Polychlorinated biphenyls (PCBs) exert thyroid hormone-like effects in the fetal rat brain but do not bind to thyroid hormone receptors.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants routinely found in human and animal tissues. Developmental exposure to PCBs is associated with neuropsychologic deficits, which may be related to effects on thyroid hormone (TH) signaling in the developing brain. However, PCBs may interfere with TH signaling solely by reducing circulating levels of TH, or they may exert direct effects on TH receptors (TRs). Therefore, we tested whether maternal exposure to a commercial PCB mixture, Aroclor 1254 (A1254), exerts effects in the fetal brain by one or both of these mechanisms. Dams were dosed daily with 0, 1, or 4 mg/kg A1254 from gestational day 6 (GD6) until they were sacrificed on GD16. A1254 significantly reduced circulating levels of tri-iodothyronine ([T.sub.3]) and thyroxine ([T.sub.4]) in pregnant rats but increased the expression of several TH-responsive genes in the fetal cortex, including neuroendocrine-specific protein A (NSP-A), RC3/neurogranin, and Oct-1. These findings are consistent with a direct action of PCBs on TRs. However, we did not identify parent PCB congeners or metabolites that bound to rat TRs isolated from hepatic nuclei. These findings indicate that PCBs can interfere with TH signaling in the fetal brain by direct actions on the fetus rather than by producing maternal hypothyroidism. Key words: brain development, endocrine disruption, NSP-A, NSP-C, Oct-1, PCBs, RC3/neurogranin, thyroid, thyroid hormone.

**********

Polychlorinated biphenyls (PCBs) are industrial chemicals consisting of paired phenyl rings with various degrees of chlorination (Chana et al. 2002). Although the production of PCBs was banned in the mid-1970s, these contaminants are routinely detected in the environment (Breivik et al. 2002). The chemical stability and lipophilicity of these compounds allow them to bioaccumulate through the food chain, and they are found in high concentrations in samples of human tissues (Fisher 1999). A number of epidemiologic studies have indicated that children developmentally exposed to PCBs suffer from neuropsychologic deficits such as a lower full-scale IQ, reduced visual recognition memory, attention deficits, and motor deficits (Ayotte et al. 2003; Huisman et al. 1995; Jackson et al. 1997; Korrick and Altshul 1998; Osius er al. 1999; Walkowiak et al. 2001). Both postnatal and prenatal exposure to PCBs contributes to these deficits, although some authors argue that prenatal exposure is more strongly associated with neurologic deficits, which indicates that fetal neurodevelopment is particularly vulnerable to PCB exposure (Jacobson and Jacobson 2002).

The specific neuropsychologic domains affected by developmental exposure to PCBs overlap with those affected by maternal thyroid hormone (TH) insufficiency, including lower IQ, visual memory deficits, and motor function and attention deficits (Haddow et al. 1999; Morreale de Escobar et al. 2000; Pop et al. 1999). Therefore, several investigators have speculated that PCBs may affect brain development by interfering with TH signaling (McKinney and Waller 1998; Porterfield 2000; Porterfield and Hendry 1998). This hypothesis is supported by the observation that the concentrations of PCBs, or of specific PCB congeners, in maternal and cord blood are associated with lower TH levels in both the mother and infant (Koopman-Esseboom et al. 1994; Schantz et al. 2003). Although several epidemiologic studies have failed to identify an association between TH and PCB body burden (Hagmar et al. 2001; Longnecker et al. 2000; Matsuura and Konishi 1990; Sala et al. 2001; Steuerwald et al. 2000), experimental studies consistently find that PCB exposure decreases circulating levels of thyroxine ([T.sub.4]) in rats (Bastomsky 1974; Bastomsky et al. 1976; Brouwer et al. 1998). Therefore, it is possible that PCB body burden is negatively associated with serum TH levels in humans but that the variability inherent in human populations makes this association difficult to reveal.

Despite the finding that PCBs uniformly reduce circulating levels of TH in experimental animals, PCBs do not exert effects in experimental animals that are fully consistent with experimentally produced hypothyroidism using goitrogens such as propylthiouracil (PTU). For example, developmental exposure to PCBs in experimental animals induces hearing loss (Crofton et al. 2000a, 2000b; Goldey et al. 1995a, 1995b), a reduction in choline acetyltransferase in the cerebral cortex (Juarez de Ku et al. 1994), and an increase in testicular growth, all consistent to some degree with effects produced by PTU. Moreover, [T.sub.4] replacement can at least partially ameliorate these effects (Goldey and Crofton 1998; Juarez de Ku et al. 1994), indicating that PCBs can influence brain development in part by causing a reduction in serum TH. However, developmental hypothyroidism induced by PTU exposure causes a significant increase in serum concentrations of thyroid-stimulating hormone (thyrotropin; TSH) (Connors and Hedge 1981), reduced body and brain weight as well as reduced brain size of rat pups (Schwartz 1983), and a delay in eye opening and tooth eruption (Varma et al. 1978). In contrast, PCB exposure at doses that lower serum TH does not always produce these effects (Goldey et al. 1995a; Hood and Klaassen 2000; Kolaja and Klaassen 1998; Zoeller et al. 2000). Therefore, there is a discrepancy between the ability of PCBs to reduce circulating levels of TH and their ability to produce symptoms of hypothyroidism.

Some authors have proposed that this discrepancy may be attributable to PCBs acting as imperfect agonists/antagonists on TH receptors (TRs) (McKinney and Waller 1994). TRs are members of the steroid/thyroid superfamily of ligand-dependent transcription factors (Mangelsdorf et al. 1995). They are encoded by two genes, designated [alpha] and [beta] c-erbA (encoding TR[alpha] and TR[beta]) (Sap et al. 1986; Weinberger et al. 1986). Both genes are alternatively spliced in a tissue-specific manner, producing a variety of functional TR isoforms (TR[beta]1, TR[beta]2, TR[beta]3, TR[alpha]1) (Flamant and Samarut 2003). TR[alpha]1 and TR[beta]1 are the predominant isoforms that are expressed throughout brain development (Bradley et al. 1989, 1992, 1994) and in other tissues such as liver, intestine, and heart (Brent 2000). However, only one report has addressed this proposal directly (Cheek et al. 1999), finding that two hydroxylated PCB congeners (4'-OH-PCB-14 and 4'-OH-PCB-106) exhibit a relatively low affinity for human TR[beta]1 ([K.sub.1], = 32 [micro]M).

Considering these findings, the present studies were initiated for two reasons. First, we tested the hypothesis that maternal PCB exposure could affect the fetal cerebral cortex by reducing the availability of TH to the fetus. We previously showed that low maternal TH, produced experimentally by goitrogen treatment, can alter gene expression in the fetal cortex before the onset of fetal thyroid function (Dowling et al. 2000, 2001; Dowling and Zoeller 2000). Therefore, if PCBs reduce circulating levels of maternal TH, then gene expression in the fetal cortex should respond in a manner consistent with hypothyroidism. Second, we tested the hypothesis that individual PCBs or their metabolites could bind to the TR. To test this, we used rat hepatic nuclei as a source of both TR[alpha]1 and TR[beta]1.

Materials and Methods

Chemicals. Aroclor 1254 (A1254; lot no. A8110048) and individual PCB congeners (PCBs 77, 105, 118, 126, 138, and 153) were purchased from AccuStandard, Inc. (New Haven, CT). Methylsulfonyl-PCBs (MeS[O.sub.2]-PCBs) were synthesized according to Haraguchi et al. (1987). The purity of these compounds was > 99% as determined by gas chromatography. The hydroxylated PCBs were synthesized using the Suzuki coupling of chlorobenzene boronic acids with bromochloro anisoles followed by demethylation with boron tribromide. The characterization and purity of these compounds have been described previously (Bauer et al. 1995; Lehmler and Robertson 2001).

Animals. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996) and were approved by the University of Massachusetts-Amherst Institutional Animal Care and Use Committee before initiating these studies. Timed-pregnant Sprague-Dawley rats (n = 18; Zivic-Miller, Inc., Pittsburgh, PA) arrived in our animal facility 2 days after insemination [gestational day (GD) 2]. The animals were individually housed in plastic cages with food and water provided continuously and were maintained on a 12:12-hr light cycle (0600 hr to 1800 hr). All dams were provided daily with a Keebler Golden Vanilla Wafer (The Kellog Company, Battlecreek, MI) dosed with A1254 on GD6 through GD16. The wafers were calibrated to provide doses of 0, 1, and 4 mg/kg (n = 6/group); details of this method have been described previously (Zoeller et al. 2000).

On GD16, dams were euthanized with carbon dioxide, and trunk blood was collected for measurement of serum total [T.sub.4] by radioimmunoassay (RIA). The pregnant uterine horns were removed and immediately placed on ice. Fetuses were dissected from the uterus, frozen on pulverized dry ice, and stored at -80[degrees]C until cryosectioned.

In situ hybridization. The NSP-A cDNA construct and NSP-C oligonucleotide probes have been described previously (Dowling et al. 2001), and the RC3/neurogranin construct (pPRC/CMV-RC3) was kindly provided by J. Bernal (Madrid, Spain; Iniguez et al. 1996). A fragment of rat Oct-1 transcript was cloned by standard polymerase chain reaction methods using primers designed to amplify a 1.2-kb region of the gene (246-1481, GenBank accession number U17013; National Center for Biotechnology Information, Bethesda, MD). The forward (5'-GCACCCAACCACCAACTTGC-3') and reverse (5'-GGTGC-CATCAGGCCTGGATT-3') primers were synthesized by Custom Primers (Invitrogen, Inc., Carlsbad, CA). The 1.2-kb fragment was then ligated into the pCRII TOPO vector using the Topo TA cloning kit according to the manufacturer's instructions (Invitrogen), and its authenticity was confirmed by sequence analysis.

Probes were generated in vitro by linearization with the restriction enzymes and transcription with the RNA polymerases specified for each construct (Table 1). Transcription reactions and in situ hybridization procedures have been described previously (Zoeller et al. 1997). After in situ hybridization, slides were arranged in X-ray cassettes and apposed to BioMax film (Eastman Kodak Co., Rochester, NY); the duration of exposure was dependent on the specific activity of the probe and the abundance of the target mRNA (2 weeks for the RC3/neurogranin cRNA probe, 1 week for the Oct-1 and NSP-A cRNA probes, and 2 days for the NSP-C oligonucleotide probe). To verify that the films were not overexposed, [sup.14]C-labeled standards (American Radiolabeled Chemicals Inc., St. Louis, MO) were simultaneously apposed to all films. The hybridization signal was analyzed as follows. First, a 5x magnified image was captured using a Scion AG-5 capture board interfaced with the public domain software NIH-Image 1.61/ppc (W. Rasband, National Institute of Mental Health, Bethesda, MD) run on a Macintosh G4 computer (Apple Computer Inc., Cupertino, CA). The optical system consisted of a Dage-MTI72 series video camera equipped with a Nikon macro lens mounted onto a bellows system over a light box. Measurements of relative mRNA levels were taken as the area of the signal over the cortex of GD16 fetal brains multiplied by the relative density of the film (Figure 1). The resulting values were averaged across eight sections for each fetus, with one fetus per litter and six litters per treatment group.

[FIGURE 1 OMITTED]

Radioimmunoassay. Total [T.sub.4] was measured in 5 [micro]L of rat serum using a barbital buffer system. Briefly, each assay tube contained 100 [micro]L barbital buffer (0.11 M barbital, pH 8.6; 0. 1% wt/vol 8-anilino-1-naphthalene-sulfonic acid ammonium salt; 15% bovine [gamma]-globulin Cohn fraction II; 0.1% gelatin), 100 [micro]L anti-[T.sub.4] (rabbit, diluted to provide a final concentration of 1:30,000; Sigma, St. Louis, MO), and 100 [micro]L [sup.125]I-labeled [T.sub.4] (Perkin-Elmer/NEN; Boston, MA). Standards were prepared from [T.sub.4] (Sigma) measured using a Cahn electrobalance; standards were run in triplicate, whereas samples were run in duplicate. Standards were calibrated to measure serum [T.sub.4] levels from 0.4 [micro]g/dL to 25.6 [micro]g/dL. Tubes were incubated at 37[degrees]C for 30 min and then chilled on ice for 30 min. Bound counts were precipitated by adding 300 [micro]L ice-cold polyethylene glycol 8000 (20% wt/wt; Sigma). Tubes were centrifuged at 1800 x g for 20 min at 4[degrees]C; the supernatant was then aspirated and the pellet counted in a gamma counter (Packard Cobra II; Global Medical Instrumentation, Inc., Albertville, MN). The assay was run at 40-50% binding; nonspecific binding was generally < 8%. The assay was validated for rat serum by demonstrating parallelism between the standard curve and a dilution series of rat serum. The two slopes did not vary significantly as evaluated by t-test for two slopes (data not shown). The variability within the assay was determined by running 10 replicates of three different standards that represent a low, medium, and high value on the standard curve. The coefficients of variance were 0.9% for 0 ng/mL; 4.7% for 3.2 [micro]g/dL, and 3.8% for 25.6 [micro]g/dL. All experimental samples were evaluated in a single assay.

Total triiodothyronine ([T.sub.3]) was measured according to the manufacturer's instructions using a [T.sub.3] RIA kit (ICN Diagnostics, Costa Mesa, CA). This assay was performed at 49% binding with detection limits of 50-800 ng/dL. All samples were evaluated in a single assay.

Isolation of hepatic nuclei. Adult male Sprague-Dawley rats were euthanized with carbon dioxide and perfused with ice-cold saline through the aorta until the liver was cleared of blood. Twenty grams of liver was then washed in 3 mM Mg[Cl.sub.2] and 0.14 M NaCl, minced, and homogenized in 3 mM Mg[Cl.sub.2], 1 mM dithiothreitol (DTT), and 0.32 M sucrose using a motor-driven Teflon mortar and glass pestle. The homogenate was centrifuged at 600 x g for 10 min, and the crude nuclear pellet was resuspended in 3 mM Mg[Cl.sub.2], 1 mM DTT, and 1.8 M sucrose. After centrifugation at 53,000 x g for 45 min, the nuclei were resuspended in binding buffer (3 mM Mg[Cl.sub.2]; 1 mM DTT; 20 mM Tris HCl, pH 7.6; 0.32 M sucrose; and 0.3% bovine serum albumin) and stored at -80[degrees]C.

TH binding assay. For saturation analysis, nuclear isolates frozen in binding buffer were thawed on ice, and triplicate aliquots (~0.1 g of the original liver) were incubated with increasing concentrations of [sup.125]I-[T.sub.3] [(1 x [10.sup.-10] to 8 x [10.sup.-9] M; 3,300 [micro]Ci/[micro]g; NEN, Boston, MA) for 30 min at 37[degrees]C. Nonspecific binding was determined at each concentration of [sup. 125]I-[T.sub.3] (and each competitor concentration) by performing the assay as described in a parallel set of tubes that included the addition of 10,000-fold excess cold [T.sub.3] (final concentration 1 x [10.sup-6] M). The reaction was terminated by placing samples on ice and by adding binding buffer/1% Triton X-100. Samples were centrifuged at 13,000 x g for 10 min, the supernatant was discarded, and the nuclear pellet was washed in 1 mL binding buffer. The bottom of the microfuge tube containing the pellet was cut off and placed in a 14 x 70 mm test tube, which was counted in a gamma counter (Packard Cobra II). For competitive binding assays, isolated nuclei were incubated with a final concentration of 1 x [10.sup.-10] M [sup.125]I-[T.sub.3] and increasing concentrations of competitors (TR agonists: [T.sub.3], [T.sub.4], Triac (triiodothyroacetic acid), and Tetrac (tetraiodothyroacetic acid, 1 x [10.sup.-12] M to 1 x [10.sup.-9] M; PCBs, 1 x [10.sup.-9] M to 1 x [10.sup.-3] M). For noncompetitive binding assays, isolated nuclei were incubated with 1 x [10.sup.-10] M [sup.125]I-[T.sub.3] and increasing concentrations (1 x [10.sup.-12] M to 1 x [10.sup.-6] M) of [T.sub.3] alone or increasing concentrations (1 x [10.sup.-12] M to 1 x [10.sup.-6] M) of [T.sub.3] in the presence of 2 x [10.sup.-6] M of competitors. Nonspecific binding was established as described above.

Statistical analysis. Results were analyzed using a one-factor analysis of variance (ANOVA), and post hoc tests, where appropriate, were performed by Bonferroni's t-test, where the mean squared error term in the ANOVA table was used as the point estimate of the pooled variance (SuperAnova Software; Abacus Concepts, Inc., Berkeley, CA). A test for outliers was performed on all data; none were identified. Some samples were lost in processes; therefore, there are some unequal cell sizes.

Results

Dams. Exposure to A1254 significantly reduced circulating levels of total [T.sub.4] and total [T.sub.3] in dams ([T.sub.4]: [F.sub.(2,15)] = 11.031, p = 0.0011; [T.sub.3]: [F.sub.(2,14)] = 5.772, p = 0.0142; Figure 2). Post hoc analysis using Bonferroni's t-test revealed that dams treated daily with 4 mg/kg A1254 exhibited [T.sub.4] levels that were significantly lower than those of control animals (Figure 2A). There was a trend in animals treated with 1 mg/kg A1254 to exhibit lower circulating levels of [T.sub.4], but this did not reach statistical significance (Figure 2A). Moreover, animals treated with either 1 or 4 mg/kg A1254 exhibited significantly lower levels of circulating [T.sub.3] compared with control animals (Figure 2B).

Fetal brains. Quantitative analysis of film autoradiograms after in situ hybridization revealed that PCB exposure selectively affected TH-responsive genes in the fetal cortex (Figure 3). We focused on four different genes. RC3/neurogranin expression was significantly higher in the cortex of fetuses derived from dams treated with 4 mg/kg A1254 ([F.sub.(2,15)] = 5.423, p = 0.0169). NSP-A expression was significantly elevated in fetuses derived from dams treated with either 1 or 4 mg/kg A1254 ([F.sub.(2,13)] = 8.212, p = 0.0049), as was Oct-1 ([F.sub.(2,14)] = 5.399, p = 0.0183). In contrast, NSP-C expression in the GD16 cortex was not affected by PCB exposure ([F.sub.(2,15)]= 0.202, p = 0.819; not significant).

[FIGURE 3 OMITTED]

TH receptor binding. To test the hypothesis that individual PCB congeners may bind to TRs to produce the observed effects on gene expression in vivo, we tested a number of parent PCBs and metabolites for their ability to bind to the TR (Table 2). To validate the assay, we first performed a saturation analysis and established that [T.sub.3] bound to TRs in isolated hepatic nuclei with a [K.sub.d] of 9.7 x [10.sup.-10] [+ or -] 2.02 x [10.sup.-10] M (Figure 4). We then tested the ability of [T.sub.3] and other TR agonists ([T.sub.4], Triac, and Tetrac) to displace [sup.125]I-[T.sub.3] from TRs in hepatic nuclei (Figure 5A); using the [K.sub.d] obtained from saturation analyses, a specific [K.sub.i] was calculated for each compound (Table 2). However, none of the tested parent PCB congeners, hydroxylated metabolites, or MeS[O.sub.2] metabolites significantly displaced [sup.125]I-[T.sub.3] in this assay (Figure 5B). Similarly, noncompetitive binding analysis revealed that parent PCB congeners, grouped according to their ortho-substitution pattern, did not alter the affinity of [T.sub.3] for TRs in isolated nuclei (Figure 6).

[FIGURES 4-6 OMITTED]

Discussion

The present study demonstrates that the commercial PCB mixture A1254 significantly reduces serum TH levels, [T.sub.4], and [T.sub.3], in pregnant rats on GD16. This developmental time occurs before the onset of thyroid function in the fetus (Fisher et al. 1977); therefore, it is reasonable to propose that this PCB-induced decrement in maternal TH would cause a reduction in the expression of genes positively regulated by TH. However, we found that PCB exposure up-regulated the expression of genes that are positively regulated by TH. These findings indicate that PCBs can activate TRs, perhaps directly, and the implication is important because inappropriate activation of TRs in the developing brain may produce adverse consequences on brain development (Kopelman i983; Rastogi and Singhal 1976, 1979; Zoeller 2003).

Our finding that A1254 decreased circulating levels of TH in pregnant rats is consistent with previous studies showing that exposure to A1254, or specific PCB congeners, causes a reduction in circulating levels of total [T.sub.4] in pregnant rats (Meerts et al. 2002; Morse et al. 1993, 1996). Additionally, we found that serum total [T.sub.3] was also reduced in the dams by A1254, indicating the degree to which serum [T.sub.4] was reduced, because nearly 80% of circulating [T.sub.3] is derived from peripheral deiodination of [T.sub.4] (Taurog 2000). Although PCB exposure reduced serum [T.sub.4] in the dams, several descriptive measures of hypothyroidism were not altered. For example, maternal body weight, weight gain, litter size, and pup weight were all unaffected by PCB treatment in this experiment (data not shown), similar to results of our previous study (Zoeller et al. 2000). Therefore, like others, we observed a discrepancy between the ability of PCB exposure to lower serum TH levels and its ability to produce symptoms of hypothyroidism.

The present finding that fetuses derived from A1254-treated dams exhibited a significant increase in RC3/neorogranin and Oct-1 expression represents strong evidence that PCBs can produce TH-like effects in the fetal brain because maternal TH increases the expression of these genes in the fetal brain (Dowling et al. 2000; Dowling and Zoeller 2000). Considering that A1254 exposure produced a significant reduction in circulating [T.sub.4] and [T.sub.3] in the dam, this finding is fully consistent with the hypothesis that PCBs can directly activate TRs in the fetal brain. Moreover, A1254 exerted selective effects on the expression of TH-responsive genes because NSP-C, which is not influenced by TH in the fetus (Dowling et al. 2000, 2001), was not affected by A1254. Furthermore, previous work in our laboratory has demonstrated that A1254 increases the expression of myelin basic protein, a known TH-responsive gene (Farsetti et al. 1991; Marta et al. 1998; Rodriguez-Pena et al. 1993), in the cerebellum and RC3/neurogranin in the forebrain of postnatal rats, despite the finding that these pups exhibited a severe reduction in the circulating levels of TH (Zoeller et al. 2000). Taken together, these data indicate that A1254 can exert agonistic effects on a variety of positively regulated TH-responsive genes in different brain regions at different developmental times.

Although A1254 produced TH-like effects on the expression of RC3/neurogranin and on Oct-1 in the fetal cortex in the present study, it did not exert these effects on NSP-A expression. We previously found that NSP-A expression is significantly increased in the brain of fetal rats derived from hypothyroid dams (Dowling et al. 2000, 2001). Therefore, the present finding that NSP-A expression is increased in the cortex of fetuses exposed transplacentally to A1254 suggests that the expression of this gene is responding to low maternal [T.sub.4] in PCB-treated dams, not to agonistic actions of PCBs on TRs. Previously, we showed that [T.sub.4] treatment of hypothyroid dams did not restore NSP-A expression in the fetal cortex (Dowling et al. 2001). Because [T.sub.4] was provided for only a short time, the interpretation was that the duration of [T.sub.4] treatment was not sufficient to produce a significant reduction in cellular levels of NSP-A mRNA. However, Chan et al. (2003) have recently reported that NSP-A expression is not directly sensitive to TH in N-Tera-2 cells, indicating that NSP-A may not be directly regulated by TH. If maternal hypothyroidism increases NSP-A expression indirectly, and NSP-A is not directly regulated by TH, then our present results indicate that PCBs produce effects on the fetal brain by exerting direct TH-like effects as well as by inducing low maternal TH.

Considering these findings, we tested a number of PCB congeners and specific metabolites for their ability to bind to TRs using a well-established binding assay (DeGroot and Torresani 1975). We found that neither the parent PCB congeners nor the hydroxylated or MeS[O.sub.2] metabolites significantly displace [T.sub.3] from rat hepatic nuclei. It is not likely that these observations are false negatives because the observed [K.sub.i] for several control ligands, including [T.sub.3], [T.sub.4], Tetrac, and Triac, were all within the published range (Evans and Braverman 1986; Goslings et al. 1976; Ichikawa and DeGroot 1987). In addition, we demonstrated in preliminary studies that the PCB diluent (dimethyl sulfoxide) does not interfere with the assay; moreover, we used different diluents in initial studies and obtained results that did not differ from those reported here. Thus, our finding that individual PCB congeners or their metabolites do not displace [T.sub.3] from its receptor indicates that these compounds do not interact with the TR in a competitive manner.

We employed hepatic nuclei to test whether individual PCB congeners could bind to rat TRs because the TR isoforms expressed in hepatocytes are also the predominant isoforms expressed in the fetal cortex (Bradley et al. 1992; Nakai et al. 1988). Therefore, the observation that individual PCB congeners did not displace [T.sub.3] from liver nuclei suggests that they also do not displace [T.sub.3] from TRs in the fetal cortex. However, Cheek et al. (1999) demonstrated that several hydroxylated PCB metabolites bind to the human TR[beta]1, although the affinity for the TR was reported to be 10,000-fold lower than that of [T.sub.3]. We evaluated two of these metabolites, 4'-OH-PCB-14 and 4'-OH-PCB-106, but did not find significant binding to the TRs in rat hepatic nuclei. These two studies may differ in their findings because we used TR isoforms from a different species, or because both TR[alpha]1 and TR[beta]1 are expressed in hepatocytes (Nakai et al. 1988). Specifically, it is possible that we may not have observed significant [T.sub.3] displacement if a PCB congener binds to only one of the two TRs with low affinity.

There are two major implications of the present study. First, the observation that PCB exposure selectively alters gene expression in the fetal cortex strongly suggests that PCBs can exert deleterious effects on fetal brain development regardless of the mechanism by which this effect is mediated. Because we used TH-responsive genes as end points for this study, it is likely that the observed effects reflect the ability of PCBs to disrupt TH action in the fetal brain. It will be important to determine whether PCB exposure can interfere with neurodevelopmental events by interfering with TH action.

The second major implication of our present results is that PCBs do not appear to bind to TRs in a competitive manner. We were surprised to find no individual PCB congeners or metabolites that exhibited strong binding to TRs, especially considering previous speculation about this (McKinney et al. 1987; McKinney and Waller 1998; Porterfield 1994, 2000; Porterfield and Hendry 1998). However, there is evidence that PCB congeners can affect TR activation without displacing [T.sub.3]. Specifically Iwasaki et al. (2002) showed that 4'-OH-PCB-106 suppressed [T.sub.3]-induced transactivation by TR in various cell lines. This appeared to be specific to the TR because it did not suppress glucocorticoid receptor-mediated transactivation. In addition, they showed that this PCB congener suppressed the ability of the TR to recruit the coactivator SRC-1. Because we found that this hydroxylated PCB did not displace [T.sub.3] from rat TRs in the present study, these observations strongly suggest that PCBs can directly alter TR action by a mechanism that is not well understood.

Our failure to identify individual PCBs that can bind with high affinity to the rat TRs requires alternate explanations for their effects on TH-responsive genes and developmental events. Individual PCB congeners may alter TH metabolism by tissue deiodinases, thereby changing the amount of hormone available to the TR. Previous studies indicate that PCBs can increase type-2 deiodinase activity in the adult (Hood and Klaassen 2000) and fetal (Meerts et al. 2002; Morse et al. 1996) rat brain. Moreover, PCBs are also known to affect second messenger signaling in the brain by affecting calcium homeostasis, receptor-mediated inositol phosphate production, and translocation of protein kinase C (Kodavanti et al. 1993, 1994). In addition, PCBs can produce toxic effects by binding either to the aryl hydrocarbon receptor (Safe 1990) or the ryanodine receptor (Schantz et al. 1997; Wong et al. 1997). These studies demonstrate that PCBs, especially as a mixture such as A1254, clearly produce multiple effects. However, there is no evidence that these other mechanisms of PCB action can exert specific effects on TH-responsive genes.

Table 1. Characteristics of plasmids used to prepare cRNA probes for
in situ hybridization.

Target                                Enzyme for
mRNA    Plasmid          Strand       linearization  Promoter

RC3     PRC/CMV          Antisense    Hind III       Sp6
                           Sense      Apa I          T7
NSP-A   PCR-II           Antisense    BamH1          T7
                           Sense      EcoRV          Sp6
Oct-1   PCR-II           Antisense    Hind III       T7
                           Sense      Xho I          Sp6
NSP-C   Synthetic oligo  Antisense    NA             NA

Target  Transcript       Gene target
mRNA    size             region       Reference

RC3     337               253-486     Iniguez et al. 1996
        344
NSP-A   202              1946-2147    Dowling et al. 2001
        210
Oct-1   1226              246-1481    Gauger et al. 2002
        1234
NSP-C   NA                230-183     Dowling et al. 2001

NA, not applicable.

Table 2. TH receptor agonists, parent PCB congeners, MeS[0.sub.2]-PCB
metabolites, and hydroxylated PCB metabolites tested in competitive TR
binding assays.

Competitor                      Abbreviation

Triiodothyronine           [T.sub.3]
Thyroxine                  [T.sub.4]
Triiodothyroacetic acid    Triac
Tetraiodothyroacetic acid  Tetrac
Parent PCBs                PCB-77
                           PCB-105
                           PCB-118
                           PCB-126
                           PCB-138
                           PCB-153
Me[So.sub.2]-PCBs          3-MeS[O.sub.2]-PCB-49
                           4-MeS[O.sub.2]-PCB-49
                           3-MeS[O.sub.2]-PCB-70
                           4-MeS[O.sub.2]-PCB-70
                           3-MeS[O.sub.2]-PCB-87
                           3-MeS[O.sub.2]-PCB-101
                           4-MeS[O.sub.2]-PCB-101
                           3-MeS[O.sub.2]-PCB-132
                           3-MeS[O.sub.2]-PCB-141
                           3-MeS[O.sub.2]-PCB-149
                           4-MeS[O.sub.2]-PCB-149
                           4-MeS[O.sub.2]-PCB-52
                           3-MeS[O.sub.2]-PCB-77
                           3-MeS[O.sub.2]-PCB-105
                           3-MeS[O.sub.2]-PCB-118
                           3-MeS[O.sub.2]-PCB-156
Hydroxylated PCBs          4'-OH-PCB-3
                           4'-OH-PCB-9
                           4-OH-PCB-14
                           4'-OH-PCB-12
                           4-OH-PCB-30
                           4'-OH-PCB-34
                           4'-OH-PCB-36
                           4'-OH-PCB-20
                           4'-OH-PCB-35
                           4-OH-PCB-39
                           4'-OH-PCB-58
                           4'-OH-PCB-72
                           4'-OH-PCB-106
                           4'-OH-PCB-112
                           4'-OH-PCB-159
                           4'-OH-PCB-165
                           3',4'-(di)0H-PCB-12
                           3-,4'-(di)0H-PCB-3
                           3'-OH-PCB-3
                           2'-OH-PCB-3

Competitor                   IUPAC nomenclature

Triiodothyronine           L-3,3',5-Triiodothyronine
Thyroxine                  L-3,3',5,5'-Tetraiodothyronine
Triiodothyroacetic acid    3,3',3-Triiodothyroacetic acid
Tetraiodothyroacetic acid  3,3',5,5'-Tetraiodothyroacetic acid
Parent PCBs                3,3',4,4'-TetraCB
                           2,3,3',4,4'-PentaCB
                           2,3',4,4',5-PentaCB
                           3,3',4,4',5-PentaCB
                           2,2',3,4',4-,5'-HexaCB
                           2,2',4,4',5,5'-HexaCB
Me[So.sub.2]-PCBs          3-MeS[O.sub.2]-2,2',4',5-tetraCB
                           4-MeS[O.sub.2]-2,2',4',5-tetraCB
                           3-MeS[O.sub.2]-2,3',4',5-tetraCB
                           4-MeS[O.sub.2]-2,3',4',5-tetraCB
                           3-MeS[O.sub.2]-2,2',3-,4',5-pentaCB
                           3-MeS[O.sub.2]-2,2',4,5,5'-pentaCB
                           4-MeS[O.sub.2]-2,2',4,5,5'-pentaCB
                           3-MeS[O.sub.2]-2,2',3,4',5,5-hexaCB
                           3-MeS[O.sub.2]-2,2',3',4',5,5'-hexaCB
                           3-MeS[O.sub.2]-2,2',4',5,5,6-hexaCB
                           4-MeS[O.sub.2]-2,2',4',5,5,6-hexaCB
                           4-MeS[O.sub.2]-2,2-,4,4'-tetraCB
                           3-MeS[O.sub.2]-3',4,4',5-tetraCB
                           3-MeS[O.sub.2]-2',3',4,4',5-pentaCB
                           3-MeS[O.sub.2]-2'4,4',5,5'-pentaCB
                           3-MeS[O.sub.2]-2',3'4,4',5,5'-hexaCB
Hydroxylated PCBs          4'-OH-4-monoCB
                           4'-OH-2,5-diCB
                           4'-OH-3',5'-diCB
                           4'-OH-3,4-diCB
                           4'-DH-2,4,6-triCB
                           4'-0H-2,3',5'-triCB
                           4'-OH-3,3',5-triCB
                           4'-OH-2,3,3'-triCB
                           4'-0H-3,3-,4-triCB
                           4-0H-3,4',5-triCB
                           4'-OH-2,3,3',5'-tetraCB
                           4'-OH-2,3',5,5'-tetraCB
                           4'-OH-2,3,3',4,5-pentaCB
                           4'-OH-2,3,3',5,6-pentaCB
                           4'-OH-2,3,3',4,5,5'-hexaCB
                           4'-OH-2,3,3',5,5',6-hexaCB
                           3',4'-OH-3,4-diDB
                           3',4'-OH-4-monoCB
                           3'-OH-4-monoCB
                           2'-OH-4-monoCB

Competitor                       [K.sub.j]        [+ or -] CV

Triiodothyronine           8.37 x [10.sup.-10] M    12.70%
Thyroxine                  3.74 x [10.sup.-9] M     14.40%
Triiodothyroacetic acid    1.29 x [10.sup.-9] M     17.30%
Tetraiodothyroacetic acid  2.37 x [10.sup.-7] M     18.40%
Parent PCBs                         ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
Me[So.sub.2]-PCBs                   ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
Hydroxylated PCBs                   ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA
                                    ND                NA

Abbreviations: CB, chlorinated biphenyl; IUPAC, International Union of
Pure and Applied Chemistry; NA, not applicable; ND, no detectable
binding.

Figure 2. Effect of A1254 treatment on serum concentrations of total
[T.sub.4] (A) and total [T.sub.3] (B) in dams at the time they were
sacrificed on GD16. Bars represent mean [+ or -] SEM; number of animals
in each group is shown within each bar. See Materials and Methods for
treatment details.

A1254 treatment

              A    B

Control       6    6
1 mg/kg *     6    6
4 mg/kg **    6    5

Note: Table made from bar graph.

* p < 0.05; ** p < 0.01 (significantly different from control group
using Bonferroni's t-test after one-way ANOVA).

REFERENCES

Ayotte P, Muckle G, Jacobson JL, Jacobson SW, Dewailly E. 2003. Assessment of pre- and postnatal exposure to polychlorinated biphenyls: lessons from the Inuit cohort study. Environ Health Perspect 111:1253-1258.

Bastomsky CH. 1974. Effects of a polychlorinated biphenyl mixture (Aroclor 1254) and DDT on biliary thyroxine excretion in rats. Endocrinology 95:1150-1155.

Bastomsky CH, Murthy PVN, Banovac K. 1976. Alterations in thyroxine metabolism produced by cutaneous application of microscope immersion oil: effects due to polychlorinated biphenyls. Endocrinology 98:1309-1314.

Bauer U, Amaro AR, Robertson LW. 1995. A new strategy for the synthesis of polychlorinated biphenyl metabolites. Chem Res Toxicol 8(1):92-95.

Bradley DJ, Towle HC, Young WS. 1992, Spatial and temporal expression of alpha- and beta-thyroid hormone receptor mRNAs, including the beta-2 subtype, in the developing mammalian nervous system. J Neurosci 12:2288-2302.

--. 1994. Alpha and beta thyroid hormone receptor (TR) gene expression during auditory neurogenesis: evidence for TR isoform-specific transcriptional regulation in vivo. Proc Natl Acad Sci USA 91:439-443.

Bradley DJ, Young WS, Weinberger C. 1989. Differential expression of alpha and beta thyroid hormone receptor genes in rat brain and pituitary. Proc Natl Acad Sci USA 96:7250-7254.

Breivik K, Sweetman A, Pacyna JM, Jones KC. 2002. Towards a global historical emission inventory for selected PCB congeners--a mass balance approach. 1. Global production and consumption, Sci Total Environ 290(1-3):181-198.

Brent GA. 2000. Tissue-specific actions of thyroid hormone: insights from animal models. Rev Endocr Metab Disord 1(1-2):27-33.

Brouwer A, Morse DC, Lans MC, Schuur AG, Murk AJ, Klasson-Wehler E, et al. 1998. Interactions of persistent environmental organohalides with the thyroid hormone system: mechanisms and possible consequences for animal and human health. Toxicol Ind Health 14(1/2):59-84.

Chan S, McCabe CJ, Visser TJ, Franklyn JA, Kilby MD. 2003. Thyroid hormone responsiveness in N-Tera-2 cells. J Endocrinol 170(1):150-167.

Chana A, Concejero MA, de Frutos M, Gonzalez MJ, Herradon B. 2002. Computational studies on biphenyl derivatives. Analysis of the conformational mobility, molecular electrostatic potential, and dipole moment of chlorinated biphenyl: searching for the rationalization of the selective toxicity of polychlorinated biphenyls (PCBs). Chem Res Toxicol 15(12):1514-1526.

Cheek AO, Kow K, Chen J, McLachlan JA. 1999. Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin. Environ Health Perspect 107:273-278.

Connors JM, Hedge GA. 1981. Feedback regulation of thyrotropin by thyroxine under physiological conditions. Am J Physiol 240(3):E308-E313.

Crofton KM, Ding D, Padich R, Taylor M, Henderson D. 2000a. Hearing loss following exposure during development to polychlorinated biphenyls: a cochlear site of action. Hear Res 144(1-2):190-204.

Crofton KM, Kodavanti PR, Derr-Yellin EC, Casey AC, Kehn LS. 2000b. PCBs, thyroid hormones, and ototoxicity in rats: cross-fostering experiments demonstrate the impact of postnatal lactation exposure. Toxicol Sci 57(1):131-140.

DeGroot LJ, Torresani J. 1975. Triiodothyronine binding to isolated liver cell nuclei. Endocrinology 96:357-369.

Dowling ALS, Iannacone EA, Zoeller RT. 2001. Maternal hypothyroidism selectively affects the expression of neuroendocrine-specific protein A messenger ribonucleic acid in the proliferative zone of the fetal rat brain cortex. Endocrinology 142:390-399.

Dowling ALS, Martz GU, Leonard JL, Zoeller RT. 2000. Acute changes in maternal thyroid hormone induce rapid and transient changes in specific gone expression in fetal rat brain. J Neurosci 20:2255-2265.

Dowling ALS, Zoeller RT. 2000. Thyroid hormone of maternal origin regulates the expression of RC3/neurogranin mRNA in the fetal rat brain. Brain Res 82:126-132.

Evans RW, Graverman LE. 1980. Use of 125I-triiodothyroacetic acid to measure nuclear thyroid hormone receptor. Endocr Res 12(1):37-47.

Farsetti A, Mitsuhashi T, Desvergne B, Robbins J, Nikodem VM. 1991. Molecular basis of thyroid hormone regulation of myelin basic protein gene expression in rodent brain. J Biol Chem 266:23226-23232.

Fisher BE. 1999, Most unwanted. Environ Health Perspect 107:A18-A23.

Fisher D, Dussault J, Sack J, Chopra I. 1977. Ontogenesis of hypothalamic-pituitary-thyroid function and metabolism in man, sheep, rat. Rec Progr Horm Res 33:56-107.

Flamant F, Samarut J. 2003. Thyroid hormone receptors: lessons from knockout and knock-in mutant mice. Trends Endocrinol Metab 14(2):85-80.

Gauger KJ, Herzig CT, Zoeller RT 2002. Developmental exposure to polychlorinated biphenyls exerts thyroid-like effects on the expression of thyroid hormone-responsive genes in the fetal rat brain, Society of Toxicology 41st annual meeting, March 17-21 2002. Nashville, TN. Abstract 1650.

Goldey ES, Crofton KM. 1998. Thyroxine replacement attenuates hypothyroxinemia, hearing loss, and motor deficits following developmental exposure to Aroclor 1254 in rats. Toxicol Sci 45:94-105.

Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM. 1995a. Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Toxicol Appl Pharmacol 135(1):77-88.

Goldey ES, Kehn LS, Rehnberg GL, Crofton KM 1998b. Effects of developmental hypothyroidism on auditory and motor function in the rat. Toxicol Appl Pharmacol 135:67-76.

Goslings B, Schwartz HL, Dillmann W, Surks MI, Oppenheimer JH. 1976. Comparison of the metabolism and distribution of L-triiodothyronine and triiodothyroacetic acid in the rat: a possible explanation of differential hormonal potency. Endocrinology 98:666-675.

Haddow JE, Palomaki GE, Allan WC, Williams JR, Knight GJ, Gagnon J, et el. 1999. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl J Med 341(8):549-555.

Hagmar L, Rylander L, Dyremark E, Klasson-Wehler E, Erfurth EM, 2001. Plasma concentrations of persistent organochlorines in relation to thyrotropin and thyroid hormone levels in women. Int Arch Occup Environ Health 74(3):184-188.

Haraguchi K, Kuroki H, Masuda Y. 1987. Synthesis and characterization of tissue-retainable methylsulfonyl polychlorinated biphenyl isomers. J Agric Food Chem 35:178-182.

Hood A, Klaassen CD. 2000. Effects of microsomal enzyme inducers on outer-ring deiodinase activity toward thyroid hormones in various rat tissues. Toxicol Appl Pharmacol 163(3):240-248.

Huisman M, Koopman-Esseboom C, Lanting CI, van der Paauw CG, Tuinstra LG, Fidler V, et al. 1995. Neurological condition in 18-month-old children perinatally exposed to polychlorinated biphenyls and dioxins. Early Hum Dev 43:165-176.

Ichikawa K, DeGroot LJ. 1987. Purification and characterization of rat liver nuclear thyroid hormone receptors. Proc Natl Acid Sci USA 84(10):3420-3424.

Iniguez MA, DeLecea L, Guadano-Ferraz A, Morte B, Gerendasy D, Sutcliffe JG, et al. 1996. Cell-specific effects of thyroid hormone on RC3/neurogranin expression in rat brain. Endocrinology 137:1032-1041.

Institute of Laboratory Animal Resources. 1996. Guide for the Care and Use of Laboratory Animals. 7th ed. Washington, DC:National Academy Press.

Iwasaki T, Miyazaki W, Takeshita A, Kuroda Y, Koibuchi N. 2002. Polychlorinated biphenyls suppress thyroid hormone-induced transactivation. Biochem Biophys Res Commun 299(3):384-388.

Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L, Horwitz KB. 1997. The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT. Mol Endocrinol 11(6):693-705.

Jacobson JL, Jacobson SW. 2002. Association of prenatal exposure to an environmental contaminant with intellectual function in childhood. J Toxicol Clin Toxicol 40(4):467-475.

Juarez de Ku LM, Sharma-Stokkermans M, Meserve LA. 1994. Thyroxine normalizes polychlorinated biphenyl (PCB) dose-related depression of choline acetyltransferase (ChAT) activity in hippocampus and basal forebrain of 15-day-old rats. Toxicology 94:19-30.

Kodavanti PR, Shafer TJ, Ward TR, Mundy WR, Freudenrich T, Harry GJ, et al. 1994. Differential effects of polychlorinated biphenyl congeners on phosphoinositide hydrolysis and protein kinase C translocation in rat cerebellar granule cells, Brain Res 662(1-2):75-82.

Kodavanti PR, Shin DS, Tilson HA, Harry DJ. 1993. Comparative effects of two polychlorinated biphenyl congeners on calcium homeostasis in rat cerebellar granule cells. Toxicol Appl Pharmacol 123(1):97-106.

Kolaja KL, Klaassen CD. 1998. Dose-response examination of UDP-glucuronosyltransferase inducers and their ability to increase both TGF-beta expression and thyroid follicular cell apoptosis. Toxicol Sci 46(1):31-37.

Koopman-Esseboom C, Morse DC, Weisglas-Kuperus N, Lutkeschiphost IJ, van der Paauw CB, Tuinstra LGMT, et al. 1994. Effects of dioxins and polychlorinated biphenyls on thyroid hormone status of pregnant women and their infants. Pediatr Res 30:468-473.

Kopelman AE. 1993. Delayed cerebral development in twins with congenital hyperthyroidism. Am J Dis Child 137(9):842-845.

Korrick SA, Altshul L. 1990. High breast milk levels of polychlorinated biphenyls (PCBs) among four women living adjacent to a PCB-contaminated waste site. Environ Health Perspect 106:513-518.

Lehmler HJ, Robertson LW, 2001. Synthesis of hydroxylated PCB metabolites with the Suzuki-coupling. Chemosphere 45(8):1119-1127.

Longnecker MP, Gladen BC, Patterson DG Jr, Rogan WJ. 2000. Polychlorinated biphenyl (PCB) exposure in relation to thyroid hormone levels in neonates. Epidemiology 11(3):249-254.

Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, et el. 1995. The nuclear receptor superfamily: the second decade, Cell 83(6):835-839.

Marta CB, Adamo AM, Soto EF, Pasquini JM. 1998. Sustained neonatal hyperthyroidism in the rat affects myelination in the central nervous system. J Neurosci Res 93(2):251-259.

Matsuura N, Konishi J. 1990. Transient hypothyroidism in infants born to mothers with chronic thyroiditis--a nationwide study of twenty-three cases. The Transient Hypothyroidism Study Group. Endocrinol Jpn 37(3):369-379.

McKinney J, Fannin R, Jordan S, Chae K, Rickenbacher U, Pedersen L, 1987. Polychlorinated biphenyls and related compound interactions with specific binding sites for thyroxine in rat liver nuclear extracts. J Med Chem 30:79-86.

McKinney JD, Waller CL. 1994. Polychlorinated biphenyls as hormonally active structural analogues. Environ Health Perspect 102:290-297.

--. 1998. Molecular determinants of hormone mimicry: halogenated aromatic hydrocarbon environmental agents. J Toxicol Environ Health B 1:27-58.

Meerts IA, Assink Y, Cenijn PH, Van Den Berg JH, Weijers BM, Bergman A, et al. 2002. Placental transfer of a hydroxylated polychlorinated biphenyl and effects on fetal and maternal thyroid hormone homeostasis in the rat Toxicol Sci 68(2):361-371.

Morreale de Escobar G, Obregon MJ, Escobar del Rey F. 2000. Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia. J Clin Endocrinol Metab 85(11):3975-3987.

Morse DC, Groen D, Veerman M, van Amerongen CJ, Koeter HB, Smits van Prooije AE, et al. 1993. Interference of polychlorinated biphenyls in hepatic and brain thyroid hormone metabolism in fetal and neonatal rats. Toxicol Appl Pharmacol 122:27-33.

Morse DC, Wehler EK, Wesseling W, Koeman JH, Brouwer A. 1996. Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). Toxicol Appl Pharmacol 136:269-279.

Nakai A, Seino S, Sakurai A, Szilak I, Bell GI, DeGroot LJ. 1989. Characterization of a thyroid hormone receptor expressed in human kidney and other tissues. Proc Natl Acad Sci USA 85(8):2781-2785.

Osius N, Karmaus W, Kruse H, Witten J. 1999. Exposure to polychlorinated biphenyls and levels of thyroid hormones in children. Environ Health Perspect 107:843-849.

Pop VJ, Kuijpens JL, van Baar AL, Verkerk G, van Son MM, de Vijlder JJ, et al. 1999. Low maternal free thyroxine concentrations during early pregnancy are associated with impaired psychomotor development in infancy. Clin Endocrinol 50(2):149-155.

Porterfield SP. 1994. Vulnerability of the developing brain to thyroid abnormalities: environmental insults to the thyroid system. Environ Health Perspect 102(suppl 2):125-130.

--. 2000. Thyroidal dysfunction and environmental chemicals--potential impact on brain development. Environ Health Perspect 108(suppl 3):433-438.

Porterfield SP, Hendry LB. 1998. Impact of PCBs on thyroid hormone directed brain development. Toxicol Ind Health 14:103-120.

Rastogi RB, Singhal RL. 1976. Influence of neonatal and adult hyperthyroidism on behavior and biosynthetic capacity for norepinephrine, dopamine and 5-hydroxy-tryptamine in rat brain. J Pharmacol Exp Ther 198:609-618.

--. 1979. Evidence that chronic apomorphine treatment enhances 5-hydroxytryptamine metabolism in brains of normal and neonatally hyperthyroid rats. Neuroendocrinology 29(6):399-405.

Rodriguez-Pena A, Ibarrola N, Iniguez MA, Munoz A, Bernal J. 1993. Neonatal hypothyroidism affects the timely expression of myelin-associated glycoprotein in the rat brain. J Clin Invest 91:812-818.

Safe S. 1990. Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and related compounds: environmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs). Crit Rev Toxicol 21(1):51-88.

Sala M, Sunyer J, Herrero C, To-Figueras J, Grimalt J. 2001. Association between serum concentrations of hexachlorobenzene and polychlorobiphenyls with thyroid hormone and liver enzymes in a sample of the general population. Occup Environ Med 53(3):172-177.

Sap J, Munoz A, Damm K, Goldberg Y, Ghysdael J, Lentz A, et al. 1986. The c-erbA brotein is a high affinity receptor for thyroid hormone. Nature 324:635-640.

Schantz SL, Seo BW, Wong PW, Pessah IN. 1997. Long-term effects of developmental exposure to 2,2',3,5',6- pentachlorobiphenyl (PCB 95) on locomotor activity, spatial learning and memory and brain ryanodine binding. Neurotoxicology 18(2):457-467.

Schantz SL, Widholm JJ, Rice DC. 2003. Effects of PCB exposure on neuropsychological function in children, Environ Health Perspect 111:357-376.

Schwartz HL. 1983. Effect of thyroid hormone on growth and development. In: Molecular Basis of Thyroid Hormone Action (Oppenheimer JH, Samuels HH, eds). New York:Academic Press, 413-444.

Steuerwald U, Weihe P, Jorgensen PJ, Bjerve K, Brock J, Heinzow B, et al. 2000. Maternal seafood diet, methylmercury exposure, and neonatal neurologic function. J Pediatr 136(5):599-605.

Taurog A. 2000. Hormone synthesis: thyroid iodine metabolism. In: The Thyroid: A Fundamental and Clinical Text (Braverman LE, Utiger RD, eds). Philadelphia:bippincott-Raven, 61-85.

Varma SK, Murray R, Stanbury JB. 1978. Effect of maternal hypothyroidism and triiodothyronine on the fetus and newborn in rats. Endocrinology 102(1):24-30.

Walkowiak J, Wiener JA, Fastabend A, Heinzow B, Kramer U, Schmidt E, et al. 2001. Environmental exposure to polychlorinated biphenyls and quality of the home environment: effects on psychodevelopment in early childhood. Lancet 358(9293):1602-1607.

Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM. 1986. The c-erbA gone encodes a thyroid hormone receptor. Nature 324:641-646.

Wong PW, Joy RM, Albertson TE, Schantz SL, Pessah IN. 1997. Ortho-substituted 2,2',3,5',6-pentachlorobiphenyl (PCB 95) alters rat hippocampal ryanodine receptors and neuroplasticity in vitro: evidence for altered hippocampal function. Neurotoxicology 18(2):443-456.

Zoeller RT. 2003. Transplacental thyroxine and fetal brain development. J Clin Invest 111(7):954-957.

Zoeller RT, Dowling AL, Vas AA. 2000. Developmental exposure to polychlorinated biphenyls exerts thyroid hormone-like effects on the expression of RC3/neurogranin and myelin basic protein messenger ribonucleic acids in the developing rat brain. Endocrinology 141(1):181-189.

Zoeller RT, Fletcher DL, Butnariu O, Lowry C, Moore FL. 1997. N-Ethylmaleimide (NEM) can significantly improve in situ hybridization results using 35S-labeled oligodeoxynucleotide or complementary RNA probes. J Histochem Cytochem 45:1035-1041.

Kelly J. Gauger, (7) Yoshihisa Kato, (2) Koichi Haraguchi, (3) Hans-Joachim Lehmler, (4) Larry W. Robertson, (4) Ruby Bansal, (1) and R. Thomas Zoeller (1)

(1) Biology Department, Molecular and Cellular Biology Program, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts, USA; (2) School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan; (3) Daiichi College of Pharmaceutical Sciences, Fukuoka, Japan; (4) Department of Occupational and Environmental Health, University of Iowa, College of Public Health, Iowa City, Iowa, USA

Address correspondence to R.T. Zoeller, Biology Department, Molecular and Cellular Biology Program, University of Massachusetts, Amherst, MA 01003 USA. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

We thank D. Darling and J. Meyer for their technical advice and R. Burch, A. Dowling, and D. Sharlin for comments on early versions of the manuscript.

This work was supported by grants ES10026 (R.T.Z.) and P42 ES 07380 (L.W.R. and H.-J.L.) from the National Institute of Environmental Health Sciences and grant T32MH47538 (K.G.) from the National Institute of Mental Health.

The authors declare they have no competing financial interests.

Received 14 August 2003; accepted 22 December 2003.