Proceedings for TestSmart -- Endocrine Disruptors

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

Chair

R. Thomas Zoeller
University of Massachusetts

Panel Members

Kevin Crofton
United States Environmental Protection Agency

Jack Leonard
University of Massachusetts Medical School

Theo Visser
Erasmus University

The goal of this session was to discuss potential in vitro assays that can be used to screen and detect chemicals that disrupt the thyroid hormone system. Specifically, determine which endpoints would be amenable for developing in vitro assays for this purpose, and to highlight all those that may be adaptable to high throughput screening.

Background

The Thyroid Hormone System. It is important to recognize the importance of detecting environmental contaminants that may interfere with the ability of thyroid hormone to function, especially in development. Thyroid hormone is a small molecule produced by the thyroid glands, which are located at the base of the neck on each side of the trachea (windpipe). Although there are several forms of thyroid hormone that circulate in the blood, thyroxine (tetraiodothyronine, T4) is the most abundant. Despite this, T4 is not believed to play a direct physiological role in regulating the activity of the thyroid hormone receptors. Instead, it is deiodinated to triiodothyronine (T3), which then acts on the receptor. The activity of the thyroid gland is regulated by a protein hormone from the pituitary gland, thyrotropin (TSH). The amount of stimulation that TSH provides to the thyroid gland is limited by thyroid hormone in a negative feedback system. Therefore, when T4/T3 levels rise in the blood, TSH will be decreased. Likewise, when T4/T3 levels decline in the blood, TSH will be increased.

What does thyroid hormone do? The role of thyroid hormone in maintaining health is often illustrated by describing the effects of too much or too little thyroid hormone. Too much thyroid hormone (hyperthyroidism) can cause adults to loose weight, become irritable, have trouble sleeping, are fatigued, have increased sweating, and can have a condition called exophalmos (protruding eyes). Interestingly, not everyone with an overactive thyroid gland exhibits all of these symptoms. Therefore, the critical piece of information to make the diagnosis of an overactive thyroid gland is to obtain measures of circulating levels of thyroid hormones. Too little thyroid hormone produces effects that are not completely the opposite of too much thyroid hormone. For example, people with an underactive thyroid gland can experience a weight gain, may be intolerant to cold, may be sleepy and lethargic, slowed heart rate, and can have constipation. Also, as in the case of an overactive thyroid gland, not everyone with an underactive thyroid gland exhibits all of these symptoms.

It is believed that the consequences of too much or too little thyroid hormone in adults is reversible, although some new information suggests that long-term follow up studies on people who have been treated for an overactive thyroid hormone may have increased risk of cardiovascular disease. In contrast, thyroid hormone plays an essential role in brain development that is not reversible if it is disrupted. For example, about 1 in 3,500 children have too little thyroid hormone at birth, largely because their thyroid gland did not form properly during development. Fifty years ago, these children were hard to identify at birth based on the clinical signs. In fact, only 10% of these infants were diagnosed within the first month, 35% within 3 months, 70% within the first year, and 100% only after they were 3 years old. The intellectual deficits as a result of this delayed diagnosis and treatment were profound. One sudy found that the average intelligence quotient (IQ) of all of these children infants was only 76. Studies now show that the long term consequences of congenital hypothyroidism are subtle if the diagnosis is made early and treatment initiated within 14 days of birth, which can be accomplished only by mandatory screening for thyroid function at birth. This medical profile has become the principal example illustrating the importance of thyroid hormone for normal brain development.

Environmental contaminants can interfere with the thyroid system. There are a large number of classes of chemicals found in the environment that can influence the biosynthesis, transport, or potentially the action of thyroid hormone. This is important because if these compounds are in high enough concentrations in the serum of a developing fetus or infant, the consequences on brain development may be measurable and permenant. In recognition of this important hormone system, the US EPA has developed potential screens and tests to identify chemicals that may interfere with this system.

Reduction in the number of animals used in these screens and tests. It is important to recognize that the proposed screens and tests for thyroid disruptors rely solely on xenobiotic effects on circulating levels of thyroid hormones, and on the anatomy of the thyroid gland itself. This latter point is important because there is some indication that overstimulation of the thyroid gland by TSH can increase the likelihood that the thyroid gland will develop cancer -- although this does not appear to be a simple concept for humans. Because these measures are included as "add-ons" in other in vivo tests (see the EDSTAC final report), one could argue that there are presently no animals being devoted exclusively to identifying thyroid disruptors. Therefore, the recommendations, described below, for new tests that could identify thyroid disruptors are difficullt to place within the EDSTAC recommendations because they would be in addition to the present proposal.

Recommendations

Proteomics. Because the structure of most of the thyroid hormone-binding proteins is known, it is possible to produce a "chip" with a variety of these proteins including the thyroid hormone receptors (TRs), serum binding proteins including transthyretin (TTR) and thyroid binding globulin (TBG), as well as metabolic enzymes such as glucuronidases, and the deiodinases. These "chips" could then be used to screen for an interaction between a xenobiotic compound and these thyroid hormone binding proteins. Obviously, this is not a technology that is functional at the moment, but has merit to consider.
Thyroperoxidase inhibition. It is well known that a large number of chemicals can inhibit this enzyme, which is important in the biosynthesis of thyroid hormone. In addition, in vitro techniques for testing TPO activity are well characterized. Therefore, it is possible that TPO activity could be employed as an in vitro measure of thyroid disrupting activity at this step.
Thyroid Activity. A functional and quantitative measure of thyroid activity could be an important supplement to screens and tests presently used to evaluate chemicals for possible effects on the thyroid system. Presently, thyroid histopathology is performed at great expense. In addition, this requires that the thyroid gland is removed (though this is performed in animals used in other in vivo assays such as the uterotrophic assay or the Hershberger assay). A weakness in thyroid histopathology is that the gland is not activated uniformly; that is, some thyroid lobules are more active than others, and a single section through the gland may not be representative of the entire gland. Therefore, it was suggested that iodide uptake tests could be performed. This test would involve the introduction of a labeled iodide (e.g., 123I) which could then be counted through the skin. Thus, this test would be equivalent to the test routinely performed in humans, and would not be invasive. Moreover, the test would be quantitative and would more accurately evaluate thyroid activity.