ozmen,et al.  The Effects of Glucocorticoids on Fetal and Placental Development , 2012 .

[55]  Robert L. Tanguay,et al.  Unraveling Tissue Regeneration Pathways Using Chemical Genetics* , 2007, Journal of Biological Chemistry.

[56]  Z. Gong,et al.  Fluorescent Transgenic Zebrafish Tg(nkx2.2a:mEGFP) Provides a Highly Sensitive Monitoring Tool for Neurotoxins , 2013, PloS one.

[57]  J. Tuckermann,et al.  Molecular Actions of Glucocorticoids in Cartilage and Bone During Health, Disease, and Steroid Therapy. , 2016, Physiological reviews.

[58]  M. Whitehouse.  Anti-inflammatory glucocorticoid drugs: reflections after 60 years , 2011, Inflammopharmacology.

[59]  Ksenia J. Groh,et al.  LC-MS/MS determination of potential endocrine disruptors of cortico signalling in rivers and wastewaters , 2014, Analytical and Bioanalytical Chemistry.

[60]  Z. Jaglic,et al.  Dexamethasone-induced immunosuppression: a rabbit model. , 2008, Veterinary immunology and immunopathology.

[61]  K. Terada,et al.  Inhibition of Nerve Growth Factor-Induced Neurite Outgrowth from PC12 Cells by Dexamethasone: Signaling Pathways through the Glucocorticoid Receptor and Phosphorylated Akt and ERK1/2 , 2014, PloS one.

[62]  R. Pictet,et al.  In vivo footprinting of rat TAT gene: Dynamic interplay between the glucocorticoid receptor and a liver-specific factor , 1991, Cell.

[63]  Z. Gong,et al.  Development of a Convenient In Vivo Hepatotoxin Assay Using a Transgenic Zebrafish Line with Liver-Specific DsRed Expression , 2014, PloS one.

[64]  Jianying Hu,et al.  Determination and source apportionment of five classes of steroid hormones in urban rivers. , 2009, Environmental science & technology.

[65]  K. Yamamoto,et al.  Regulatory Actions of Glucocorticoid Hormones: From Organisms to Mechanisms. , 2015, Advances in experimental medicine and biology.

[66]  M. Minnetti,et al.  Mechanisms in endocrinology: The spectrum of haemostatic abnormalities in glucocorticoid excess and defect. , 2015, European journal of endocrinology.

[67]  Leonard I Zon,et al.  Of fish and men: using zebrafish to fight human diseases. , 2013, Trends in cell biology.

[68]  Charles A. Harris,et al.  Animal Models of Altered Glucocorticoid Signaling. , 2015, Advances in experimental medicine and biology.

[69]  A. Odermatt,et al.  Suppression of the Nrf2-Dependent Antioxidant Response by Glucocorticoids and 11β-HSD1-Mediated Glucocorticoid Activation in Hepatic Cells , 2012, PloS one.

[70]  I. Colditz,et al.  The Effect of Dexamethasone on Some Immunological Parameters in Cattle , 1999, Veterinary Research Communications.

[71]  S. Ru,et al.  Impairment of the cortisol stress response mediated by the hypothalamus-pituitary-interrenal (HPI) axis in zebrafish (Danio rerio) exposed to monocrotophos pesticide. , 2015, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[72]  Hélène Budzinski,et al.  Identification of synthetic steroids in river water downstream from pharmaceutical manufacture discharges based on a bioanalytical approach and passive sampling. , 2014, Environmental science & technology.

[73]  John P Sumpter,et al.  Metabolic and reproductive effects of relatively low concentrations of beclomethasone dipropionate, a synthetic glucocorticoid, on fathead minnows. , 2013, Environmental science & technology.

[74]  Christian Lawrence,et al.  The husbandry of zebrafish (Danio rerio): A review , 2007 .

[75]  H. Spaink,et al.  The zebrafish as a model system for glucocorticoid receptor research. , 2009, Comparative biochemistry and physiology. Part A, Molecular & integrative physiology.

[76]  S. Swendeman,et al.  The effects of serum depletion and dexamethasone on growth and differentiation of human neuroblastoma cell lines. , 2000, Journal of pediatric surgery.

[77]  A. Odermatt,et al.  Retinoic acid reduces glucocorticoid sensitivity in C2C12 myotubes by decreasing 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor activities. , 2009, Endocrinology.

[78]  K. Clark,et al.  A transgenic zebrafish model for monitoring glucocorticoid receptor activity , 2014, Genes, brain, and behavior.

[79]  H. Vandenburgh,et al.  Glucocorticoid-induced skeletal muscle atrophy in vitro is attenuated by mechanical stimulation. , 1992, The American journal of physiology.

[80]  Gerald Brenner-Weiss,et al.  A Chemical Screening System for Glucocorticoid Stress Hormone Signaling in an Intact Vertebrate , 2012, ACS chemical biology.

[81]  G. Hager,et al.  Prevalent Glucocorticoid and Androgen Activity in US Water Sources , 2012, Scientific Reports.

[82]  S. Conzen,et al.  Activation of the glucocorticoid receptor is associated with poor prognosis in estrogen receptor-negative breast cancer. , 2011, Cancer research.