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Fish Screening Assay Detailed Review Paper NACEPT Endocrine Disruptor Methods Validation Subcommittee March 2002 Les Touart. Detailed Review Paper: FISH SCREENING ASSAYS FOR ENDOCRINE DISRUPTION. WORK PERFORMED BY On behalf of the United States Environmental Protection Agency

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Fish Screening Assay

Detailed Review Paper

NACEPT

Endocrine Disruptor Methods Validation Subcommittee

March 2002

Les Touart


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Detailed Review Paper:FISH SCREENING ASSAYS FOR ENDOCRINE DISRUPTION

WORK PERFORMED BY

On behalf of the United States Environmental Protection Agency

EPA CONTRACT NUMBER 68-W-01-023


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METHODS USED IN THIS ANALYSIS

  • On-line Literature Search (August 9th 2001)

    • “Dialog” On-Line search with database Biosis Previews Aquatic Science and Fisheries Abstracts

    • Endocrine disruptor screening methods for fathead minnows, zebrafish, and Japanese medaka

    • Key Words “estrogen* or testosteron* or endocrin* or antiandrogen* or androgen* or hormon* or thyroxin* or * thyroid * method, protocol etc…

    • Approximately 10000 records were refined down to 500 papers that were reviewed


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OVERVIEW AND SCIENTIFIC BASIS OF FISH REPRODUCTIVE SCREENING ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

  • Fish differ in steroid profiles from mammals

  • Estrogen receptor in fish differ structurally and functionally from mammals

  • Steroid receptors in eggs and hepatic vitellogenin (VTG) have no known analogous receptors in mammals

  • Fish Reproductive Screen Assay is essential to address these known endocrine differences.


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Test Species ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

  • Fathead minnow (Pimephales promelas)

  • Japanese medaka (Oryzias latipes)

  • Zebrafish (Danio rerio)

    • small size at maturity

    • ease of culture

    • maintenance costs

    • asynchronous spawners


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Fathead Minnow ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)Family Cyprinidae

  • 35 to 75 mm length

  • Extensive aquatic toxicity in USA

  • generation time about 4 months

  • sexually dimorphic

  • can be kept in breeding condition all year

  • females produce 50 to 250 embryos per spawn


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Strengths ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

Large enough to collect individual blood plasma samples

Distinct secondary sex characteristics in both sexes

Large historical regulatory database

Many laboratories are familiar with culture and testing

Spawn on a substrate

High fertilization rate

Indigenous to North America

Weaknesses

Relatively long life cycle

Relatively high variability in fecundity

Relative size of the fish requires more space for culture and testing

Intersex condition is less frequently observed compared to other fishes.

Genome poorly characterized

Fathead Minnow


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Japanese medaka ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)Family Cyprinodontidae

  • indigenous to Japan, Taiwan, and southeastern Asia

  • Generation interval of 2 to 3 months

  • sexually dimorphic

  • 25 mm to 50 mm length

  • females produce 10 to 30 eggs per spawn

  • estimated to be over 500 cultivated strains

    • Genetically Engineered / Inbred Strains in Toxicity Testing


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Strengths ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

Relatively short life cycle

Relatively small fish, making culture and testing possible in smaller space

Female sex determined during embryo stage vs. male sex determined after hatch

Sex-linked color strain

Weaknesses

Smaller size reduces individual blood sample volumes compared to fathead minnow

Less distinctive secondary sex characteristics

Regulatory data base less extensive compared to fathead minnow.

Limited use in short-term tests in the U.S.A.

Japanese medaka


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Zebrafish ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)Family Cyprinidae

  • Native to East India and Burma

  • 4 cm to 5 cm in length

  • Extensive aquatic toxicity in Europe

  • Difficult to sex zebrafish

  • Sexual maturity in 10 to 12 weeks

  • 150 to 400 eggs per female

  • Development of transgenic zebrafish


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Strengths ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

Short life cycle

Small fish, making culture and testing possible in smaller spaces

Male fish go through a hermaphroditic phase as juveniles

Widely used in other medical and genetic research

Frequently used in Europe for regulatory purposes

Transgenic fish increasingly available

Anticipated that entire genome will be sequenced soon.

Weaknesses

Small size makes individual blood plasma samples not likely

Minimal secondary sex characteristics

Limited US regulatory data base

Limited testing experience in the US

Zebrafish


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Routes of Administration of Chemical Exposure ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

  • Aqueous

  • Dietary exposures

  • Direct injection techniques

    -intraperitoneal

    -intramuscular

    -dorsal sinus


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Measurement Endpoints ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

  • Growth and Morphological Alterations

    • Gonadosomatic Index

    • Histology Techniques

    • Sexual Differentiation

    • Secondary Sex Characteristics

  • Biochemical Measures

    • Vitellogenin Induction

    • Tissue Steroid Concentrations

    • Thyroid hormones

  • Measures of Reproductive Performance

    • Fecundity

    • Gamete Viability

    • Fertilization Success


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MEASUREMENT OF BIOCHEMICAL ENDPOINTS ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

  • Sex Steroids in Tissues Estrogens/Androgens/Progestins

    • Radioimmunoassay (RIA)

    • Enzyme-linked Immunosorbent Assay (ELISA)

    • Liquid/Gas Chromatography with Mass Selective Detection (LC/GC-MS)


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Measurement of Vitellogenin ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

  • Indirect Quantification of Vitellogenin Protein

    • Alkaline-labile Phosphate Assay

  • Direct Quantification of Vitellogenin Protein

    • RIA

    • Enzyme-linked Immunosorbent Assay

      • Antibody-capture

      • Antigen-capture

    • Direct Enzyme-linked Immunosorbent Assay

    • Sandwich Enzyme-linked Immunosorbent Assay

  • Quantifying Vitellogenin mRNA

    • Ribonuclease Protection Assay (RPA)

    • Quantitative Reverse Transcription-Polymerase Chain Reaction (QRT-PCR)

  • Mass spectrometry (MS)


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CANDIDATE PROTOCOLS ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

1) 21-day reproductive assay (as described in Ankley et al., 2001)

2) 14-day fish reproductive assay (modified from the version described in Ankley et al., 2001)

3) 14-day fish non-reproductive screen (OECD Draft-31 December 2001)


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21-Day Fish Reproductive Assay ASSAY (ENDOCRINE CONTROL OF REPRODUCTION) (See Hand Out)

  • Chemical Exposure – 21 Days

  • 14 day pre-exposure spawning quantitative measures

  • Exposure Measurement Endpoints

    • Adult survival, reproductive behavior, secondary sexual characteristics, GIS and gonadal history, plasma VTG and sex steroids (b-estradiol, testosterone, 11-KT) concentrations, fecundity, fertility, embryo hatch, and larval survival

  • Acceptance Criteria: D.O. ≥ 60% saturation; Mean temp. 25°C ± 2°C; 90% survival in the controls and successful egg production in controls


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14-Day Fish Reproductive Assay ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)(See Hand Out)

  • Chemical Exposure – 14 Days

  • 14 day pre-exposure spawning observations

  • Exposure Measurement Endpoints

    • Adult survival, reproductive behavior, secondary sexual characteristics, GIS and gonadal history, plasma VTG and sex steroids (b-estradiol, testosterone, 11-KT) concentrations, fecundity, fertility, embryo hatch, and larval survival

  • Acceptance Criteria:

    • D.O. ≥ 60% saturation; Mean temp. 25°C ± 2°C; 90% survival in the controls and successful egg production in controls

  • Similar to the 21 day Fish Reproductive Assay with out quantitative measures of fecundity or embryo viability (e.g., hatchability) are made during the 14 day pre-exposure


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14-Day Fish Non-Reproductive Assay (See Hand Out) ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

  • Chemical Exposure - 14 Days

  • Exposure Measurement Endpoints

    • Non-specific:

      • Survival,

      • Behavior

    • Endocrine specific:

      • Gross morphology (GSI)

      • VTG

      • Gonadal history

  • Acceptance Criteria: D.O. ≥ 60% saturation; Mean temp. 25°C ± 2°C; 90% survival in the controls

  • (No reproductive Endpoints)


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Significant Data Gaps ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

• Male-specific effects of estrogen agonists other than VTG induction.

• The effects of anti-estrogens, especially pure or Type II anti-estrogens in sexually mature test species.

• The effects of androgenic and anti-androgens in sexually mature test species, specifically, endpoints other than secondary sex characteristics that may be more sensitive to (anti-) androgens.

• Baseline data for thyroid hormone levels during reproduction in test species.

• The effects of thyroid hormone agonists (or thyroid stimulation) on reproduction.


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IMPLEMENTATION CONSIDERATIONS ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

  • Pre-validation studies following the ICCVAM validation process

    • Recommend Comparison of three candidate protocols with like chemicals

    • Recommend Evaluation of Vtg methods

    • Recommend Consideration be given to the lack of data on Thyroid – none of candidate protocols adequately measure thyroid function/performance. Consider smoltification assay

  • Validation of the study design through interlaboratory comparisons


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Questions ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)

  • Does the DRP provide adequate background and rationale to support further validation of a fish reproduction assay for screening potential endocrine disrupting chemicals?

  • Does the EDMVS have suggestions to improve the DRP?