Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin.

Organochlorine compounds, particularly polychlorinated biphenyls (PCBs), alter serum thyroid hormone levels in humans. Hydroxylated organochlorines have relatively high affinities for the serum transport protein transthyretin, but the ability of these compounds to interact with the human thyroid receptor is unknown. Using a baculovirus expression system in insect cells (Sf9 cells), we produced recombinant human thyroid receptor ss (hTRss). In competitive binding experiments, the recombinant receptor had the expected relative affinity for thyroid hormones and their analogs. In competitive inhibition experiments with PCBs, hydroxylated PCBs (OH-PCBs), DDT and its metabolites, and several organochlorine herbicides, only the OH-PCBs competed for binding. The affinity of hTRss for OH-PCBs was 10,000-fold lower (Ki = 20-50 microM) than its affinity for thyroid hormone (3,3',5-triiodothyronine, T3; Ki = 10 nM). Because their relative affinity for the receptor was low, we tested the ability of OH-PCBs to interact with the serum transport proteins--transthyretin and thyroid-binding globulin (TBG). With the exception of one compound, the OH-PCBs had the same affinity (Ki = 10-80 nM) for transthyretin as thyroid hormone (thyroxine; T4). Only two of the OH-PCBs bound TBG (Ki = 3-7 microM), but with a 100-fold lower affinity than T4. Hydroxylated PCBs have relatively low affinities for the human thyroid receptor in vitro, but they have a thyroid hormonelike affinity for the serum transport protein transthyretin. Based on these results, OH-PCBs in vivo are more likely to compete for binding to serum transport proteins than for binding to the thyroid receptor. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6Figure 7

transthyretin. Environ Health Perspect 107:273-278 (1999). [ Online 9 March 1999] 'p://ehpnnl.niehs. nib.gov/docs/l999107p273-27acbeek/abstra html An important question in endocrine disruption is the mechanism by which a xenobiotic compound alters the action of endogenous hormones. One possible mechanism is direct interaction with the hormone receptor, either as an agonist or as an antagonist. In the case of thyroid hormone, a second important mechanism may be the ability of compounds to alter serum transport of thyroid hormones (TH). In nonmammalian vertebrates, the major transport protein is prealbumin (transthyretin); while some mammals, induding humans, have a second binding protein, thyroid-binding globulin (TBG) (1). Assessing the relative affinity of the thyroid receptor and the serum transport proteins for xenobiotics should help clarify one of the mechanisms by which xenobiotics alter thyroid homeostasis. Alterations in thyroid homeostasis by organochlorine compounds have been documented for many species, induding humans. In most cases, exposure to organochlorine compounds is correlated with decreased serum levels of thyroid hormone, particularly thyroxine (T4). Exposure to polychlorinated biphenyls (PCBs) has been correlated with decreased serum T4 concentrations in rats (2)(3)(4)(5)(6)(7)(8)(9)(10) and humans (6,11,12). Evidence from rat studies indicates that PCB-induced decreases in serum T4 are the result of increased metabolism by uridine diphosphate glucuronysyltransferase (UDPGT), a hepatic enzyme that glucuronidates T4 (5,(13)(14)(15). Another class of organochlorines, the choroacetanilides acetochlor and alachlor, elevates UDPGT activity and concomitandy decreases serum T4 levels in rats (16,1A. Acetochlor also alters thyroid hormone (3,3',5-triiodothyronine; T3) action in amphibians, accelerating T3-induced metamorphosis (18). DDT and its metabolites alter serum T4 levels in birds (19) and humans (20). DDT also alters thyroid metabolism in rats by increasing hepatic UDPGT activity (21).
Because of their physiological effects and their structural resemblance to thyroid hormones ( Fig. 1), several studies have investigated the ability of PCBs to bind to the serum transport proteins transthyretin (3,8,(22)(23)(24) and TBG (22) and to the rat thyroid receptor (23). Transthyretin (TTR) and TBG have similar affinities for the natural ligand, T4 (50-90 nM) (22), but have different affinities for PCBs. Hydroxylated PCBs are potent ligands for TTR, having affinities in the 1 nM range, 50-fold greater than that of T4 (8,22,24). Few hydroxylated PCBs bind TBG (22) and few unmetabolized PCBs have strong affinities for either TTR or TBG (8,22,23). Like the transport proteins, the rat thyroid receptor appears to have a higher affinity for hydroxylated versus parent PCBs (23).

Preparation of Protein
Extract 7-S-2;mi t E Protocols for preparing cell extracts were modified from '! ,~sToscano (25), Bres and Eales (26), and Sullivan et al. (27). ited polychlo-Transfected Sf9 cells were scraped from the flask and centrifuged at 5,000 rpm for 5 min. The supernatant was harvested and stored at 40C. The cell pellet was resuspended in 2.5 ml buffer A [10 mM Tris-HCI (pH 7.6), 10% glycerol, 3 mM MgCI2, 2 mM CaCI2, 5 mM dithiothreitol (DYl), 1 mM Pefabloc, 1 pg/ml aprotinin, and 20 pM leupeptin], incubated for 20 min on ice, and homogenized in a glass dounce. KCI was added tO a concentration of 0.4 M and the homogenate was incubated on ice for 30 min, with shearing through a pasteur pipet every 10 min. The homogenate was then centrifuged at 25,000 rpm for 15 min at 40 C. The supernatant (cell extract) was aliquotted and stored at -80°C until use.

Immunodetection ofTR Protein
Cell extracts were heated (95°C) in sodium dodecyl sulfate (SDS) loading buffer and electrophoresed through 10% SDS-PAGE. Gels were electroblotted onto polyvinylidene difluoride membranes (Sigma) at 25 V overnight. Membranes were rinsed in Trisbuffered saline (TBS; 10 mM Tris-HCl, pH 7.6, and 150 mM NaCl) and blocked in TBS supplemented with 3% bovine serum albumin, fraction V (Sigma). Membranes were incubated with a polyclonal antiserum to amino acids 62-82 of human TR31 To characterize the specificity of the recombinant receptor, the known TR agonists T3, T4, Triac, and Tetrac, and the inactive T3 metabolite, rT3, were tested in competitive inhibition experiments. We selected organochlorine compounds for testing based on their reported ability to alter in vivo responses or to bind serum transport proteins. PCB mixtures and some specific PCBs decrease serum T4 levels, but PCB 126 and PCB 77 markedly enhance spatial learning in rats exposed in utero, while only slightly altering serum T4 (2). Because the enhancement of learning is similar to that in hyperthyroid rat pups, Schantz et al. (2) proposed that PCBs 126 and 77 might be thyroid receptor agonists. We examined the interaction of these two compounds with the human thyroid receptor. Because several OH-PCBs bind TTR as effectively as T4 (22), we also tested the ability of OH-PCBs to bind the receptor. DDTs and chloroacetanilides alter serum T4 levels and catabolism, but their ability to bind TR is unknown.

Thyroid-binding Protein and Transthyretin Binding Assays
Conditions for these binding assays were modified from Lans et al. (22). Briefly, purified TBG or TTR was added to 200 pl assay buffer (TR assay buffer discussed previously) for a final concentration of 30 nM. We used 55 nM L-T4 containing 100,000 cpm [125I]L-T4 to estimate total binding. Nonsaturable binding was estimated by preincubating protein solutions with 100fold molar excess unlabeled L-T4 for 15 min. Varying concentrations of unlabeled competitors were also pre-incubated with the protein before the addition of [1251]L-T4. All other conditions were as described for TR saturation analysis, except the volumes of HAP slurry and of slurry buffer for resuspension were doubled to account for the twofold larger assay volume.

Immunodetection
Infected Sf9 cells expressed a protein of approximately 51 kDa that cross-reacted with a polyclonal antibody specific to hTRfI (Fig. 2). This protein is similar in size to the 52-55 kDa proteins recombinantly expressed in Escherichia coli (28,29 (Fig. 3). The binding affinity observed with recombinant hTR, expressed in Sf9 cells is similar to that reported for other recombinant thyroid receptors (28,29), but is lower than that reported for thyroid receptors extracted from tissues (approximately 0.1 nM) (30).

Discussion
Of the four groups of compounds examined, only the hydroxylated PCBs bound to human TRj 1 with affinities ranging from 30 affinities for TTR than for TR, making them competitors for the natural ligand L-T4. Half of the hydroxylated PCBs tested had higher affinities for TTR than did T4.
Previous work by McKinney et al. (23) indicated that two other coplanar PCBs, PCB 169 and PCB 80, bound to a rat nuclear extract with 100-fold lower affinities than L-T4, whereas PCB 54, an orthosubstituted congener, did not bind at all. Although the present study did not examine the binding affinity of PCBs 169 and 80, reported differences in affinity for coplanar PCBs may be due to several factors. First, the current study used a protein extract of insect cells producing recombinant human TRf1. Therefore, only a single TR isoform was available to interact with compounds in competitive binding experiments. In contrast, the rat liver nuclear extract probably contained not only rat TR,1, but also TRal and TRat2 (30). Second, because only a single TR isoform was present in recombinant cell extracts, only TR homodimers could form, while in rat liver nuclear extracts, retinoid X receptors (RXRs), the heterodimeric partners of TR, were probably also present (30,32). In vitro, TR-RXR heterodimers exhibit different affinities for ligands than do TR-TR homodimers, although both appear to form spontaneously in cells (30).
Third, species-specific differences in TR affinity for coplanar PCBs may exist.
DDTs and chloroacetanilide herbicides cause hypothyroidlike effects in animals, decreasing serum T4 (16,17,(19)(20)(21). None of these compounds bound to the thyroid receptor, indicating that they are unlikely to disrupt the thyroid axis via receptor interaction. DDOH bound to TTR and to TBG, but with such low affinity that concentrations in serum are unlikely to be high enough to compete for T4 binding. o,p'-DDD bound to TBG with a fairly low affinity (5 gM) and is unlikely to reach such high concentrations in serum because of environmental exposure. However, clinical treatment of adrenal carcinomas resulted in 100-600 pM doses of o,p'-DDD (20). One consequence of o,p'-DDD treatment was decreased serum T4, purportedly due to direct competition with o,p'-DDD for TBG binding (20). Our results support that hypothesis. Neither of the chloroacetanilides acetochlor nor alachlor bound TTR or TBG, but they enhance hepatic metabolism of T4 in rats (16,17). Alteration of metabolism is probably the major mechanism by which chloroacetanilides affect thyroid axis function.
Our results suggest that disruption of thyroid hormone transport is one of the Log-molar concentration mechanisms by which organochlorine compounds alter thyroid homeostasis. In particular, hydroxylated PCBs compete effectively for T4 binding to TTR, but few compounds compete for TBG binding, even at pM concentrations. TBG is found only in some mammals, including primates, ungulates (cattle, sheep, goats, pigs, water buffalo, and horses), and carnivores (dog), but not in rodents (rat) or lagomorphs (rabbit). Depending on species, TBG binds 60-90% of serum T4. Interestingly, TBG deficiency in humans does not interfere with euthyroid status, suggesting that TTR is also important for T4 transport in humans (1). TTR is a highly conserved TH binding protein in all vertebrate species (1), so disruption of thyroid hormone transport by hydroxylated PCBs could potentially occur in all vertebrates, not only in humans.