Fish Screening Assay Detailed Review Paper

NACEPTEndocrine Disruptor Methods Validation

SubcommitteeMarch 2002

Les Touart

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

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

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.

Test Species

• Fathead minnow (Pimephales promelas)• Japanese medaka (Oryzias latipes) • Zebrafish (Danio rerio)

– small size at maturity – ease of culture– maintenance costs– asynchronous spawners

Fathead MinnowFamily 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

Fathead MinnowStrengths

• 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

Japanese medakaFamily 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

Japanese medaka

Strengths

• 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.

ZebrafishFamily 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

ZebrafishStrengths

• 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

Routes of Administration of Chemical Exposure

• Aqueous • Dietary exposures• Direct injection techniques

-intraperitoneal-intramuscular-dorsal sinus

Measurement Endpoints

• 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

MEASUREMENT OF BIOCHEMICAL ENDPOINTS

• Sex Steroids in Tissues Estrogens/Androgens/Progestins– Radioimmunoassay (RIA)– Enzyme-linked Immunosorbent Assay

(ELISA) – Liquid/Gas Chromatography with Mass

Selective Detection (LC/GC-MS)

Measurement of Vitellogenin• 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)

CANDIDATE PROTOCOLS

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)

21-Day Fish Reproductive Assay (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

14-Day Fish Reproductive Assay(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

14-Day Fish Non-Reproductive Assay (See Hand Out)

• 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)

Significant Data Gaps

• 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.

IMPLEMENTATION CONSIDERATIONS

• 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

Questions

• 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?