Hypothesis-Based Weight of Evidence Approach for Evaluating Endocrine Activity (EDSP Data)

Transcription

1 Hypothesis-Based Weight of Evidence Approach for Evaluating Endocrine Activity (EDSP Data) Lessons Learned, Challenges, and Opportunities: the US Endocrine Disruptor Screening Program April 23-24, Research Triangle Park, NC Christopher J. Borgert, PhD 1

2 Co-Authors & Collaborators Rick Becker (ACC) Karin Bentley (DuPont) Ann Blacker (Bayer) John Brausch (BASF) Katie Coady (Dow) Sean Gehen (Dow) David Geter (Bayer) Richard Green (Syngenta) Elliot Gordon (E.Gordon LLC) Pat Guiney (SCJohnson) Matt Kern (Bayer) Fred Hess (BASF) Katie Holmes (BASF) Claire Iorizzo (Manna) Matt LeBaron (Dow) Steve Levine (Monsanto) Sue Marty (Dow) Ellen Mihaich (ER 2 ) Sandeep Mukhi (FMC) Barb Neal (Exponent) Lisa Ortego (Bayer) David Saltmiras (Monsanto) Mark Shocken (Exponent) Suzanne Snjadr (DuPont) Pramila Singh (Syngenta) Abe Tobia (Bayer) 2

3 Endocrine Disrupter Screening Program Tier 1 to identify the potential of chemicals to interact with the estrogen, androgen and thyroid systems in vivo. Tier 2 to identify and characterize adverse effects on reproductive function and development and the exposure required to produce them NOTE: no component of the EDSP seeks to establish whether adverse effects in tier 2 arise from endocrine mechanisms: no causal link between tiers 1 and 2. 3

4 Hyopthesis-Based WoE for ESB (Borgert et al., 2011) WoE Evalua)ons Will Be Needed for Determining: [a] from Tier 1 ESB and OSRI whether a substance exhibits the poten)al for interac)on with androgen, estrogen, or thyroid pathways in vivo; [b] from Tier 1 ESB, OSRI and other informa9on whether the substance should be further evaluated in Tier 2 toxicity tests; [c] from Tier 2 toxicity tests whether a substance exhibits adverse effects poten9ally mediated by androgen, estrogen, or thyroid pathways; [d] from Tier 1 ESB, OSRI, Tier 2 toxicity tests, and as necessary, addi9onal mode- of- ac9on experiments, whether the adverse effects observed in Tier 2 toxicity tests are a consequence of endocrine ac9vity, and; [e] whether endocrine- mediated adverse effects on humans or wildlife are possible at environmentally relevant exposure levels. 4

5 Hyopthesis-Based WoE for ESB (Borgert et al., 2011) Summary 1. Define explicit hypotheses 2. Probe literature via rules for systematic review 3. Evaluate primary, secondary, & tertiary validity 4. Weight endpoints quantitatively or rank-ordered based on explicit criteria and data 5. Weight results within the context of known positives and negatives 6. Develop narrative interpretation listing all assumptions 5

6 Hyopthesis-Based WoE for ESB (Borgert et al., 2011) Agonist Antgonist 6

7 Hyopthesis-Based WoE for ESB (Borgert et al., 2011) [a] Chemical X exhibits the poten9al to: [a]- 1 interact as an agonist with components of estrogen pathways [a]- 2 interact as an antagonist with components of estrogen pathways [a]- 3 interact as an agonist with components of androgen pathways [a]- 4 interact as an antagonist with components of androgen pathways [a]- 5 interact as an agonist with components of thyroid pathways [a]- 6 interact as an antagonist with components of thyroid pathways [a]- 7 interact with components of aromatse enzyme system [a]- 8 interact with components of steroidogenesis enzyme system 7

8 Why Hypotheses? 1. Clarity of purpose and analysis defines the activity under consideration separates different endocrine activities that may be exhibited by the test substance 2. Requires testing mode rather than speculation mode hypothesis testing is not the same as hypothesis generation 3. Discourages assay-by-assay, + / approach Tier 1 designed to utilize patterns of response, not isolated assays. 8

9 Hyopthesis-Based WoE for ESB (Borgert et al., 2011) 2. Relevance Weight (W REL ) for each endpoint / assay based on minimal epistemic status (Gori s tenets) Ideally, the analyst understanding would also include the posi9ve and nega9ve predic9ve value of each end point in each assay - i.e., its sensi9vity and specificity - for predic9ng endocrine ac9vity in vivo. May require rank ordering of endpoints un9l sufficient data are available to provide numerical rankings. 3. Result Weight (W RES ) scores the results obtained with the test substance according to a scale derived from the posi9ve and nega9ve control data for the assay. Preserves con9nuous nature of data 9

10 Endpoint Ranking Relevance Weight (W REL ) Descrip)on of Ranking Rank #1 W REL Endpoint is specific & sensi9ve for the hypothesis; relevance to hypothesis is interpretable without other endpoints; in vivo endpoints rarely confounded by ar4facts or non- specific ac4vity. Rank #2 W REL Rank #3 W REL NR Endpoint is specific & sensi9ve for the hypothesis; relevance to hypothesis is interpretable without other endpoints, but less informa9ve than Rank #1, oben due to sensi9vity & poten9al confounding; in vitro assays (Ques9on: steroidogenesis & aromatase?); many in vivo endpoints Endpoint is relevant for the hypothesis but only when corrobora9ve of Rank #1 and #2 endpoints; many apical in vivo endpoints. Not Relevant 10

11 Justification: Rank 1: Estrogenic Endpoints classically used to define the hormonal activity. See medical dictionaries, etc. Estrogenic: estrus-producing in animals; similar to that of an endogenous estrogen; induction of sexual behavior; feminizing; cellular proliferation of tissues of sexual organs and other tissues related to reproduction. Uterotrophic assay in rodents Vitellogenin production in male fish 11

12 Justification: Rank 1: Androgenic Endpoints classically used to define the hormonal activity. See medical dictionaries, etc. Androgenic: growth, development, and function of the male reproductive system. Herberger concordance endpoints: Cowper s gland, seminal vesicles, LABCM, glan penis, ventral prostate Fish screen: tubercles in females 12

13 Justification: Rank 1: Thyroidal Endpoints classically used to define the hormonal activity. See medical dictionaries, etc. Thyroidal: controlling growth and development AMA: asynchronous development; thyroid histopathology 13

14 Justification for Rankings Rank 2 Expected to respond to the hormonal activity, but not necessarily the defining endpoints. Also, specific components of the defining response, e.g., transactivation. Rank 3 Known to respond to the hormonal activity, but may lack specificity. Also, specific components of the defining response, e.g., receptor binding. 14

15 Estrogen Agonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints Fish Screening Assay ER TA Assay AM Assay Vitellogenin: increased in males ER agonism Asynchronous Development Uterotrophic Assay Fish Screening Assay Delayed Development Increased uterine weight (wet/blotted) Secondary Sexual Characteristics: decreased tubercle score: males Gonad Histopathology: males Behavior: males Pubertal Female Assay Age & vaginal opening Uterus Weight Ovary Weight Uterus Histopathology Ovary Histopathology Age at first estrous Testes Weight Pubertal Male Assay ER RB Assay ER competitive binding affinity Fecundity Estradiol Testosterone Fish Screening Assay Gonad Somatic Index: decreased in males, increased in females Behavior: females Fertilization Success: males & females Growth Estrous Cyclicity Pubertal Female Assay Testes Histopathology: atrophy Uterotrophic Assay Conversion to Estrous-Supplemental Pubertal Male Assay Growth Ventral Prostate Weight Epididymides Histopathology Estradiol Levels 15 Steroidogenesis Assay

16 Estrogen Antagonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints ER RB Assay Aromatase Assay ER competitive binding affinity Aromatase Inhibition Pubertal Female Assay Steroidogenesis Assay Age and Weight at vaginal opening Estradiol Levels Uterus Weight Pubertal Female Assay Ovaries Weight Estrous Cyclicity Uterus Histopathology Fish Screening Assay Ovary Histopathology Fecundity Age at first estrous Estradiol Fish Screening Assay Testosterone Vitellogenin: decreased in females Gonad Somatic Index Gonad Histopathology: in females Behavior Fertilization Success 16

17 Androgen Agonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints Hershberger Assay AR RB Assay Aromatase Assay Cowper s Gland Weight: for concordance of all endpoints AR competitive binding affinity Aromatase inhibition Seminal Vesicle Weight: for concordance of all endpoints Hershberger Assay Steroidogenesis Assay Levator Ani-bulbocavernosus Muscle Weight: for concordance of all endpoints Cowper s Gland Weight: if unique response Testosterone Levels Glans Penis Weight: for concordance of all endpoints Seminal Vesicle Weight: if unique response Pubertal Male Assay Ventral Prostate Weight: for concordance of all endpoints Levator Ani-bulbocavernosus Muscle Weight: if unique response Growth Fish Screening Assay Glans Penis Weight: if unique response Testosterone Levels Secondary Sexual Characteristics: for tubercles in females Ventral Prostate Weight: if unique response Pubertal Female Assay Pubertal Male Assay Age and Weight at Preputial Separation: if accelerated Seminal Vesicle + Coagulating Gland Weight (wet/ blotted) Ventral Prostate Weight Dorsolateral Prostate Weight Levator Ani + Bulbocavernosus Muscle Complex Weight Epididymis Weight Growth Age and Weight at vaginal opening Uterus Weight Ovaries Weight Adrenals Weight Uterus Histopathology Ovary Histopathology Testes Weight Testes Histopathology Fecundity Fish Screening Assay 17 Epididymides Histopathology Testosterone

18 Androgen Antagonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints Hershberger Assay AR RB Assay Steroidogenesis Assay Cowper s Gland Weight: for concordance of all endpoints AR Competitive binding affinity Testosterone Levels Seminal Vesicle Weight: for concordance of all endpoints Hershberger Assay Pubertal Male Assay Levator Ani-bulbocavernosus Muscle Weight: for concordance of all endpoints Cowper s Gland Weight: if unique response Testosterone Levels Glans Penis Weight: for concordance of all endpoints Seminal Vesicle Weight: if unique response Fish Screening Assay Ventral Prostate Weight: for concordance of all endpoints Levator Ani-bulbocavernosus Muscle Weight: if unique response Glans Penis Weight: if unique response Ventral Prostate Weight: if unique response Fecundity Estradiol Testosterone Pubertal Male Assay Age and Weight at preputial separation: if delayed Gonad Somatic Index Behavior Seminal Vesicle + Coagulating Gland Weight (wet/ blotted) Fertilization Success Ventral Prostate Weigh Dorsolateral Prostate Weight AM Assay Wet Weight: increase in males; decrease in females Levator Ani + Bulbocavernosus Muscle Complex Weight Epididymis Weight Testes Weight 18 Testes Histopathology

19 Thyroid Agonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints AM Assay Pubertal Male Assay Pubertal Male Assay Asynchronous Development Thyroid Weight Growth Thyroid Histopathology T4 Age and Weight at Preputial Separation TSH Pituitary Weight Pubertal Female Assay Pubertal Male Assay Ovary Histopathology Growth TSH Age and Weight at Vaginal Opening Estrous Cyclicity Blood Chemistry AM Assay Fish Screening Assay Advanced Development Vitellogenin AM Assay Snout-Vent Length Hind Limb Length Wet Weight 19

20 Thyroid Antagonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints Pubertal Male Assay Pubertal Male Assay Pubertal Male Assay Thyroid Weight Liver Weight Growth Thyroid Histopathology T4 Age and Weight at Preputial Separation Pubertal Female Assay TSH Pituitary Weight Thyroid Weight Pubertal Female Assay Pubertal Female Assay Kidney Weight Age and Weight at Vaginal Opening Growth Ovary Histopathology Ovaries Weight Blood Chemistry Thyroid (colloid area and follicular cell height) T4 Fish Screening Assay AM Assay TSH Testosterone Asynchronous Development Estrous Cyclicity Gonad Somatic Index Thyroid Histopathology AM Assay Delayed Development Snout-Vent Length Hind Limb Length Wet Weight 20

21 Aromatase Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints Pubertal Female Aromatase Steroidogenesis Uterus Weight Aromatase Inhibition Estradiol Levels Pubertal Female Testosterone Levels Age and Weight at vaginal opening Pubertal Female Age at first estrous Ovaries Weight Steroidogenesis Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints Steroidogenesis Steroidogenesis Aromatase Testosterone Values Estradiol Levels Aromatase Inhibition Pubertal Female Assay Uterus Weight Ovaries Weight 21

22 Interpretation of WREL + WRES Testing Hypotheses Require the Battery: All Ranks Rank 1 Rank 2 Preliminary indication of support or refutation of hypothesis Consider not only whether endpoints respond, but the strength of the response (WRES) Consistency with Rank 1 (response direction and strength) is sufficient to conclude support or refutation of hypothesis Consult Rank 3 for additional support Inconsistencies Ranks 1 & 2 Refute / Rank 3 responds = Hypoth. Refuted Rank 1 Supports / Rank 2 Refutes? Rank 1 Refutes / Rank 2 Supports?? 22

23 Isoflavone present in plant foods like soy and fava beans Identified as an estrogen agonist Example : Genistein Fertility issues in sheep feeding on fields of clover Asian diet high in soy and phytoestrogens 10-4 potent as estradiol Cline et al. 2001* Cardiovascular benefits Not estrogenic in humans at dietary doses Impedes E2-proliferation, but PR not down-regulated, thus, effects may not be ER-mediated. *Cline JM, Söderqvist G, Register TC, Williams JK, Adams MR, Von Schoultz B Assessment of hormonally active agents in the reproductive tract of female nonhuman primates. Toxicol Pathol.29:

24 Endpoint Negative or Vehicle Control Genistein: Estrogen Agonism, R1 Positive Control Genistein Response Weight (WRES) Increased VTG (23) Vehicle Control 17-β estradiol 300 ng/d ip, significant increase in VTG 75 to 3000 ng/d No increase in VTG 0 Increased VTG (24) Males fed genistein-free diet - Significant increase in VTG (but lower than )? Uterine wet weight (1) mg 0.3µg/kg/d = mg 1.0µg/kg/d = mg 35mg/kg/d = mg 0.3 (3.5E-4) Uterine blotted weight (1) mg 0.3µg/kg/d = mg 1.0µg/kg/d = mg 35mg/kg/d = mg (3.5E-4)

25 Endpoint Negative or Vehicle Control Genistein: Estrogen Agonism, R2 Positive Control Genistein Response Weight (WRES) ERTA agonism (12) Negative control no response 17-β estradiol 0.1nM 7.52X - ERα 7.82X - ERβ 10 nm 8X ERα 6X ERβ 1.0 (1E-2) Fish Screen Gonad Histopath (24) - - Increase in Stage 4 (<20% spermatozoa) with increased genistein in the diet? Pubertal male:testes Weight - 17-β estradiol Decreased No significant difference (2 studies) 0 Testes Histopath (18) - 17-β estradiol Atrophy in seminiferous tubules Hyperplasia of Leydig cells (18) 25?

26 Endpoint Negative or Vehicle Control Genistein: Estrogen Agonism, R2 Positive Control Genistein Response Weight (WRES) Uterus Weight (20, 22) - - Dose-dependent increase (2 studies)? Age & Weight at Vaginal Opening - - Linear trend toward an accelerated time to VO (20)? Ovary Histopath - - Abnormal epithelial changes and persistent corpora lutea (20)? 26

27 Estrogen Receptor Binding ER competitive binding affinity Genistein: Estrogen Agonism, R3 Response Effect Steroidogenesis Estradiol levels Effect (5), No Effect (7) Pubertal Female Growth Effect (20) Estrous Cyclicity Did not measure Pubertal Male Growth Effect (18), No Effect (20) Ventral prostate weight Effect (20) - decrease Epididymides histopathology Effect (18) Amphibian Metamorphosis Assay Asynchronous development Not measured Delayed development Effect (25) 27

28 Fish Screening Assay Genistein: Estrogen Agonism, R3 Response Fecundity No effect (24) Estradiol (Female) Effect (24) Testosterone (Male) Effect (23, 24) Gonadal-Somatic Index: Decreased in male Increased in female Behavior (Male) No effect (24) Not measured Fertilization Success Effect (24) 28

29 Rank 1 RUA: potency [E2] ~ 3.4E -04 VTG: inconsistent (1 study + / 1 study ) Rank 2 ERTA: potency ~ 1E -02 Fish histopath Rat testes E2-like responses in female pubertal Rank 3 ER Binding Some corroboration from pubertal screens Some corroboration from E2 and T levels in fish Genistein (WREL + WRES) WoE Narrative: Rank 1 & 2 generally consistent; Rank 3 corroborative. Based on these studies, Genistein shows moderate potential for estrogenic activity in vivo. 29

30 Hypothesis-Based WoE Framework is NOT: box-checking falsely quantitative onerously time-consuming a filibuster Concluding Remarks Hypothesis-Based WoE Framework IS: Transparent - hypotheses, methods, assumptions defined explicitly Testable - methods and assumptions can be challenged, altered to determine sensitivity to assumptions, etc. Objectively Reproducible - unlike professional judgment Streamlined - interpretation focuses on most relevant endpoints (WREL); obviates time spent on least critical information. Biologically Plausible: Potency Consideration (WRES) Updatable: WREL and other assumptions can be altered with better information Generalizable: can address other WoE determinations in EDSP & additional endocrine and non-endocrine activities. (e.g., McCarty, Borgert, Mihaich, EHP 120: ) 30