Endocrine Disrupting Chemicals: New Tools for Assessing Ecological Risk

Transcription

1 Endocrine Disrupting Chemicals: New Tools for Assessing Ecological Risk G. Ankley Office of Research and Development NHEERL, Mid-Continent Ecology Division, Duluth, MN February 25, 2009

2 What are Endocrine-Disrupting Chemicals (EDCs)? Exogenous agents that interfere with the production, release, transport, metabolism, binding, action or elimination of natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental process (Kavlock et al. 2007) Emphasis, to date, on mechanisms affecting reproduction and development via the hypothalamic-pituitary-gonadal (HPG) and HPT (thyroidal) axes 1

3 Compartment Brain Pituitary GABA? PACAP Follistatin? GABA A R GABA B R GnRH Neuronal System GnRH D2 R Y 2 R D1 R Y 2 R NPY? Dopamine Generalized Vertebrate HPG Axis Activin PAC 1 R GnRH R Y 1 R Activin R Gonadotroph GPα D2 R FSHβ LHβ Blood Circulating LDL, HDL Circulating LH, FSH LDL R HDL R Outer mitochondrial membrane LH R FSH R Cholesterol StAR Gonad (Generalized, gonadal, steroidogenic cell) Activin Inhibin Inner mitochondrial membrane pregnenolone 17 α -hydroxyprogesterone P450scc P450c17 3βHSD progesterone 20βHSD 17α,20β-P (MIS) androstenedione 17βHSD testosterone P45011β. P450arom 11βHSD 11-ketotestosterone estradiol Blood Circulating Sex Steroids / Steroid Hormone Binding Globlulin 2 Androgen / Estrogen Responsive Tissues ER AR

4 Postulated EDC Effects: Humans Cervical cancer (DES daughters) Breast Cancer Prostate/testicular cancers Learning/neurological deficits Decreases in sperm quality Precocious maturation 3

5 Postulated EDC Effects: Wildlife Hermaphrodism in gastropods Developmental abnormalities in GL fish and birds Malformations in amphibians Feminization of reptiles Behavioral changes in birds Reproductive abnormalities in fish-eating mammals Feminization/masculinization of fish 4

6 Assessing Ecological Risks of EDCs Emphasis has been on predicting possible effects of new chemicals rather than diagnosing impacts in field, where several very challenging questions exist -Analytical (What to measure at what detection?) -Evaluation of mixtures (Endocrine MOA of concern? Interactive effects?) -Biological endpoints (Biomarker vs. apical?) -Population-level responses (How to discern?) 5

7 Tools Needed for Assessing Risk of EDCs in the Field Short-term in vitro and in vivo assays suitable for complex mixtures Analytical/fractionation approaches to identify specific chemicals causing biological effects Diagnostic endpoints (biomarkers) indicative of chemical MOA Approaches to link diagnostic responses to alterations in individuals and populations 6

8 Assessing EDC Mixtures: A Pulp Mill Case Study 7 Buckeye Plant, Fenhalloway River, FL Collaborators: G. Ankley, L. Durhan (EPA, MED); E. Gray, P. Hartig, C. Lambright, L. Parks, V. Wilson (EPA, RTD); L. Guillette (Univ. Florida)

9 PME Masculinizes Fish in the Field 8 Exposed

10 Cell-Based Assays for Detecting EDCs in Mixtures I. Field Sample Media made with Field Sample CV-1 cells har MMTV-luciferase Cells Exposed to Field Sample II. Measure Luciferase Activity 9

11 10 PME Androgenic Activity 1.5 Con PME 1 PME 2 PME 2 Up Fen Econfina DHT Log10 Fold induction over Media Con

12 Fractionation (TIE) Analysis to Identify EDCs in Complex PME PME C18 SPE % Methanol / Water Elutions 25% 50% 75% 80% 85% 90% 95% 100% 11

13 Linking EDC Activity in Cells to Fractionation of PME 7 6 Androgenic Activity Androstenedione Media 12 0 DHT HPLC Fraction Number

14 A Short-Term in vivo Test for Detecting Reproductive Toxicity of EDCs Conducted with fathead minnow, a model species used for research and regulatory work in labs throughout the world Three-week test includes endpoints reflective of specific classes of EDCs and apical responses useful to risk assessment -Vitellogenin, steroids, SSC, histology -Egg production, fertility, hatch Developed for EPA s EDSP (Endocrine Disruptor Screening and Testing Program), but applicable to assessment of complex mixtures as well 13

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16 Effects of an Estrogen on Male Secondary Sex Characteristics 15 Miles-Richardson et al. (1999) Aquat. Toxicol.

17 Effects of 17β-Trenbolone on Fathead Minnow Fecundity 4000 Trenbolone (µg/l) Cumulative Number of Eggs Spawned Control * * * 16 Exposure (d)

18 Example Applications to Diagnostic Assessments/Monitoring Monitoring Nebraska CAFO (Concentrated Animal Feeding Operation) samples for effects on reproduction and endocrine function (Kolok, Sellin et al.) Evaluating UK municipal effluents for estrogenicity (Thorpe, Tyler et al.) Monitoring Canadian pulp/paper mill effluents for reproductive and endocrine impacts (Kovacs, Parrott et al.) 17

19 Linkage of Exposure and Effects Using Genomics, Proteomics, and Metabolomics in Small Fish Models USEPA Cincinnati, OH D. Bencic, M. Kostich, D. Lattier, J. Lazorchak, G. Toth, R. Wang, USEPA Duluth, MN, and Grosse Isle, MI G. Ankley, E Durhan, M Kahl, K Jensen, E Makynen, D. Martinovic, D. Miller, D. Villeneuve USEPA Athens, GA T. Collette, D. Ekman, M. Henderson, Q. Teng USEPA-RTP, NC M. Breen, R. Conolly USEPA STAR Program N. Denslow (Univ. of Florida), E. Orlando, (Florida Atlantic University), K. Watanabe (Oregon Health Sciences Univ.), M. Sepulveda (Purdue Univ.) USACE Vicksburg, MS E. Perkins, N. Garcia-Reyero Other partners Joint Genome Institute, DOE (Walnut Creek, CA) Sandia, DOE (Albuquerque, NM) Pacific Northwest National Laboratory (Richland, WA) O. Mekenyan (University of Bourgas) 18

20 Project Objectives Investigate effects of EDCs with different MOA using the fathead minnow 21-d test to establish toxicity pathways and ecologically- relevant responses Identify the genomic responses to the same chemicals using shorter-term exposures to support development of specific biomarkers indicative both toxic MOA and adverse outcome Concurrently integrate data in a systems and network modeling contexts, as well as relevant population modeling, as a basis for prediction 19

21 Molecular Cellular Organ Individual Population Gene/Protein Expression Metabolite Profiles Alterations in production of signaling molecules Functional changes Structural changes (pathology) Altered reproduction or development Decreased numbers of animals Conceptual Toxicity (Adverse Outcome) Pathway Office of Research and Development NHEERL, Mid-Continent Ecology Division, Duluth, MN 2/25/2009

22 Compartment Chemical Probes Brain GABA? GnRH Neuronal System GnRH D1 R Y 2 R NPY? Dopamine 1 2 Fipronil (-) Muscimol (+)? GABA A R D2 R Pituitary PACAP Follistatin Activin Activin R PAC 1 R GABA B R GnRH R Y 2 R Gonadotroph GPα Y 1 R D2 R 3 4 Apomorphine (+) Haloperidol (-) FSHβ LHβ Blood Circulating LDL, HDL LDL R HDL R Outer mitochondrial membrane Circulating LH, FSH LH R FSH R Cholesterol StAR 5 6 Trilostane (-) Ketoconazole (-) Gonad (Generalized, gonadal, steroidogenic cell) Activin Inhibin Inner mitochondrial membrane pregnenolone 17 α -hydroxyprogesterone 20βHSD P450scc P450c17 androstenedione 3βHSD progesterone 7 8 Fadrozole (-) Prochloraz (-,-) 17α,20β-P (MIS) testosterone 17βHSD P45011β. P450arom 9 Vinclozolin (-) 11βHSD 11-ketotestosterone estradiol 10 Flutamide (-) Blood Circulating Sex Steroids / Steroid Hormone Binding Globlulin 11 β-trenbolone (+) 21 Androgen / Estrogen Responsive Tissues (e.g. liver, fatpad, gonads) ER AR 12 Ethynyl estradiol (+)

23 Types of Genomic Data Transcriptomics Fathead Minnow Microarray Proteomics Peptide Mass Fingerprinting Intens. [a.u.] x Representative protein expression profile in testes of control zebrafish Data from EPA/ EcoArray CRADA Metabolomics Fathead Minnow Liver NMR Scan Data from EPA-Cincinnati Fathead Minnow (male) m/z 22 Data from EPA-Athens

24 Effects of Aromatase Inhibition on Reproduction in the Fathead Minnow Cumulative Number of Eggs (Thousands) Fadrozole (ug/l) Control Exposure (d) * * * N Fadrozole N CN 8 Aromatase Activity (fmol/mg -1 hr -1 ) a b Male Female c c 0 50 Fadrozole (µg / L) 6 E2 (ng/ml) 4 2 * 0 * Vtg (mg/ml) * 23 0 * * Control Fadrozole (µg/l)

25 Key Nodes in Toxicity Pathways: Illustration from the HPG Axis in Fish Vitellogenein (vtg), egg yolk protein, is produced normally by oviparous female vertebrates in response to stimulation of the ER by 17β-estradiol Commonly used exposure biomarker in males for exposure to exogenous estrogens Effective production of vtg in females critical to successful egg production Vtg production in females can hypothetically be decreased via several discreet mechanisms within the HPG axis 24

26 Compartment Brain? GABA? GnRH Neuronal System GnR H GAB A A R D2 R D1 R Y 2 R NPY? Dopamine Molecular Mechanisms of Inhibition of VTG Production Pituitary PACAP Follistatin GAB A B R Y 2 R PAC 1 R FSHβ GnR H R Gonadotroph GP α Y 1 R LHβ D2 R Activin R Activi n Blood Gonad (Generalized, gonad, steroidogenic cells and oocytes) Circulating LDL, HDL LDL R HDL R Outer mitochondrial membrane Inner mitochondrial membrane Circulating LH, FSH LH R Cholestero l P450scc FSH R StAR Activin Inhibin pregnenolone 3βHSD (oocytes) 17 α -hydroxyprogesterone progesterone P450c17 Fadrozole 20βHSD 17α,20β-P (MIS) androstenedion e 17βHSD testosterone P45011β P450arom Prochloraz (steroidogenic cells) 11βHSD 11-ketotestosterone estradiol α trenbolone Blood Circulating Sex Steroids / Steroid Hormone Binding Globlulin AR β trenbolone 25 Androgen / Estrogen Responsive Tissues (e.g. liver, fatpad, gonads) Vtg Estradiol + ER Fenarimol

27 Linking Molecular Responses to Apical Effects: VTG and Fecundity Chemical Exposure Concentrations 17β-trenbolone 0.005µg/l, 0.05µg/l, 0.5µg/l, 5µg/l, and 50µg/l 17α-trenbolone 0.003µg/l, 0.01µg/l, 0.03µg/l, and 0.1µg/l Prochloraz 0.03mg/l, 0.1mg/l, and 0.3mg/l Fenarimol 0.1mg/l and 1mg/l Fadrozole 2µg/l, 10µg/l, and 50µg/l Fecundity = * Vtg (R 2 = 0.88) Relative Fecundity Fathead Minnow Fecundity vs Vtg Relative Vitellogenin 26

28 Population Forecasts Based on Molecular Responses Fecundity 17β-trenbolone 17α-trenbolone prochloraz fenarimol Measurement of vtg concentrations and fecundity for female fathead minnows Life table with age specific vital rates of survival and fecundity for the fathead minnow population Projection of density dependent logistic population trajectories for the fathead minnow population based upon change in vtg fadrozole Vtg Carrying capacity for the fathead minnow population Forecast Population Trajectories Population projection for populations at carrying capacity exposed to stressors that depress vitellogenin production 27 Average Average Population Population Size Size (Proportion (Proportion of Carrying of Carrying Capacity) Capacity) 1 1 A A 0% B B 25% C 50% C E D 0 >95% E 75% D Time (Years) Time (Years)

29 Tools Needed for Assessing Risk of EDCs in the Field Short-term in vitro and in vivo assays suitable for complex mixtures Analytical/fractionation approaches to identify specific chemicals causing biological effects Diagnostic endpoints (biomarkers) indicative of chemical MOA Approaches to link diagnostic responses to alterations in individuals and populations 28