# Presentation: 003353

## Amphibian Metamorphosis Assay 

**Detailed Review Paper**
**Endocrine Disruptor Methods Validation Subcommittee**
**July 2002**
**Leslie Touart**

## Detailed Review Paper:AMPHIBIAN METAMORPHOSIS ASSAY FOR ENDOCRINE DISRUPTION

**WORK PERFORMED BY:**
**	****	****	****	****	****	****	**
**	**
**	****	**_**Fort Environmental **_**	****	**** ****	****       ****	**_**Laboratories, Inc.**_
**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 (December 2001)
**Encompassed searching of traditional literature databases, contacting specific experts in the field, and evaluating other presonal communication**
**Databases included, Medline/PubMed, biological Abstracts, Chemical Abstracts, and Toxline using key word and author search strategies**
**General search was performed initially using the key phrases amphibian metamorphosis and amphibian thyroid**
**Search was refined to Key Words - endocrine disruptor, thyroid impairment, TH analysis, cDNA techniques, and culture methods using Boolean operators “and” and “or” **
**Approximately 10000 records were refined down to 1000 papers that were reviewed**

## METHODS USED IN THIS ANALYSIS Cont. 

- Telephone/Email Consultations:
- Drs. Tyrone Hayes, James Burkhart, Robert Denver, and Robert Granger
- Yielded 9 new documents not originally included in initial literature search
- Each were included in various sections of the DRP

## METHODS USED IN THIS ANALYSIS Cont.

- Interviews With The Following Experts:
- 			Dr. Robert Denver
- 			Dr. Brent Palmer
- 			Dr. Joe Bidwell
- 			Dr. Jim Burkhart

## METHODS USED IN THIS ANALYSIS Cont.

- External/Internal Peer Review
- Dr. Joe Bidwell – Oklahoma State Univ.
- Dr. Brent Palmer – U of Kentucky
- Mr. Michael Blanton - Battelle
- EPA Technical Experts
- (principally Mr. Joe Tietge, MED)

## OVERVIEW AND SCIENTIFIC BASIS OF AMPHIBIAN METAMORPHOSOSIS ASSAY (ENDOCRINE CONTROL OF THYROID AXIS)

**Amphibian metamorphosis is well understood and generally involves morphological, biochemical, and molecular changes **
**These changes result in resorption, remodeling, and creation of new tissues**
**Thyroid axis control of metamorphosis in amphibians involves the CNS, hypothalamus, pituitary gland, thyroid gland, TH transport proteins, TRs, and transcriptional elements**
**Many aspects of the thyroid axis are conserved amongst chordates at both the morphological and molecular levels enhancing the use of an amphibian as a general vertebrate model for evaluating thyroid disruption     **
**The Amphibian Metamorphosis Assay will be useful in evaluating thyroid perturbation.**

## Overview of Metamorphic Periods in Anurans

- Premetamorphosis
- Characterized as phase of embryogenesis and early tadpole growth
- Thyroid gland is developing
- Prometamorphosis
- Larvae acquires TH synthesis capability and is characterized by low circulating TH levels which stimulate early metamorphic changes
- Metamorphic Climax
- Characterized by peak levels of endogenous TH and rapid and drastic morphological changes (e.g., tail resorption)

**Notes:**
Early metamorphic changes = hindlimb elongation and differentiation, etc

## Test Species

- Anurans
- Pipids
**South African clawed frog (*****Xenopus sp*****.)**
***X. laevis***
***X. tropicalis***
- Ranids
**Leopard frog (*****Rana pipiens*****)**
- Hyperoliids
*Hyperolius sp.*
- Urodeles

## Xenopus laevis

- Native to Africa, south of Sahara desert
- Extensively used in scientific research and can be obtained from US vendors
- Adult males are ca. 10 cm and females 15 cm in length (metamorphic tadpoles are 2-3 cm in length)
- Can be kept in breeding condition all year and are bred in the lab using hCG
- Females produce 1500 embryos per breeding
- May be re-bred every two months and are productive for 3 to 5 years
- Purely aquatic species

## Xenopus tropicalis

- Native to southern tip of Africa
- Similar to X. laevis with the following exceptions:
- Physically smaller
- Develop more rapidly (can be cultured at higher temperature)
- Diploid genome
- Somewhat greater embryo production
- Reproductively mature in 4-5 months (X. laevis requires 1-2 years)

## Rana pipiens

- Native to North America
- Breeding season ranges from March to April
- Aquatic and terrestrial life phase – more difficult to raise and breed in lab
- Sexual maturity in 1 to 2 years
- Produces Eggs per egg masses of 1500 to 4000 in the wild

## Hyperoliid sp.

- Native to Africa, known as the reed frog
- Undergoes ontogenic color change as the result of secondary sexual development
- No commercial source of species
- Limited culturing information available

## Urodeles

- Includes “non-frog” species, such as salamanders, newts, and axolotls
- Native to North America and other locations across the globe.
- Breeding occurs in the Early Spring months depending on temperature
- Produce 100-200 relatively large embryos per breeding
- Limited culturing information available

## Xenopus laevis

- _Strengths_
- Large database on all aspects of development, reproduction, metamorphosis, including information at molecular level
- Ease of culture
- Breeds in laboratory repeatedly with hormonal stimulus
- High productivity
- Mapped genome
- Many laboratories are familiar with culture and testing

- _Weaknesses_
- Relatively long life cycle
- Oligotetraploid genome

## Xenopus tropicalis (compared to X. laevis only)

- _Strengths_
- Relatively short life cycle
- Rapid development
- Diploid genome
- Good transgenic capacity
- Greater breeding output

- _Weaknesses_
- More challenging animal husbandry
- Less scientific information availablecs
- Disease susceptibility?
- Availability

## Rana pipiens

- _Strengths_
- Native species
- Reasonable database
- Relatively short metamorphic period for native species
- Terrestrial and aquatic life phases

- _Weaknesses_
- More difficult animal husbandry and breeding
- Limited testing experience

## Urodeles

- _Strengths_
- Represent different Order
- Native species
- Terrestrial and aquatic life phases

- _Weaknesses_
- More difficult animal husbandry and breeding
- Limited testing experience
- Limited database

## Hyperolius sp.

- _Strengths_
- External endpoints (morphological)
- Suitable animal husbandry
- Connection to sexual development

- _Weaknesses_
- Availablity
- Limited testing experience
- Limited database
- Does not directly measure thyroid dysfunction

## Routes of Administration of Chemical Exposure 

- Aqueous
- Dietary exposures
- Parenteral
- -intravenous
- -intramuscular

## Potential Exposure Periods

- Exposure from premetamorphosis through metamorphic climax
- 35+ days
- Exposure from prometamorphosis through metamorphic climax
- 28+ days
- Exposure during prometamorphosis
- ca. 14 days
- Exposure during metamorphic climax
- ca. 14+ days

## Exposure Period Considerations

- Premetamorphosis
- No thyroidal activity
- This phase only considers development of the thyroid gland
- Prometamorphosis
- Potentially the most sensitive stage as the organism is acquiring thyroid activity
- Hind limb development occurs
- Metamorphic climax
- Substantial morphological changes occur
- TH saturation
- May be difficult to distinguish effects due to TH saturation
- Reduced sensitivity anticipated

## Measurement Endpoints Considered

- Morphological and Histological
- Rate of Development (developmental delay)
- Must discriminate between thyroidal and non-thyroidal responses
- Hind Limb Development and Differentiation
- Tail Resorption
- Thyroid gland histopathology
- Hyper- and hypotrophy, and hyper- and hypoplasia
- Biochemical
- TH
- TH precursors

## Measurement Endpoints Considered (continued)

- Molecular
- cDNA Techniques
- Single Gene Expression
- RPA
- Q-PCR (RT-PCR)
- Multiple Gene Expression
- Differential Display
- Gene Arrays
- Transgenic Lines
- Organ and Cell Culture
- Receptor binding Assays

## MEASUREMENT OF BIOCHEMICAL ENDPOINTS

- TH and TH precursors
- Radioimmunoassay (RIA)
- Enzyme-linked Immunosorbent Assay (ELISA)
- Liquid/Gas Chromatography with Mass Selective Detection (LC/GC-MS)

## Relevant Gene Expression Techniques

- Single Gene Expression
- Q-PCR
- Genes Considered:  TRbeta, ST3, transthyretin
- Multiple Gene Expression
- Gene Arrays
- Gene Familes Considered:  many including those associated with synthesis, regulation, metabolism, and TH action

**Notes:**
Q-PCR = Real time PCR vs. reverse transcriptase (RT-PCR)
Note that Gene Arrays are not very quantitative but do qualitatively cover a wide MOA

## Anticipated Endpoint Sensitivity

- Molecular/Biochemical (most sensitive)
- Histological
- Morphological (least sensitive)

## Species Selection Criteria

- Species must be amenable to continuous culture in the lab
- Reproduction must be continuous throughout the year or be inducible through hormonal treatments
- Larvae must be able to be routinely reared to predetermined developmental stages
- Relatively fast rate of development
- The endpoints that will be used must be supported by a sufficient database clearly indicating their relevance

## Additional Beneficial Criteria

- Knowledge of genetic information
- Biochemical information known about the endocrine axis (HPT in this case)
- Metabolism information (TH homeostasis)

## Recommended Test Species

- The only species that meets the minimal criteria established is *X. laevis.*

## CANDIDATE PROTOCOLS

*Xenopus* 28-day full metamorphosis assay (proposed by OECD, 2001)
- Note – theoretically this assay should require 50+ days
- 2)  14-day metamorphic climax assay (as described in Fort et al., 2000)
- 3)  14-day prometamorphosis assay (as described by Tietge et al., personal communication)

## Metamorphosis in X. laevis

- (adapted from Shi, et.al., 1996)

## 14-Day Prometamorphosis Assay

- Chemical Exposure – 14 Days through end of premetamorphosis and onset of prometamorphosis
- NF Stage 51 to 54
- Exposure Measurement Endpoints
- Thyroid gland histopathology, hind limb emergence (rate and normalcy), TH and TH precursor levels, and differential gene expression via gene array from tissue punch samples (i.e., TR beta family)
- Interpretation of Results

## Significant Data Gaps

- Responses, at the organismal and suborganismal levels, to known thyroid agonists and antagonists
- Which endpoints provide most meaningful information regarding effects due to thyroid-based mechanisms
- The time course of responses
- Sensitivity of the measurement endpoints
- Relationship between quantitative changes in molecular activity and observed changes in thyroid homeostasis
- Dynamic range of thyroid axis homeostasis and its relationship to gross morphological, histological, molecular, and biochemical changes

## IMPLEMENTATION CONSIDERATIONS

- Animal Welfare Considerations
- Test Facility Capablities
- Prevalidation Studies (in general accordance with ICCVAM guidelines)
- Phase I – Final definition and development of the recommended endpoints
- Most work will be required with molecular techniques including construction of the gene arrays
- Phase II – Preliminary protocol development for exposure and measurement endpoints.

## IMPLEMENTATION CONSIDERATIONS (continued)

- Prevalidation Studies
- Phase II (cont.) – Evaluate a set of three known thyroid agonists and antagonists using preliminary protocols
- Phase III – Evaluate data, review and revise preliminary protocols as necessary
- Phase IV – Test and additional set of three test substances with anecdotal or unknown information regarding thyroid axis activity
- Phase V – Review data, revise protocol accordingly to generate Final Protocol for use in interlaboratory GLP validation studies

## Questions

- Does the EDMVS agree that the Prometamorphosis Assay with Xenopus is the appropriate amphibian assay to recommend for further implementation?

## Questions (continued)

- 2)	Does the EDMVS agree that pre-validation efforts should be phased as described in the DRP and targeted to address the following:
- establish organismal and sub-organismal responses to established thyroid agonists and antagonists,
- determine which endpoints are diagnostic of thyroid-based mechanisms,
- ascertain the general time course of responses,
- establish the sensitivity of the measurement endpoints, and
- establish the dynamic range of thyroid axis related responses?

## Questions (continued)

- 3)	Does the EDMVS agree with using thyroxine as a thyroid agonist and perchlorate, propylthiouracil, and amiodarone as thyroid antagonists for evaluating the performance of this assay?
- 4)	Does the EDMVS have suggestions to improve the DRP?

**Notes:**
Perhaps Methimazole should be considered = MOA is synthesis inhibition via TPO inhibition