# Presentation: 174851

## 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**
**	****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

**Notes:**
These fish share several attributes that make them ideal test species for reproductive toxicity testing, including small size at maturity which reduces maintenance costs, and overall ease of culture.  An additional important attribute is that all three species are asynchronous spawners, meaning the ovaries contain oocytes at all stages of maturity, allowing spawning to occur repeatedly over an extended time period.  This reproductive attribute is important as it allows groups of fish to be tested for spawning success before and after chemical treatments during a relatively short time period.  It should be recognized, however, that the presence of gametes in all developmental stages may make identification of gonadal cellular targets more difficult.  

## Fathead Minnow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

## Fathead Minnow

- _Strengths_
- 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 medaka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

**Notes:**
.  Spawning activity normally occurs through 14 to 15 months of age

## 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.

## Zebrafish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

**Notes:**
In recent years, reliable methods for the development of transgenic zebrafish has become widely available (Moav et al., 1993).  The use of transgenic fish in aquatic toxicological research has been limited until recently, but offers great potential for the development of sensitive biomarkers and aid in the identification of mode of action.  One approach has been to transfect zebrafish with DNA motifs that once activated will also activate a reporter gene such as luciferase that can be readily detected

Among the motifs successfully introduced into the zebrafish include dioxin, heavy metal and estrogen responsive motifs (Carvan et al., 2000a; Legler et al., 2000).  This approach would be particularly valuable in the development of an androgen sensitive biomarker. 

## Zebrafish

- _Strengths_
- 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

**Notes:**
Water
	Besides being ecologically relevant, water is the most common laboratory route for exposing fish to EACs.  The delivery of a toxicant to water at different concentrations is well established. 

## 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)

**Notes:**
Currently, assays for individual steroids based on competitive binding with a specific antibody (e.g., RIA, ELISA) are most widely used. 

## 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.

**Notes:**
There were gaps of data found during the literature review, particularly a lack of information on the reproductive endocrinology of the test species.  This paucity of information is especially true for the medaka and zebrafish.  More specific data gaps include the following:

•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?