In recent years, lower-chlorinated, airborne congeners of polychlorinated biphenyls (PCBs) have evolved as an emerging class of potentially hazardous environmental contaminants. Previous work has demonstrated that sulfation is a major metabolic pathway for these PCBs in vitro and in vivo; however, their metabolic fate and toxicities have not been explored. Hypothyroxinemia is among the most prevalent adverse health effects associated with PCB exposure in human populations and is an assumed cause of a variety of neurodevelopmental effects observed in infants following prenatal PCB exposure. The displacement of L-thyroxine (T4) from binding sites on transthyretin (TTR), a major T4 transport protein and trans-placental carrier of thyroid hormones, is thought to be a significant contributing factor in PCB-induced hypothyroxinemia. Structural similarities between sulfated metabolites of PCBs and T4 led to the central hypothesis that PCB sulfates are bioactive metabolites that exhibit high affinity binding to T4 binding sites on human TTR. An examination of the ability of six lower-chlorinated PCB sulfates to bind to human TTR in vitro, as well as subsequent computational modeling, revealed that these compounds interact with the high-affinity binding site in a non-covalent manner and with affinities comparable to T4. Corroborating evidence for the binding of PCB sulfates stems from their ability to inhibit the formation of TTR amyloid fibrils through stabilization of the protein's native conformation. Fibrillar TTR aggregates are the cause of amyloidoses like senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. All PCB sulfates examined were effective inhibitors of TTR fibrillogenesis with equal or higher efficiencies than some of the best previously described inhibitors. In vivo exposure of male Sprague-Dawley rats to a model PCB sulfate, 4-PCB 11 sulfate, resulted in rapid and widespread distribution of the metabolite to various organs, including the brain. Consequently, there is a strong indication for a potential role of PCB sulfates in thyroid disruption and inter-tissue transport of PCBs, and the binding of PCB sulfates to TTR may also provide structural information for improved design of anti-amyloid therapeutics. To date there are no analytical procedures for the quantification of PCB sulfates available, and exposure levels in human populations remain unknown. This study provides, for the first time, evidence that PCB sulfates, if present in human serum samples, are not extracted by current standard protocols for the analysis of PCBs and their metabolites. Consequently, PCB sulfates may have been overlooked in the past decades resulting in potential underestimation of total PCB exposure levels in exposed populations. Based on this finding, an efficient approach for the quantitative extraction of PCB sulfates from a variety of biological samples was developed. This procedure, coupled with quantitative mass spectrometry, has been validated for the future screening of human serum samples, and it was successfully applied to determine the tissue distribution and elimination profile of 4-PCB 11 sulfate in male Sprague-Dawley rats.

Thyroid disruptions by xenobiotics have been associated with a broad spectrum of severe adverse human health effects, such as impaired brain development and metabolic syndrome. Ingestion of indoor dust and contact with industrial chemicals are two significant human exposure routes of thyroid hormone disrupting chemicals (THDCs), raising serious concerns for human health. However, it is a laborious and costly process to identify THDCs using conventional experimental methods, due to the number of chemicals in commerce and the varieties of potential disruption mechanisms. In this thesis, we are aimed at in silico identification of novel THDCs targeting transthyretin (TTR) and thyroid hormone receptor (THR) among dust contaminants and commonly used industrial chemicals. In vitro assays were used to validate the in silico prediction results. Co-crystallization and molecular dynamics (MD) simulations were applied to reveal binding modes of THDCs at the studied biological targets and to explain their intermolecular recognition. The main findings presented in this thesis are: 1. Over 144 environmental pollutants have been confirmed as TTR-binders in vitro and these cover a wide range of environmental pollutants and show distinct chemical profiles including a large group of halogenated aromatic compounds and a second group of per- and polyfluoroalkyl substances. (Paper I) 2. In total 485 organic contaminants have been reported to be detected in household dust. The developed QSAR classification model predicted 7.6% of these dust contaminants and 53.1% of their metabolites as potential TTR-binders, which emphasizes the importance of metabolic bioactivation. After in vitro validation, four novel TTR binders with IC50 ≤ 10 µM were identified, i.e. perfluoroheptanesulfonic acid, 2,4,2',4'-tetrahydroxybenzophenone (BP2), 2,4,5-trichlorophenoxyacetic acid, and 3,5,6-trichloro-2-pyridinol. (Paper II) 3. The development of a robust structure-based virtual screening (VS) protocol resulted in the prediction of 31 dust contaminants as potential binders to THRβ1 including musk compounds, PFASs, and bisphenol A derivatives. The in vitro experiments confirmed four compounds as weak binders to THRβ1, i.e. 2,4,5-trichlorophenoxyacetic acid, bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether, 2,4,2',4'-tetrahydroxybenzophenone, and 2,4-dichlorophenoxyacetic acid. (Paper III) 4. We revealed the binding conformations of perfluorooctanesulfonic acid, perfluorooctanoic acid, and BP2 in the thyroxine binding sites (TBSs) of TTR by co-crystallizing TTR with the three compounds. A VS protocol was developed based on the TTR complex structures that predicted 192 industrial chemicals as potential binders to TTR. Seven novel TTR binders were confirmed by in vitro experiments including clonixin, 2,6-dinitro-p-cresol (DNPC), triclopyr, fluroxypyr, bisphenol S, picloram, and mesotrione. We further co-crystallized TTR with PBS, clonixin, DNPC, and triclopyr, and their complex structures showed that the compounds bind in the TBSs as proposed by the VS protocol. In summary, 13 indoor dust contaminants and industrial chemicals were identified as THDCs using a combination of in silico and in vitro approaches. To the best of our knowledge, none of these compounds has previously been reported to bind to TTR or THR. The identifications of these THDCs improve our understanding on the structure-activity relationships of THDCs. The crystal structures of TTR-THDC complexes and the information on THDC-Target intermolecular interactions provide a better understanding on the mechanism-of-actions behind thyroid disruption. The dataset compiled and in silico methods developed serve as a basis for identification of more diverse THDCs in the future and a tool for guiding de novo design of safer replacements.

Perchlorate ion interferes with thyroid function by competitively inhibiting the sodium-iodide symporter, thus blocking iodide uptake into the thyroid gland. In this study, the effect of perchlorate exposure on thyroid function and thyroid-responsive gene expression were examined in (1) embryos from eggs laid by perchlorate-treated Japanese quail hens and (2) perchlorate-treated young Japanese quail. I hypothesized that perchlorate exposure would decrease thyroid function and that the consequent hypothyroidism would alter the expression of thyroid sensitive genes. Laying Japanese quail hens were treated with 2000 mg/l and 4000 mg/l ammonium perchlorate in drinking water. Eggs from these hens were incubated. Embryos, exposed to perchlorate in the egg, were sacrificed at day 14 of the 16.5 day incubation period. Japanese quail chicks, 4-5 days old, were treated with 2000 mg/l ammonium perchlorate in drinking water for 2 and 7.5 weeks. Thyroid status was evaluated by measuring plasma thyroid hormone concentrations, thyroid gland weight and thyroidal thyroid hormone storage. Expression of thyroid-responsive genes was evaluated by measuring the mRNA levels of Type 2 deiodinase (D2) in the brain and liver, RC3/neurogranin mRNA level in the brain and Spot 14 mRNA level in the liver. Maternal perchlorate exposure led to embryonic hypothyroidism, demonstrated by thyroid hypertrophy and very low embryonic thyroidal TH storage. Embryonic hypothyroidism decreased body growth and increased D2 mRNA level in the liver (a presumed compensatory response to hypothyroidism) but did not affect the mRNA levels of D2 and RC3 in the brain. Spot 14 mRNA was not detected in embryonic liver. In the second part of the study, quail chicks showed early signs of hypothyroidism after two weeks of 2000 mg/l ammonium perchlorate exposure; plasma concentration and thyroid gland stores of both T4 and T3 were significantly decreased. After 7.5 weeks of perchlorate exposure, all thyroid variables measured indicated that the chicks had become overtly hypothyroid. D2 mRNA level was increased, a compensatory response to hypothyroidism, and spot 14 mRNA level was decreased, a substrate-driven response in the liver of quail chicks after two weeks of perchlorate exposure. However, no difference was observed in the mRNA levels of D2 and spot 14 in the liver after 7.5 weeks of perchlorate exposure, suggesting there was some adaptation to the hypothyroid condition. The mRNA level of D2 and RC3 in the brain was not affected by perchlorate-induced hypothyroidism in quail chicks after either 2 or 7.5 weeks of perchlorate exposure. As in the embryos, this suggests the brain of chicks was "protected" from the hypothyroid body conditions. / Ph. D.