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

Abstract for TestSmart -- Endocrine Disruptors

Challenges of Confronting a Screening and Testing Program for Thyroid Disruptors

Thomas Zoeller
University of Massachusetts

Because thyroid hormone is essential for normal brain development, there is legitimate concern about the possibility that environmental chemicals interfere with thyroid hormone action. However, there are few well-defined end-points of thyroid disruption, especially in development that can be recruited for use by an Endocrine Disruptor Screening and Testing Program (EDSTP). The goal of this discussion is to describe the challenges inherent in any approach to identify potential thyroid disruptors in EDSTPs.

The Role of Thyroid Hormone in Brain Development - Humans. Thyroid hormone (TH) is essential for normal brain development. The most serious consequence of TH deficit during development is a condition known as cretinism. However, there are two very different forms of cretinism - neurological and myxedematous. It is postulated that the different forms represent the consequences of TH deficit at different times during development, and it is very likely that many mixed or intermediate forms exist. Although cretinism is the result of profound maternal and/or neonatal hypothyroidism, there are also consequences of milder forms of hypothyroidism. For example, congenital hypothyroidism can produce effects on full scale IQ and other neurological measures, which is why all states in the U.S. provide neonatal T4 screening. It is also clear that there are measurable neurological effects of TH deficit even when circulating TH levels are within the normal range. Recent studies clearly show that the offspring of women whose T4 levels were in the lowest 10th to 20th percentile of the normal range exhibited a variety of neurological deficits including lower full scale IQ and attention deficit.

The Role of Thyroid Hormone in Brain Development - Experimental Systems. Profound hypothyroidism in experimental models (usually rodents) is known to affect a number of neurodevelopmental processes including proliferation, migration, synaptogenesis, and apoptosis. Much of this work has been focused on the cerebellum. However, developmental processes occur at different times in different brain regions. For example, cortical neuroblasts undergo proliferation long before cerebellar granule cells. Studies are lacking that clearly delineate the effects of thyroid hormone on developmental processes occurring in all brain areas as well as to identify the "critical period" over which thyroid hormone may exert its effects. Moreover, dose-response curves have largely been ignored in the thyroid field, so there is little appreciation for the responsiveness of these measures to changes in circulating levels of thyroid hormone.

The Role of Thyroid Hormone in Development of Gut and Lung. There is far less known about the role of thyroid hormone in the development of various epithelia include that lining the lungs and the gut. However, it is clear that thyroid hormone plays important roles in these important events. Specific elements of gut and lung development may offer important quantitative end-points for EDC screening and testing, though this would require development.

Thyroid Hormone Action. Thyroid hormone receptors (TRs) are ligand-dependent transcription factors. Thus, these proteins regulate gene transcription in a manner controlled or modified by thyroid hormone. TRs can also form a partnership (heterodimer) with other proteins such as the retinoid X receptors, so it is generally held that thyroid hormone interacts with other factors to regulate gene expression. Studies on thyroid hormone-responsive genes in the developing brain have revealed several important features relevant to an EDSTP. For example, thyroid hormone-responsive genes are not affected by thyroid hormone in all brain areas even if the TR is present. Moreover, the direction of regulation (up-regulated or down-regulated) can be different in different brain areas or at different times during development. However, no work has clearly linked thyroid hormone-regulated gene expression to specific developmental events in thyroid hormone-responsive cells. Moreover, there has been no clear link established between thyroid hormone-regulated events and CNS function of the adult.

Specific Challenges for an EDSTP. The goal of an EDSTP is to identify potential endocrine disruptors. Screens and tests for endocrine disrupting chemicals (EDCs) have been designed based on studies that originally demonstrated "prototypical" chemicals (e.g., DES) to be EDCs. For example, the uterotrophic assay can detect estrogenic or antiestrogenic compounds with reasonable sensitivity. However, there are simply no validated, sensitive and specific bioassays for thyroid disruptors. There are very few, if any, environmental chemicals that have been shown to bind to the TR with an affinity that might be construed to be toxicologically important, and these kinds of studies are rarely performed. Therefore, the greatest source of information about thyroid disruptors is derived from animal studies in which circulating levels of hormones and thyroid histopathology has been measured. The reasoning behind these measurements is as follows. Thyroid hormone exerts a negative feedback effect on circulating levels of the pituitary glycoprotein, thyrotropin (TSH). Therefore, low circulating TH should produce elevated levels of TSH. Likewise elevated TH should produce low circulating TSH. Therefore, one indicator of a chemical that alters thyroid status would be altered TH and TSH concentrations. Because TSH stimulates thyroid function, it is possible for a chemical to produce a situation where thyroid hormone levels are normal and TSH is slightly elevated (i.e., "compensated"). This situation is confirmed by evaluating thyroid histopathology. For example, TSH levels could be elevated to a very small degree but could increase the size of thyroid follicles (hypertrophy) or could induce proliferation of thyroid cells (hyperplasia). Only thyroid hyperplasia is generally considered to be "adverse" because it is "pre-cancerous".

Therefore, any chemical that acts to change circulating levels of thyroid hormone, and there are many known, these blood measurements and histopathology are highly significant. However, among the measures described above, only TSH levels represent a direct action of thyroid hormone. Therefore, in the condition where TSH is only slightly elevated resulting in thyroid hypertrophy, it must be assumed that the pituitary is more sensitive to thyroid hormone deficit than any other tissue, including the developing brain. This assumption is not even true for humans in which has been shown that the offspring of women with thyroid hormone concentrations in the low-normal range during pregnancy have a variety of neurological deficits including lower overall IQ and attention deficit. Moreover, for chemicals that influence TH action without affect circulating levels of TH or TSH, these end-points are completely inadequate.

Because we do not fully understand the mechanism(s) by which TH regulates gene expression in the developing brain, it is difficult to envision in vitro screens for thyroid EDCs that protect against a high probability of a false-negative result. However, it is also unclear whether the thyroid hormone-responsive genes that have been identified in the fetal brain offer characteristics important to EDSTP end-points (e.g., amenable to dose-response experiments). We must learn more about the mechanisms of TH action on the developing brain and the consequences of those actions, to design EDSTPs that minimize false-negative results.