The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Proceedings for TestSmart -- Endocrine Disruptors

General Discussion and Summary

Ellen K. Silbergeld
Johns Hopkins University

Pamela Lein
Johns Hopkins University

SUMMARY

Session I - Thoughtful Science

Approaches for reducing animal numbers:

  1. Repeat Tier I testing for equivocal results obtained in Tier I testing;
  2. sequence of in vivo testing should be driven by in vitro test results, e.g., if test chemical is positive in ER test, go directly to uterotropic tests, do not do Hershberger test;
  3. use all available existing data - may not need to do Tier I testing;
  4. increase the number of pups used per litter in Tier II testing which will more efficiently use animals and also increase the power and sensitivity of the tests;
  5. apply range finding - up and down approach;
  6. as increase knowledge of Tier I and Tier II relationship, may be able to circumvent Tier II testing, e.g., if know that T4 is altered, not necessary to test for this in Tier II testing;
  7. prioritization for screening based on QSAR, HTPS and human exposure data.

Other general points:

  1. Diet composition can impact findings, especially phytoestrogens in the diet;
  2. strain considerations
  3. report coefficient of variation
  4. time of dosing and route of exposure present a dilemma: pick most sensitive route or most relevant route? suggest most sensitive route for screening and most relevant route for testing
  5. steady state

Most pressing/potentially high impact research question: Re-evaluate litter as the unit in experimental design (focus on statistics).

Session II - Interspecies Extrapolation

Interspecies extrapolation is dependent on determining the degree of similarity in endocrine signaling mechanisms across species. Endocrine signaling includes physiological processes that regulate release, transport and metabolism of hormones, as well as binding of hormones to receptors, and signal transduction pathways activated downstream of receptor binding.

The more complex the endpoint of interest, the less conversed it tends to be across species. Thus, receptor binding is relatively conserved across species, however, metabolism and signal transduction pathways tend to be less well-conserved across species.

Other issues that need to be considered when extrapolating from wildlife to humans include susceptibility factors and exposure.

Most pressing/potentially high impact research question: How conserved are endocrine signaling pathways, particularly hormone metabolism, across species and across life stages.

Session III - In Vivo Imaging Techniques

A number of in vivo imaging modalities are well developed and could be potentially used to significantly reduce the numbers of animals required for EDSTP, or if used in conjunction with the existing Tier I/Tier II tests could yield significant mechanistic data. The biggest deterrent to incorporating these modalities into EDSTP is cost.

Most pressing/potentially high impact research question: What is the predictability of novel techniques and costs!!!

Session IV - In Vitro Assays for Disruption of the Thyroid Hormone System

The group reached consensus on the following:

  1. Tools are available to develop and validate an in vitro Tier I battery;
  2. with respect to in vivo assays, measuring levels of T4, TSH and thyroid histopathology are not sufficient;
  3. need to develop markers for effects of thyroid hormone disruption in the developing brain.

In vitro methods for detecting thyroid hormone disruption:

  1. inhibition of T4 binding to TTR, TBG, thyroid hormone receptors (this could potentially be performed as a HTPS using a chip)
  2. induction or inhibition of metabolic enzymes, e.g., glucorinidases or sulfotransferases, deiodinase, TPO

In vivo methods for detecting thyroid hormone disruption include:

  1. rate of iodine uptake (non-invasive)
  2. markers of effects on postnatal brain, especially the cerebellum, including apoptosis, PCNA, or laminin immunocytochemistry

Most pressing/potentially high impact research question: What is the relevance between presently used markers of thyroid function and brain development?

Session V: In Vitro Assays for Non-Receptor-Mediated Endpoints

The group reached consensus on the following:

  1. In vitro assays for non-receptor-mediated endpoints exist that assess endpoints of relevance to EDSTP and are fairly well-characterized
  2. It is unlikely that in vitro assays will replace in vivo assays, but in vitro assays can be a powerful tool for screening and prioritizing potential EDs, and for investigating mechanisms of action.

In vitro assays that are fairly well characterized and feasible for translation studies include:

  1. aromatase
  2. thyroid hormone biosynthesis (TPO)
  3. steroidogenesis (STAR)
  4. QSAR

Most pressing/potentially high impact research question: Identification of relevant in vitro mechanisms and feasibility of translation.

Session VI - Application of Genomics and Proteomics to Endocrine Disruptor Screening and Testing

The group agreed that there is potential for 'omics to be useful in EDSTP. However, this methodology is still very much in the Discovery phase and there are significant questions regarding technical procedure, timing of sample collection relative to exposure, and analysis and interpretation of the data.

Most pressing/potentially high impact research question: Testing effects of methods (sampling, storage, etc.) on array data.

GENERAL DISCUSSION

Endocrine Disruptor Screening and Testing: Forcing New Paradigms

  1. mechanism driven
  2. conservation of endpoints across species and across life stages
  3. low dose issues
  4. 3Rs as a goal

Are there other opportunities?

Emerging technologies and methodologies to be investigated for their application in EDSTP include:

  1. 'omics (genomics, proteomics, metabonomics) analysis of either tissue/cell extracts or tissue arrays
  2. detecting signals in the intact organism using in vivo imaging modalities
  3. cell systems (more apical then biochemical assays)

Traditionally, toxicology testing has proceeded in a linear fashion, starting with discovery then moving to translation. We recommend that instead discovery and translation occur as parallel processes. Examples of integrating discovery into ongoing translational studies include:

  1. applying 'omics to existing cell systems used for screening and testing Eds
  2. applying 'omics in T1/T2 (e.g., archive tissue samples from animals being used in T1/T2 tests for later analysis by genomics, proteomics or metabonomics)
  3. apply in vivo imaging modalities to animals being used in T2 testing
  4. mine information on potential EDs from human data being collected, e.g., epidemiological studies such as the Long-Range Children's studies and from clinical studies

Where Are We Now?

What is the current status of in vitro assays vis á vis Discovery and Translation?

  1. in Translation
    Receptor-mediated assays (ER, AR binding assays) - this endpoint is relatively conserved across species and across life stages; these assays are in validation
  2. transitioning from Discovery into Translation
    Non-receptor-mediated assays (aromatase, steroidogenesis) - some conservation across species and life stages; some assays are well-developed, ready for validation (aromatase); others are still in discovery stage but are promising (STAR)
  3. in Discovery
    Identification of relevant target cells in addition to reproductive tissues (e.g., neurons, bone cells, splenocytes) - these endpoints are much less conserved across species and across life stages.

Stage-specific Extrapolation and Validation

Figure 1

Example: QSARs

Figure 2

Opportunities in the Framework

Figure 3

Discovery → Translation

How do we facilitate 3Rs goals?