In search for obesogens. Ewa Szalowska BDS Amsterdam 2012

Transcription

1 In search for obesogens Ewa Szalowska BDS Amsterdam 212

2 Obesogens-an environmental link to Obesogens (B. Blumberg 26)- a subset of endocrine disrupting chemicals (EDCs) that activate PPARγ obesity PPARγ is one of the nuclear receptors ligandactviated transcription factor that mediates adipogenesis and maintenance of mature adipocytes PPAR γ gene targets: PPAR γ 2, FABP4, LPL, CD36, adiponectin, Klf15 Adipocytes differentiation & maintenance

3 Adipose tissue energy homeostasis Chemerin Adiponectin Leptin Omentin Visfatin Resistin Vaspin TNFa/IL-6 RBP4 Glucose and insulin homeostasis Lipid homeostasis Reproduction Immunity

4 MEHP and rosiglitazone bind similarly to the PPAR ligandbinding domain Feige JN, et al. The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor g modulator that promotes adipogenesis. JBC 282:26: (27)

5 MEHP regulates only a subset of genes compared with rosiglitazone MEHP induces adipogenesis through PPARγ Feige JN, et al. The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor g modulator that promotes adipogenesis. JBC 282:26: (27)

6 Obesogens selection Compound Concentrations µm Product Rosiglitazone (positive control) > µm Anti Diabetes 15d-PGJ2 prostaglandin (positive control) > µm Natural agonist of PPARγ Flavone (positive control) > µm Natural compound Ethinyl-Estradiol (negative control) > 3, 1 µm Hormone DDT (dichlorodiphenyltrichloroethane) > 2 µm Organochlorine, insecticide DDE (Dichlorodiphenyldichloroethylene) > 2 µm Product of DDT β HCH (β-hexachlorocyclohexane) >1 1-3 µm Pesticide product, organochloride DES (Diethylstilbestrol) > 2 µm Synthetic estrogen Bisphenol A/B/C > µm Plasticizers PCB 153 2,2',4,4',5,5'-Hexachlorobiphenyl > µm Environmental contaminant DEHP (Bis(2-ethylhexyl)phthalate) > µm Plasticizer MEHP (mono-2-ethylhexyl phthalate) > µm Plasticizer TPTC tri-n-propyltin chloride > µm Biocides, organotin TBTC Tributyltin chloride.1 >.1% Biocides, organotonin HCB β-hexachlorocyclohexane > µm Fungicide, organochloride, insecticide

7 PPARγ2 CALUX

8 Luminescence % Luminescence % DEHP, PCB, Flavone-PPARγ CALUX DEHP, PCB 153 and Flavone interaction with PPARy compaired to Rosiglitazone Roziglitazone DEHP PCB 153 Flavone 2,1,1,1,1 1 Concentrations µm HCB, TBTC and MEHP interaction with PPARy compaired to Rosiglitazone Roziglitazone HCB TBTC MEHP,1,1,1,1 1 Concentrations µm -2

9 Bisphenol A -PPARγ CALUX

10 Obesogens beyond PPARγ Casals-Calas C, Desvergne B. 211 Annu. Rev. Physiol.73:135-62

11 Human Simpson-Golabi-Bhemel-Syndrome (SGBS) cells Advantages: Human More efficient to differentiate Unlimited source

12 Luminescence No direct effect of TCDD on PPARγ PPARγ CALUX 1nM coexposure 1nM Rosi 1nM Rosi No antagonistic effect Conc. TCDD pm 1nM coexposure

13 Relative cell viability (%) Cytotoxicity Seed SGBS cells in 96- well plate 24h 24h Expose adhered cells to TCDD Measure ATP, WST1 and protein 2 Relative cell viability 15 WST-1 1 ATP 5.2% Conc. TCDD (nm) Protein

14 Dose dependent acute effect of TCDD on gene in undifferentiated SGBS cells 4 days 24 h Seed cells in 6-well plate Expose confluent cells Isolate RNA Synthesise cdna qpcr TCDD target genes: AhR, CYP1A1, CYP1B1 Inflammatory genes: MCP1, IKKβ, Serpin B2, IL-6, IL-1β, TNFα

15 TCDD target gene,4,3,2,1 AhR Conc. TCDD nm,6 CYP1A1,6 CYP1B1,4,4,2,2,1,1, ,1,1, Conc. TCDD nm Conc. TCDD nm 1nM TCDD down-regulates AhR and up-regulates CYP1A1 and CYP1B1

16 Inflammatory gene,8,6,4,2 MCP ,4,3,2,1 IL Conc. TCDD nm Conc. TCDD nm,15,1,5 IL-1β ,8,6,4,2 Serpin B Conc. TCDD nm Conc. TCDD nm 1nM TCDD and higher induces dose-dependent up-regulation of inflammatory marker gene

17 Chronic effect of 1nM TCDD on gene during differentiation Isolate RNA at different time points (store at -8 C) Synthesise cdna qpcr TCDD target genes Inflammatory marker genes Adipogenic (metabolic) genes: PPARγ, Adiponectin, Leptin, LPL, FABP4

18 TCDD target gene,25,2,15,1,5 AhR Days of differentiation nm 1nM,8,6,4,2 CYP1A Days of differentiation nm 1nM 2 1,5 1,5 CYP1B Days of differentiation nm 1nM TCDD down-regulates AhR and up-regulates CYP1A1 and CYP1B1

19 Inflammatory gene 1,5 MCP Days of differentiation nm 1nM,6,4,2 IL-1β Days of differentiation nm 1nM,3,15,2,1,5,1 IL Days of of differentiation nm 1nM 1nM,3,2,1 Serpin B Days of differentiation nm 1nM TCDD up-regulates most of the inflammatory marker gene

20 Adipogenic (metabolic) gene,1 PPARγ, Days of differentiation nm 1nM,1,5 2 1 Leptin Days of differentiation LPL Days of differentiation nm 1nM nm 1nM 1,5 4 2 Adiponectin FABP Days of differentiation Days of differentiation nm 1nM nm 1nM

21 Oil Red O staining nm 1nM 5nM 1nM Dose dependent decrease of TCDD on the amount of lipid droplets

22 Oil Red O (%) Oil Red O quantification % TCDD (nm) Significantly more lipid droplets Significantly less lipid droplets

23 Conclusion TCDD despite clear toxicity significantly alters gene These changes could lead to functionally altered adipocytes that are predisposed to metabolic disorders

24 Conclusion PPARg CALUX is successful to identify subset of obesogens SGBS cell line can be used to identify larger number of obesogens/edcs

25 Thanks to : Dionne Heezius (RIKILT) Marieke Jans (RIKILT) Dr. Ad Peijnenburg & Dr. Ron Hoogenboom (RIKILT) Prof. Bram Brouwer (BDS) Dr. Peter A. Behnisch (BDS) Prof. Martin Wabitsch (University of Ulm) ewa.szalowska@wur.nl