ENDOCRINE DISRUPTORS RESEARCH PROGRAM:

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ENDOCRINE DISRUPTORS RESEARCH PROGRAM Long Term
Goal 2 Progress Report
Susan A. Laessig, Ph.D. ORD National Center for
Environmental Research EDCs Research Program
Mid-Cycle BOSC Review September 14, 2007
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Multi-Year Plan Long-Term Goals

• LTG 1 - Reduction in uncertainty to Provide a
  better understanding of the science underlying
  the effects, exposure, assessment, and management
  of endocrine disruptors so that EPA has a sound
  scientific foundation for environmental
  decision-making
• LTG 2 - Determination of the extent of the impact
  of endocrine disruptors on humans, wildlife, and
  the environment to better inform the federal and
  scientific communities
• LTG 3 - OPPTS uses endocrine disruptor screening
  and testing assays developed by ORD to create
  validated methods that evaluate the potential for
  chemicals to cause endocrine-mediated effects in
  order to reduce or prevent risks to humans and
  wildlife from exposure to EDCs (Support EPAs
  screening and testing program)

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Outline

• LTG 2 2003 Annual Performance Goals
• Characterize sources of exposure and
  environmental fates of EDCs
• Determine whether adverse developmental/reproducti
  ve effects are occurring in human populations
• Determine extent to which EDCs contribute to the
  severity or onset of disease
• Future Directions

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LTG 2

• 1. Characterize sources of exposure and
  environmental fates of EDCs
• Sources of EDCs from Concentrated Animal Feeding
  Operations (CAFOs)
• ORD Collaborative Project NERL, NHEERL, NRMRL,
  NCER
• Goal Characterize occurrence and ecological
  impacts of estrogenic and androgenic chemicals
  from CAFO waste
• Approach
• Develop in vitro and analytical methods to
  identify and quantify compounds responsible for
  endocrine activity
• Assess ecological impacts using a combination of
  laboratory and field studies
• Identify ecologically-relevant biomarkers in
  aquatic species using genomic approaches
• Evaluate the environmental fate, transport and
  metabolism of CAFO-derived EDCs in surface and
  ground waters
• Evaluate capability of existing risk management
  technologies for CAFOs to reduce exposure to EDCs

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LTG 2

• 1. Characterize sources of exposure and
  environmental fates of EDCs
• ORD Collaborative Project NERL, NHEERL, NRMRL,
  NCER
• Sources of EDCs from Concentrated Animal Feeding
  Operations (CAFOs)
• ORD EDC Projects
• Developed analytical methods for determining
  estrogens and conjugates in swine waste
  characterized ground water contamination from
  swine lagoons
• Developed cell-based assays for estrogens and
  androgens
• Developed biomarkers of androgen exposure in
  fathead minnows
• Determined androgenicity in runoff from beef
  feedlots
• Developed reproductive fecundity models (Ankley
  et al, 2003 Jensen et al, 2006) and a population
  dynamics model for effects of trenbolone in
  fathead minnows (Miller and Ankley, 2004)

Control Male
Control Female
bTB - Female
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LTG 2

• 1. Characterize sources of exposure and
  environmental fates of EDCs
• Sources of EDCs from Concentrated Animal Feeding
  Operations (CAFOs) ORD Collaborative Project
  NERL, NHEERL, NRMRL, NCER
• Impacts
• Both a and b-TB masculinize females and produce
  similar response profiles in plasma steroids and
  vitellogenin
• Water samples associated with a typical,
  operating beef CAFO consistently exhibit
  androgenic activity (Durhan, et al 2006)
• Both a and b-trenbolone (TB) were consistently
  measured and confirmed in samples
• Simple estimate of risk ratio (exposure/effects)
  indicates sufficient TB to adversely affect egg
  production in fish

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LTG 2

• Population Dynamics Modeling
• Determining Population-level Ecological Effects
  of EDCs
• Joint project between EDC and Computational
  Toxicology Program
• Modeling construct toxicity, life history,
  carrying capacity, and habitat data.

• Applications of model
• Predict population dynamics of fathead minnow
  exposed to the endocrine disruptor 17ß-trenbolone
  
• Relate changes in biomarkers (vitellogenin,
  steroids) to changes in fecundity from exposures
  to multiple chemicals
• Impact
• 3 publications to date
• Forecasted population trajectories for fathead
  minnow population initially at carrying capacity
  and subsequently exposed to chemicals that
  depress vitellogenin or 17ß-estradiol (E2)

(A) No reduction in VTG (B) 25 reduction in
VTG (C) 50 reduction in VTG(D) 75 reduction in
VTG (E) 96 reduction in VTG
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LTG 2

• 1. Characterize sources of exposure and
  environmental fates of EDCs
• ORD Collaborative Project NERL, NHEERL, NRMRL,
  NCER
• STAR Awards
• 7 university grants and cooperative agreements
  awarded to study the fate and effects of hormones
  in waste from CAFOs. (3-year projects begin in
  2007)
• Research Goals
• Increase knowledge of the amount and fate of
  steroid hormones associated with animal waste
  under various conditions
• Determine the extent of the impact of hormones in
  CAFO waste on aquatic and terrestrial habitats
• Evaluate new or improved animal waste handling
  systems and risk management options for steroid
  hormones in animal waste

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LTG 21. Characterize sources of exposure and
environmental fates of EDCs

• ORD Collaborative Project NERL, NHEERL, NRMRL,
  NCER
• Aug. 2007 Workshop on fate and effects of
  hormones in waste from CAFOs
• EPA, FDA, USDA, USGS
• Participants from federal, state, industry, and
  academic communities
• Initial meeting between 7 STAR awards, EPA, and
  other collaborators
• Impact
• Obtained stakeholder input on proposed and
  ongoing research
• Facilitated discussion and resolution of common
  research issues
• Produced workshop summary report
• Improved interactions with other agencies

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LTG 2

• 1. Characterize sources of exposure and
  environmental fates of EDCs
• Sources of EDCs from Concentrated Animal Feeding
  Operations (CAFOs) ORD Collaborative Project
  NERL, NHEERL, NRMRL, NCER
• Anticipated Impacts
• Understanding of risks associated with CAFOs as
  potential sources for hormones in manure, soil,
  surface water, and groundwater
• Support the activities of the EPA Office of Water
  (OW) and Regional Offices with respect to the
  regulation of CAFOs
• Support site-specific risk assessments and
  development of risk management options for
  hormones in waste from CAFOs

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LTG 2

• 1. Characterize sources of exposure and
  environmental fates of EDCs
• STAR awards on Exposure Methods and Applications
  to Monitoring
• 5 STAR projects awarded in 2006 are developing
• Analytical methods for mixtures of EDCs in
  environmental media
• Rapid chemical or biological detection methods
  for environmental exposure monitoring
• Activity-based biological indicators (androgen,
  estrogen, and/or thyroid) for assessing EDC
  exposure in environmental media
• Methods under development include
• Bioaffinity-based nanomechanical sensors
• A passive concentrator and spectral analysis
  technique for rapid in situ detection
• Biomarkers of survival and reproduction in
  aquatic vertebrates exposed to treated waste
  water
• Receptor-to population-level analytical tools for
  assessing EDC exposure in wastewater-impacted
  estuarine systems
• Gene expression biomarkers in aquatic
  invertebrates

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LTG 2

• 1. Characterize sources of exposure and
  environmental fates of EDCs
• STAR awards on Exposure Methods and Applications
  to Monitoring
• Examples of Progress
• Evaluated 5 polymers for the ability to extract
  EDCs from water
• Tested an array of known EDCs in relevant
  matrices for SERS response characteristics to
  create a quantitative spectral library
• Established methodology for determining exposure
  effects through a newly developed bioassay, the
  physiological response of diatoms to estrogens -
  - 4-nonylphenol, but not 17b-estradiol, results
  in cell mortality
• Cloning and characterization of the retinoid X
  receptor from Daphnia magna
• The environmental-endocrine basis of intersex in
  a crustacean
• Evaluated estrogens singularly (E1, E2, EE2) and
  in mixture for effects on reproduction and brain
  of fathead minnows
• PIs presented progress at 2006 review workshop

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LTG 2

• 1. Characterize sources of exposure and
  environmental fates of EDCs
• Molecular biomarkers of EDC exposure in
  invertebrates
• Approach
• Genome in aquatic invertebrate, Daphnia pulex,
  sequenced and analyzed
• Identify differentially expressed genes after
  exposure to atrazine or nonylphenol, previously
  identified as EDCs in Daphnia sp.
• Progress
• Picked 960 clones for initial screening for each
  exposure
• Atrazine no outstanding candidates from initial
  screening analysis of 10 candidates showed no
  differential expression
• Nonylphenol - screening revealed a few candidates
• Continuing to test candidate genes, develop PCR
  primers, and test lower concentrations of exposure

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Experimental Lakes Study (1999-2004)Whole Lake
Additions of Ethinylestradiol (EE2)
Collaborators NERL-Cincinnati, Fisheries and
Oceans Canada, USGS
Goal apply genomic tools to assess exposure of
wildlife to EDCs in a field exposure study

• Major Findings
• Fathead minnows and pearl dace had elevated whole
  body concentrations of vitellogenin within 7
  weeks of EE2 additions to Lake 260
• Egg development delayed in fathead minnows and
  pearl dace testes development impaired
  testes-ova observed in males
• Reproductive failure was observed in both of
  these minnow species during the second year of
  additions
• Kidd KA, Blanchfield PJ, Mills KH, Palace VP,
  Evans RE, Lazorchak JM, Flick RW. Collapse of a
  fish population after exposure to a synthetic
  estrogen. Proc Natl Acad Sci U S A. 2007 May
  22104(21)8897-901.
• Impact
• Extended the capabilities of EPA/ORD EDC
  monitoring efforts
• EPA cites the study for developing Ambient WQC
  for EDCs
• FDA has cited paper for Environmental Assessment
  testing

Vitellogenin gene expression in male fathead
minnows
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LTG 2

• 2007 Co-sponsored separate interagency workshops
  on impacts of EDCs on ecosystems and human health
• Goal document progress and identify potential
  interagency collaborations
• EPA, USGS, USDA, FDA, NOAA, USFWS, OMB, NIEHS,
  DOD, CPSC
• Assessed progress since CENR 1996 Framework for
  Research on EDCs
• Biomarkers of ecological effects and exposure
  and effects monitoring
• Translation of basic and animal research to
  understand human disease
• Impact
• Improved awareness and inventory of federal
  activities
• Identified potential interagency collaborations
• Identified research priorities
• Improved interactions with other agencies

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LTG 2

• 2007 Report on EPA Research Accomplishments
• Exposure and Effects of EDCs in wildlife
• Purpose Synthesize results and communicate
  progress
• Topics include
• Identifying sources of EDCs in the environment
• Effects of exposure to EDCs in vertebrate
  wildlife
• Exposure and effects in invertebrates
• Species differences
• Effects in wild populations
• Anticipated Impact
• Improved understanding of EPAs progress in
  critical areas
• Improved communications and outreach to Congress
  and general public

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LTG 2

• 2. Determine the extent of the impact of EDCs in
  humans
• STAR grants on EDCs Epidemiology Approaches
• 2001 EPA, NIEHS, CDC, NCI jointly funded grants
  for 12 epidemiology studies to examine the human
  health effects of EDCs
• Progress
• 2004 Meeting of all grantees supported under
  interagency RFA
• 2006 EPA grantees met at EPA EDC Progress Review
• No cost extensions given to complete sample and
  data analyses
• Grantees will be invited to full BOSC review in
  early 2009

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LTG 2

• 2. Determine the extent of the impact of EDCs in
  humans
• STAR grants on EDCs Epidemiology Approaches
• Impacts
• Key results highlighted in 2007 EDC
  Accomplishments Report
• Phthalates in pregnant women and children linked
  to altered male reproductive development
• Consumption of local produce increased exposure
  to dioxins in a population of Russian boys
• PBDEs may affect thyroid hormone homeostasis
  through effects on thyroid binding globulin.
  Effects vary by gender and menopausal status in
  women

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LTG 2

• 2. Determine the extent of the impact of EDCs in
  humans
• STAR grants on EDCs Epidemiology Approaches
• Impacts (continued)
• Hot paper
• Swan SH, et al. Decrease in anogenital distance
  among male infants with prenatal phthalate
  exposure. Environ Health Perspect. 2005
  Aug113(8)1056-61.
• Subsequent grant awards received for continued
  study of cohorts
• Positive impacts on communities

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LTG 2
3. Determine extent to which EDCs contribute to
the severity or onset of disease Effects of EDCs
on mammary gland development
Fenton. 2006. Endocrine Disrupting Compounds
(EDCs) and Mammary Gland Development Early
exposure and later life consequences.
Endocrinology. 147 (Supplement)S18-24.
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LTG 2
3. Determine extent to which EDCs contribute to
the severity or onset of disease Effects of EDCs
on mammary gland development
Effects Observed Delayed Development Atrazine Di
oxin PFOA AMM Impaired Lactation Atrazine Diox
in PFOA Transgenerational Effects Atrazine Dioxin
Precocious Development 4-Nonylphenol Estradiol
( control) Atrazine metabolite mixture (AMM)

• Impacts
• Defined critical window of mammary gland
  development for atrazine, mixture of atrazine
  metabolites, dioxin, and PFOA
• Publications on the effects of atrazine on
  mammary gland morphology
• Toxicological Sciences paper won Best paper of
  the year award from Reproductive and
  Developmental Toxicology Specialty Section at the
  2006 SOT meeting
• Pending publications on mammary gland
  development as a sensitive endpoint, body burden
  of atrazine metabolites following exposure, and
  atrazine disposition in pregnant and lactating
  rats

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LTG 2 Future Directions

• 2007 MYP Implementation
• Complete epidemiology studies
• Complete exposure methods and application
  studies
• Complete fate and effects of hormones in waste
  from CAFOs
• Participation in Pharmaceuticals in the
  Environment Workgroup
• Global Water Research Coalition collaboration

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LTG 2 Future Directions
Linkage and Timeline for APGs to Meet Long Term
Goal 2 (2007 MYP)
2009
2010
2011
2012
2013
2015
2014
2011- Provide OPPTS, OW, the Regions and other
organizations with data to determine the
potential for adverse developmental/reproductive
effects in human populations following exposures
to EDCs
2012 - Provide OPPTS, OW, the Regions and other
organizations with information from the
application of cross-disciplinary tools to
characterize the occurrence and potential
biological effects from and develop management
approaches for EDCs in complex environmental media
Determination of the extent of the impact of
endocrine disruptors on humans, wildlife and the
environment to better inform the federal and
scientific communities
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LTG 2 Future Directions

• Pharmaceuticals in the Environment (PiE)
  Interagency Workgroup
• EPA, FDA, USGS (Co-leads), CDC, NIEHS, NOAA,
  USDA, FWS
• White House OSTP Toxics and Risks Subcommittee
• Progress
• Interactive project inventory database
• Developing 2 strategy documents outlining data
  gaps/priorities/areas of collaboration
• Research strategy for human and veterinary
  pharmaceuticals in the environment (12/2007)
• Research strategy for antibiotics in the
  environment focusing on the question of
  antibiotic resistance (12/2008)

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LTG 2 Future Directions

• Global Water Research Coalition
• Tools for Analyzing Estrogenicity in
  Environmental Waters
• International collaboration
• Analytical round-robin comparing cell-based
  endocrine assays
• Water samples collected from participating
  countries around the world
• EPA Transcriptional activation assays in stable
  cell lines
• T47D-KBluc estrogen responsive (ERa ERb)
• Positive estrogenic response demonstrated with
  tertiary treated sewage effluent samples
• Anticipated Impact
• Recommendations for selecting appropriate
  bioassays for a given water source