Grantee Research Project Results
2006 Progress Report: Low-Dose Effects of Thyroid Toxicants on Neurodevelopment
EPA Grant Number: R832137Title: Low-Dose Effects of Thyroid Toxicants on Neurodevelopment
Investigators: Zoeller, R. Thomas
Institution: University of Massachusetts - Amherst
EPA Project Officer: Hahn, Intaek
Project Period: December 1, 2004 through November 30, 2008
Project Period Covered by this Report: December 1, 2005 through November 30,2006
Project Amount: $738,971
RFA: Development and Characterization of Biological Systems for Studying Low Dose Effects of Endocrine Disrupting Chemicals (2004) RFA Text | Recipients Lists
Research Category: Environmental Justice , Endocrine Disruptors , Safer Chemicals
Objective:
The overall goal of this project is to test the hypothesis that thyroid hormone (TH) produces non-linear dose-dependent effects on endpoints within the developing brain, heart and liver, but that some endpoints are more sensitive than others to thyroid hormone. In addition, we propose that thyroid toxicants disrupting the HPT axis by different mechanisms will produce different dose-response curves on these endpoints. And finally, a principle mechanism shaping the dose-response curve to thyroid hormone – or by extension, thyroid disrupters – are changes in tissue metabolism of thyroid hormone in response to perturbations in the HPT axis.
Approach:
To accomplish these goals we will: 1) characterize the dose-response of a variety of developmental endpoints in rats to thyroid hormone levels ranging in degrees of insufficiency; 2) test whether known thyroid toxicants – perchlorate, propylthiouracil, or polybrominated diphenyl ethers – produce dose-dependent effects on these endpoints that can be predicted by their effects on serum thyroid hormones; and 3) investigate mechanisms accounting for the shape of the dose-responses across these endpoints. We have chosen developmental endpoints that will provide a comprehensive picture of thyroid hormone action during development, including endpoints of gene expression and developmental anatomy. To obtain a comprehensive picture of thyroid "economy", we will measure serum total and "free" T4 and T3, TSH, the binding proteins transthyretin (TTR) and thyroxine binding globulin (TBG, which is expressed in rats especially during development). We will develop new radioimmunoassays for measurement of these two binding proteins, as well as for serum thyroglobulin, which is a measure of thyroid function. Four potential mechanisms accounting for non-linear dose-responses include differential effects on hormonal profiles (T4/T3), relationship to serum thyroid hormone binding proteins (TTR/TBG), deiodinase activity in tissues, and thyroid hormone receptors.
Progress Summary:
We have made significant progress toward the stated goals, especially the formation of a cooperative agreement with the EPA and with investigators at the University of Georgia and now at the University of Illinois.
Perchlorate
All animal procedures were performed following the NIH guidelines for the Care and Use of Laboratory Animals and were approved by the University of Massachusetts-Amherst Institutional Animal Care and Use Committee. Timed pregnant Sprague Dawley rats arrived at our facility on gestational day 2 (G2) in 2 cohorts. All animals were housed individually in plastic cages with food and water provided continuously and maintained on a 12:12hr light schedule (0600-1800hr lights on). Animals were randomly assigned to one of six treatment groups in which they were exposed to potassium perchlorate dissolved in drinking water at concentrations of 0, 10ppb (μg/L), 100ppb, 250ppb, 500ppb, and 5,000ppb. Treatment was initiated on G6 and continued through postnatal day 21 (P21). On P5, litters were culled to 5 males and 5 females where possible. One male and one female pup were sacrificed from each litter on P5, P15, P21, and P90. One extra male and one extra female was taken on P21 for tissue measurements. Perchlorate levels in the water were measured by Dr. Ben Blount (CDC). The actual concentration of the water was very close to the target concentrations. Neither maternal nor body weight was not affected by treatment.
Interestingly, serum T4 was not affected by treatment in the pups at any time during development. We have focused on P21 because the pups are large enough to provide enough serum for measurements of hormones, TBG and perchlorate. Despite the lack of effect perchlorate on serum T4, serum TBG levels were significantly increased in perchlorate-treated animals, with the exception of the highest dose group. Total thyroidal iodide (measured by Braverman in Boston) exhibited a complex relationship to perchlorate exposure in both the dams and the P21 pups. In both cases, total thyroidal iodide declined precipitously at low doses of perchlorate, but then recovered at higher doses of perchlorate. These data may well reflect the increase in thyroid sensitivity to TSH in low iodine settings. Serum perchlorate levels were nearly two-fold greater in P21 pups than in the dams. This likely reflects the ability of the lactating mammary gland to concentrate perchlorate. We are currently working on brain tissues to complete this part of the study.
Propylthiouracil
This experiment was performed in the laboratory of Dr. Mary Gilbert at the U.S. EPA in North Carolina. In general, PTU was administered in the drinking water from G6 to P21. Concentrations of PTU included 0 ppm, 1 ppm, 2 ppm, and 3 ppm. Body weight was not affected, and serum T4 (in P15 pups) was diminished in a stepwise fashion.
TRH mRNA in the PVN was increased by 2 and 3 ppm PTU. Regression of TRH mRNA data against log serum T4 revealed a significant (r2=0.3) but weak correlation. I predict that this correlation would be improved if TRH mRNA were measured in PVN punches (Palkovits’ punch technique) using real-time PCR. RC3 mRNA was significantly reduced in CA1 and CA3 of Ammon’s horn in the animals exposed to 2 and 3 ppm PTU. These data are unique in that other studies (ours included) do not find that RC3 mRNA is affected by thyroid status in this brain region. However, all previous studies have been performed with MMI, not with PTU. Therefore, there may be an additional effect of PTU that is separate from causing a reduction in serum T4 (perhaps inhibition of D1). RC3 mRNA was significantly reduced in the dentate of animals exposed on 2 and 3 ppm PTU. The degree to which RC3 mRNA is reduced was considerably greater than in CA subfields (nearly 20% in dentate). RC3 mRNA was also reduced in the retrosplenial cortex in animals exposed on 2 and 3 ppm PTU. Regression of RC3 mRNA in Dentate with serum T4 (either using historical control data or not) reveals a highly significant relationship between RC3 expression in the dentate gyrus and serum total T4. These data are improved when adding historical control data, indicating that this relationship will hold across studies. MCT8 is a T3-specific transporter in the brain, pituitary and heart. We incorporated this measure into our studies since the time of the grant because it has become clear that MCT8 may represent a key mechanism by which different parts of the brain may adapt to low TH (i.e., the shape of the dose-response curve for a thyroid toxicant is explained in part by MCT8). MCT8 expression was weakly affected by PTU at the 3 ppm dose. MCT8 expression is not affected by PTU in the dentate gyrus. There is also a significant correlation between MCT8 expression and log T4. MCT8 expression in hippocampus is significantly correlated with serum T4 in a region-specific manner. Specifically, MCT8 expression in Ammon’s horn (especially the CA3 subfield) is correlated with serum T4, and this relationship is improved with the addition of historical control data. It is important to recognize that RC3 expression is affected by PTU in the Dentate, not in Ammon’s horn. Thus, the response of MCT8 to PTU in Ammon’ horn may in part be responsible for the site-specific effect of PTU on RC3 expression in these brain regions. Type 2 deiodinase (D2) expression in the “Hippocampal field”. D2 is expressed in type-2 astrocytes. As a result, the pattern of expression of this gene in the hippocampus is quite different from the pattern of expression of RC3 or MCT8, both of which are neuron-specific in their expression. Thus, we refer to this as the hippocampal field. PTU increased D2 expression, but this response is small. There is a significant correlation between D2 expression and serum T4. It is important to recognize that developmental hypothyroidism produces an increase in type-2 astrocytes; therefore, it is unclear whether the PTU-induced increase in D2 mRNA is related only to the increase in cells that express it, or if it also represents an increase in cellular levels of D2 mRNA. D2 mRNA levels in the glial layer of the Dentate reveals a more robust response to PTU and a higher degree of correlation between D2 and serum T4. D2 expression in the retrosplenial granular cortex is also significantly increased by 2ppm and 3ppm of PTU and exhibit a still higher degree of correlation with serum T4.
Final work. We are currently evaluating TH-regulated endpoints in the liver and heart of these animals, as well as the cerebellum. Moreover, we are measuring TBG levels in serum.
Polybrominated Biphenyl Ethers
For this experiment, 36 timed-pregnant animals were purchased from Zivic-Miller in two cohorts, arriving in our facility on gestational day 2 (G2). The animals were provided an untreated wafer daily from G2 to G5. On G6, the animals were provided with a wafer that had been treated with diluent (toluene), or with DE-71 calibrated to deliver 0.1, 1.0, 10, or 30 mg/kg. Animals were weighed daily and the dose adjusted. The wafers were treated in the morning and allowed to dry under a fume hood throughout the day so that residues of diluent evaporated. Pups were sacrificed on postnatal day (P) 5, 15, 21, and 90. The following data have been collected.
Maternal body weight and body weight gain were not different among the treatment groups. Litter size or sex ratio also was not different among the treatment groups (data not shown). PBDE (1 mg/kg-day) was associated with a dose-dependent decrease in serum total T4 in P21 pups. PBDE exposure (10 and 30 mg/kg-day) was also associated with a dose-dependent decrease in serum free T4. Pituitary TSH beta mRNA was elevated also in response to 10 and 30 mg/kg-day PBDE. Interestingly, liver T4 levels were not affected by treatment whereas heart T4 levels were decreased by 10 and 30 mg/kg-day PBDE exposure. We are currently evaluating tissue levels of T3, deiodinase expression, and the expression of TH-responsive genes in these tissues.
Final goals
The data we have collected for PTU (correlations between serum total T4 and various measures in the brain) will be performed for all serum measures and all tissue endpoints. This will allow us to fully characterize which serum measurement is most uniformly linked (across toxicants) to predictions of effects on various tissues (brain, pituitary, liver, heart). In addition, our measurements of TBG and of other potential adaptive responses will help us explain differences in these relationships across toxicants.
Expected Results:
The proposed studies will identify critical temporal windows of sensitivity to these effects, which are likely to be endpoint specific, gender differences, and long-term consequences of thyroid toxicants on brain development. These studies will aid in risk assessment by providing appropriate screens and tests for identifying thyroid toxicants. The detailed dose-response curves generated from these studies will form the basis for evidence-based design of such screens and tests.
Future Activities:
All animal manipulations are now performed and we are systematically evaluating the endpoints described in the original proposal. We expect to begin publishing elements of this work in the summer of ’07 and to continue throughout the next academic year as the collaborations become completed.
Journal Articles:
No journal articles submitted with this report: View all 36 publications for this projectSupplemental Keywords:
T4, T3, TSH, TTR, thyroxine binding globulin, TBG,, RFA, Health, Scientific Discipline, POLLUTANTS/TOXICS, Health Risk Assessment, Chemicals, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Biochemistry, Biology, Endocrine Disruptors - Human Health, neurotoxic, bioindicator, EDCs, thyroid toxicants, exposure studies, endocrine disrupting chemicals, sexual development, endocrine disrupting chemcials, human growth and development, toxicity, invertebrates, estrogen receptors, hormone production, ecological risk assessment modelRelevant Websites:
http://www.bio.umass.edu/biology/zoeller/ Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.