The mammary gland is a hormone sensitive organ that is susceptible to endocrine-disrupting chemicals (EDCs) during the vulnerable periods of parous reorganization (ie, pregnancy, lactation, and involution). Pregnancy is believed to have long-term protective effects against breast cancer development; however, it is unknown if EDCs can alter this effect. We examined the long-term effects of propylparaben, a common preservative used in personal care products and foods, with estrogenic properties, on the parous mouse mammary gland. Pregnant BALB/c mice were treated with 0, 20, 100, or 10 000 µg/kg/day propylparaben throughout pregnancy and lactation. Unexposed nulliparous females were also evaluated. Five weeks post-involution, mammary glands were collected and assessed for changes in histomorphology, hormone receptor expression, immune cell number, and gene expression. For several parameters of mammary gland morphology, propylparaben reduced the effects of parity. Propylparaben also increased proliferation, but not stem cell number, and induced modest alterations to expression of ERα-mediated genes. Finally, propylparaben altered the effect of parity on the number of several immune cell types in the mammary gland. These results suggest that propylparaben, at levels relevant to human exposure, can interfere with the effects of parity on the mouse mammary gland and induce long-term alterations to mammary gland structure. Future studies should address if propylparaben exposures negate the protective effects of pregnancy on mammary cancer development.

Polychlorinated biphenyls (PCBs) pose significant risk to the developing human brain; however, mechanisms of PCB developmental neurotoxicity (DNT) remain controversial. Two widely posited mechanisms are tested here using PCBs identified in pregnant women in the MARBLES cohort who are at increased risk for having a child with a neurodevelopmental disorder (NDD). As determined by gas chromatography-triple quadruple mass spectrometry, the mean PCB level in maternal serum was 2.22 ng/mL. The 12 most abundant PCBs were tested singly and as a mixture mimicking the congener profile in maternal serum for activity at the thyroid hormone receptor (THR) and ryanodine receptor (RyR). Neither the mixture nor the individual congeners (2 fM to 2 mu M) exhibited agonistic or antagonistic activity in a THR reporter cell line. However, as determined by equilibrium binding of [H-3]ryanodine to RyR1-enriched microsomes, the mixture and the individual congeners (SO nM to 50 mu M) increased RyR activity by 2.4-19.2-fold. 4-Hydroxy (OH) and 4-sulfate metabolites of PCBs 11 and 52 had no TH activity; but 4-OH PCB 52 had higher potency than the parent congener toward RyR These data support evidence implicating RyRs as targets in environmentally triggered NDDs and suggest that PCB effects on the THR are not a predominant mechanism driving PCB DNT. These findings provide scientific rationale regarding a point of departure for quantitative risk assessment of PCB DNT, and identify in vitro assays for screening other environmental pollutants for DNT potential.

The CLARITY-BPA experiment, a large collaboration between the National Institute of Environmental Health Sciences, the National Toxicology Program, and the US Food and Drug Administration, is designed to test the effects of bisphenol A (BPA) on a variety of endocrine systems and end points. The specific aim of this subproject was to test the effect of BPA exposure on thyroid functions and thyroid hormone action in the developing brain. Timed-pregnant National Center for Toxicological Research Sprague-Dawley rats (strain code 23) were dosed by gavage with vehicle control (0.3% carboxymethylcellulose) or one of five doses of BPA [2.5, 25, 250, 2500, or 25,000 µg/kg body weight (bw) per day] or ethinyl estradiol (EE) at 0.05 or 0.50 µg/kg bw/d (n = 8 for each group) beginning on gestational day 6. Beginning on postnatal day (PND) 1 (day of birth is PND 0), the pups were directly gavaged with the same dose of vehicle, BPA, or EE. We also obtained a group of animals treated with 3 ppm propylthiouracil in the drinking water and an equal number of concordant controls. Neither BPA nor EE affected serum thyroid hormones or thyroid hormone‒sensitive end points in the developing brain at PND 15. In contrast, propylthiouracil (PTU) reduced serum T4 to the expected degree (80% reduction) and elevated serum TSH. Few effects of PTU were observed in the male brain and none in the female brain. As a result, it is difficult to interpret the negative effects of BPA on the thyroid in this rat strain because the thyroid system appears to respond differently from that of other rat strains.

Hormones and endocrine-disrupting chemicals are generally thought to have permanent "organizational" effects when exposures occur during development but not adulthood. Yet, an increasing number of studies have shown that pregnant females are disrupted by endocrine-disrupting chemical exposures, with some effects that are permanent. Here, we examined the long-term effects of exposure to oxybenzone, an estrogenic chemical found in sunscreen and personal care products, on the morphology of the mammary gland in mice exposed during pregnancy and lactation. Female mice were exposed to vehicle or 30, 212, or 3000 µg oxybenzone/kg/d, from pregnancy day 0 until weaning. A nulliparous group, receiving vehicle treatment, was also evaluated. Mammary glands were collected 5 weeks after involution for whole-mount, histological, immunohistochemical, and molecular analyses. Exposure to 3000 µg oxybenzone/kg/d induced permanent changes to ductal density that was significantly different from both the nulliparous and vehicle groups. The two highest doses of oxybenzone similarly induced an intermediate phenotype for expression of progesterone receptor. A monotonic, dose-dependent increase in cell proliferation was also observed in the oxybenzone-treated females, becoming statistically significant at the highest dose. Finally, oxybenzone exposure induced an intermediate phenotype for Esr1 expression in all oxybenzone-treated groups. These data suggest that oxybenzone, at doses relevant to human exposures, produces long-lasting alterations to mammary gland morphology and function. Further studies are needed to determine if exposure to this chemical during pregnancy and lactation will interfere with the known protection that pregnancy provides against breast cancer.

High doses of estrogenic pharmaceuticals were once prescribed to women to halt lactation. Yet, the effects of low-level xenoestrogens on lactation remain poorly studied. We investigated the effects of bisphenol S (BPS), an estrogen receptor (ER) agonist, on the lactating mammary gland; the arcuate nucleus, a region of the hypothalamus important for neuroendocrine control of lactational behaviors; and nursing behavior in CD-1 mice. Female mice were exposed to vehicle, 2 or 200 µg BPS/kg/d from pregnancy day 9 until lactational day (LD) 20, and tissues were collected on LD21. Tissues were also collected from a second group at LD2. BPS exposure significantly reduced the fraction of the mammary gland comprised of lobules, the milk-producing units, on LD21, but not LD2. BPS also altered expression of Esr1 and ERα in the mammary gland at LD21, consistent with early involution. In the arcuate nucleus, no changes were observed in expression of signal transducer and activator of transcription 5, a marker of prolactin signaling, or ERα, suggesting that BPS may act directly on the mammary gland. However, observations of nursing behavior collected during the lactational period revealed stage-specific effects on both pup and maternal nursing behaviors; BPS-treated dams spent significantly more time nursing later in the lactational period, and BPS-treated pups were less likely to initiate nursing. Pup growth and development were also stunted. These data indicate that low doses of BPS can alter lactational behaviors and the maternal mammary gland. Together, they support the hypothesis that pregnancy and lactation are sensitive to low-dose xenoestrogen exposures.

Polybrominated diphenyl ethers (PBDEs) are routinely found in human tissues including cord blood and breast milk. PBDEs may interfere with thyroid hormone (TH) during development, which could produce neurobehavioral deficits. An assumption in experimental and epidemiological studies is that PBDE effects on serum TH levels will reflect PBDE effects on TH action in tissues. To test whether this assumption is correct, we performed the following experiments. First, five concentrations of diphenyl ether (0-30 mg/kg) were fed daily to pregnant rats to postnatal day 21. PBDEs were measured in dam liver and heart to estimate internal dose. The results were compared with a separate study in which four concentrations of propylthiouracil (PTU; 0, 1, 2, and 3 ppm) was provided to pregnant rats in drinking water for the same duration as for diphenyl ether. PBDE exposure reduced serum T4 similar in magnitude to PTU, but serum TSH was not elevated by PBDE. PBDE treatment did not affect the expression of TH response genes in the liver or heart as did PTU treatment. PTU treatment reduced T4 in liver and heart, but PBDE treatment reduced T4 only in the heart. Tissue PBDEs were in the micrograms per gram lipid range, only slightly higher than observed in human fetal tissues. Thus, PBDE exposure reduces serum T4 but does not produce effects on tissues typical of low TH produced by PTU, demonstrating that the effects of chemical exposure on serum T4 levels may not always be a faithful proxy measure of chemical effects on the ability of thyroid hormone to regulate development and adult physiology.

Context: Thyroid hormone (TH) is essential for normal development; therefore, disruption of TH action by a number of industrial chemicals is critical to identify. Several chemicals including polychlorinated biphenyls are metabolized by the dioxin-inducible enzyme CYP1A1; some of their metabolites can interact with the TH receptor. In animals, this mechanism is reflected by a strong correlation between the expression of CYP1A1 mRNA and TH-regulated mRNAs. If this mechanism occurs in humans, we expect that CYP1A1 expression will be positively correlated with the expression of genes regulated by TH. Objective: The objective of the study was to test the hypothesis that CYP1A1 mRNA expression is correlated with TH-regulated mRNAs in human placenta. Methods: One hundred sixty-four placental samples from pregnancies with no thyroid disease were obtained from the GESTE study (Sherbrooke, Québec, Canada). Maternal and cord blood TH levels were measured at birth. The mRNA levels of CYP1A1 and placental TH receptor targets [placental lactogen (PL) and GH-V] were quantitated by quantitative PCR. Results: CYP1A1 mRNA abundance varied 5-fold across 132 placental samples that had detectable CYP1A1 mRNA. CYP1A1 mRNA was positively correlated with PL (r = 0.64; P < .0001) and GH-V (P < .0001, r = 0.62) mRNA. PL and GH-V mRNA were correlated with each other (r = 0.95; P < .0001), suggesting a common activator. The mRNAs not regulated by TH were not correlated with CYP1A1 expression. Conclusions: CYP1A1 mRNA expression is strongly associated with the expression of TH-regulated target gene mRNAs in human placenta, consistent with the endocrine-disrupting action of metabolites produced by CYP1A1.

This work tests the mode-of-action (MOA) hypothesis that maternal and developmental triclosan (TCS) exposure decreases circulating thyroxine (T4) concentrations via up-regulation of hepatic catabolism and elimination of T4. Time-pregnant Long-Evans rats received TCS po (0–300 mg/kg/day) from gestational day (GD) 6 through postnatal day (PND) 21. Serum and liver were collected from dams (GD20, PND22) and offspring (GD20, PND4, PND14, PND21). Serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) concentrations were measured by radioimmunoassay. Ethoxy-O-deethylase (EROD), pentoxyresorufin-O-depentylase (PROD) and uridine diphosphate glucuronyltransferase (UGT) enzyme activities were measured in liver microsomes. Custom Taqman® qPCR arrays were employed to measure hepatic mRNA expression of select cytochrome P450s, UGTs, sulfotransferases, transporters, and thyroid hormone-responsive genes. TCS was quantified by LC/MS/MS in serum and liver. Serum T4 decreased approximately 30% in GD20 dams and fetuses, PND4 pups and PND22 dams (300 mg/kg/day). Hepatic PROD activity increased 2–3 fold in PND4 pups and PND22 dams, and UGT activity was 1.5 fold higher in PND22 dams only (300 mg/kg/day). Minor up-regulation of Cyp2b and Cyp3a expression in dams was consistent with hypothesized activation of the constitutive androstane and/or pregnane X receptor. T4 reductions of 30% for dams and GD20 and PND4 offspring with concomitant increases in PROD (PND4 neonates and PND22 dams) and UGT activity (PND22 dams) suggest that up-regulated hepatic catabolism may contribute to TCS-induced hypothyroxinemia during development. Serum and liver TCS concentrations demonstrated greater fetal than postnatal internal exposure, consistent with the lack of T4 changes in PND14 and PND21 offspring. These data support the MOA hypothesis that TCS exposure leads to hypothyroxinemia via increased hepatic catabolism; however, the minor effects on thyroid hormone metabolism may reflect the low efficacy of TCS as thyroid hormone disruptor or highlight the possibility that other MOAs may also contribute to the observed maternal and early neonatal hypothyroxinemia.

Polychlorinated biphenyls (PCB) are industrial chemicals linked to developmental deficits that may be caused in part by disrupting thyroid hormone (TH) action by either reducing serum TH or interacting directly with the TH receptor (TR). Individual PCB congeners can activate the TR in vitro when the metabolic enzyme cytochrome P4501A1 (CYP1A1) is induced, suggesting that specific PCB metabolites act as TR agonists. To test this hypothesis in vivo, we compared two combinations of PCB congeners that either activate the TR (PCB 105 and 118) or not (PCB 138 and 153) in the presence or absence of a PCB congener (PCB 126) that induces CYP1A1 in vitro. Aroclor 1254 was used as a positive control, and a group treated with propylthiouracil was included to characterize the effects of low serum TH. We monitored the effects on TH signaling in several peripheral tissues by measuring the mRNA expression of well-known TH-response genes in these tissues. Aroclor 1254 and its component PCB 105/118/126 reduced total T4 to the same extent as that of propylthiouracil but increased the expression of some TH target genes in liver. This effect was strongly correlated with CYP1A1 expression supporting the hypothesis that metabolism is necessary. Effects were gene and tissue specific, indicating that tissue-specific metabolism is an important component of PCB disruption of TH action and that PCB metabolites interact in complex ways with the TR. These are essential mechanisms to consider when evaluating the health risks of contaminant exposures, for both PCB and other polycyclic compounds known to interact with nuclear hormone receptors.

Thyroid hormone (TH) is essential for normal brain development, and polychlorinated biphenyls (PCBs) are known to interfere with TH action in the developing brain. Thus, it is possible that the observed neurotoxic effects of PCB exposure in experimental animals and humans are mediated in part by their ability to interfere with TH signaling. PCBs may interfere with TH signaling by reducing circulating levels of TH, acting as TH receptor analogs, or both. If PCBs act primarily by reducing serum TH levels, then their effects should mimic those of low TH. In contrast, if PCBs act primarily as TH agonists in the developing brain, then they should mimic the effect of T(4) in hypothyroid animals. We used a two-factor design to test these predictions. Both hypothyroidism (Htx) and/or PCB treatment reduced serum free and total T(4) on postnatal d 15. However, only Htx increased pituitary TSHbeta expression. RC3/neurogranin expression was decreased by Htx and increased by PCB treatment. In contrast, Purkinje cell protein-2 expression was reduced in hypothyroid animals and restored by PCB treatment. Finally, PCB treatment partially ameliorated the effect of Htx on the thickness of the external granule layer of the cerebellum. These studies demonstrate clearly that PCB exposure does not mimic the effect of low TH on several important TH-sensitive measures in the developing brain. However, neither did PCBs mimic T(4) in hypothyroid animals on all end points measured. Thus, PCBs exert a complex action on TH signaling in the developing brain.

BACKGROUND: Polychlorinated biphenyls (PCBs) may interfere with thyroid hormone (TH) signaling by reducing TH levels in blood, by exerting direct effects on TH receptors (TRs), or both.OBJECTIVE: Our objective was to identify individual PCBs that directly affect TH signaling by acting on the TR.METHODS: We administered a mixture of six PCB congeners based on their ortho substitution pattern, including PCBs 77 and 126 (non-ortho), PCBs 105 and 118 (mono-ortho), and PCBs 138 and 153 (di-ortho), to pregnant Sprague-Dawley rats from gestational days (G) 6 to 16. This mixture, or various combinations of the components, was also evaluated in a transient transfection system using GH3 cells.RESULTS: The mixture reduced serum TH levels in pregnant rats on G16 but simultaneously up-regulated the expression of malic enzyme in liver. It also functioned as a TR agonist in vitro; however, none of the individual PCB congeners comprising this mixture were active in this system. Using the aryl hydrocarbon receptor (AhR) antagonist alpha-naphthoflavone, and the cytochrome P450 (CYP)1A1 antagonist ellipticine, we show that the effect of the mixture on the thyroid hormone response element required AhR and CYP 1A1.CONCLUSIONS: We propose that PCB 126 induces CYP1A1 through the AhR in GH3 cells, and that CYP1A1 activates PCB 105 and/or 118 to a form a compound that acts as a TR agonist. These data suggest that some tissues may be especially vulnerable to PCBs interfering directly with TH signaling due to their capacity to express CYP1A1 in response to coplanar PCBs (or other dioxin-like molecules) if sufficient mono- ortho PCBs are present.

Polychlorinated biphenyls (PCBs) may interfere with thyroid hormone (TH) action by interacting directly with the TH receptor (TR). We found that the hydroxylated PCB metabolite, 4-OH-CB106, bound to the human TRβ1 and significantly elevated endogenous growth hormone (GH) expression in GH3 cells in a manner similar to that of T3 itself. This effect was also observed using a consensus TH response element (TRE) in a luciferase expression system, and was blocked by a single base-pair substitution in this TRE. In addition, we found that 4-OH-CB106 did not alter the ability of TRβ1 to physically interact with the TRE in the GH promoter, or with SRC1 or NCoR. These effects were directly parallel to effects of T3, indicating that 4-OH-CB106 exerts a direct agonistic effect on the TRβ1.

Thyroid hormone is known to be essential for normal brain development both before and after birth, but much less is known about the role of thyroid hormone development before birth. In rodents, thyroid hormone of maternal origin can selectively regulate gene expression in the fetal cortex; HES1 was identified as a putative thyroid hormone responsive gene in the fetal cortex. Using in situ hybridization, we now confirm that thyroid hormone administration to pregnant rats can increase the abundance of HES1 mRNA in the fetal cortex on gestational day 16 (G16). In separate experiments, we found that maternal exposure to polychlorinated biphenyls (PCBs) increases HES expression similarly. Western analysis of proteins extracted from fetal cortex did not confirm that Notch-1 or Notch-3 activation was associated with treatment effects on HES expression. However, considering the role of HES proteins in fate specification of cortical neurons, these findings suggest that thyroid hormone, and PCB exposure, may influence fate specification of cortical neurons.

Developmental exposure to polychlorinated biphenyls (PCBs) is associated with a variety of cognitive deficits in humans, and recent evidence implicates white matter development as a potential target of PCBs. Because PCBs are suspected of interfering with thyroid hormone (TH) signaling in the developing brain, and because TH is important in oligodendrocyte development, we tested the hypothesis that PCB exposure affects the development of white matter tracts by disrupting TH signaling. Pregnant Sprague Dawley rats were exposed to the PCB mixture Aroclor 1254 (5 mg/kg), with or without cotreatment of goitrogens from gestational d 7 until postnatal d 15. Treatment effects on white matter development were determined by separately measuring the cellular density and proportion of myelin-associated glycoprotein (MAG)-positive, O4-positive, and glial fibrillary acidic protein (GFAP)-positive cells in the genu of the corpus callosum (CC) and in the anterior commissure (AC). Hypothyroidism decreased the total cell density of the CC and AC as measured by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and produced a disproportionate decrease in MAG-positive oligodendrocyte density with a simultaneous increase in GFAP-positive astrocyte density. These data indicate that hypothyroidism reduces cellular density of CC and AC and fosters astrocyte development at the expense of oligodendrocyte density. In contrast, PCB exposure significantly reduced total cell density but did not disproportionately alter MAG-positive oligodendrocyte density or change the ratio of MAG-positive oligodendrocytes to GFAP-positive astrocytes. Thus, PCB exposure mimicked some, but not all, of the effects of hypothyroidism on white matter composition.

Considering the importance of thyroid hormone (TH) in brain development, it is of potential concern that a wide variety of environmental chemicals can interfere with thyroid function or, perhaps of greater concern, with TH action at its receptor (TR). Recently bisphenol-A (BPA, 4,4' isopropylidenediphenol) was reported to bind to the rat TR and act as an antagonist in vitro. BPA is a high production volume chemical, with more than 800 million kg of BPA produced annually in the United States alone. It is detectable in serum of pregnant women and cord serum taken at birth; is 5-fold higher in amniotic fluid at 15-18 wk gestation, compared with maternal serum; and was found in concentrations of up to 100 ng/g in placenta. Thus, the human population is widely exposed to BPA and it appears to accumulate in the fetus. We now report that dietary exposure to BPA of Sprague Dawley rats during pregnancy and lactation causes an increase in serum total T4 in pups on postnatal d 15, but serum TSH was not different from controls. The expression of the TH-responsive gene RC3/neurogranin, measured by in situ hybridization, was significantly up-regulated by BPA in the dentate gyrus. These findings suggest that BPA acts as a TH antagonist on the beta-TR, which mediates the negative feedback effect of TH on the pituitary gland, but that BPA is less effective at antagonizing TH on the alpha-TR, leaving TRalpha-mediated events to respond to elevated T4.

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 triiodothyronine (T3) and thyroxine (T4) 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.

Thyroid hormone is essential for normal brain development. Therefore, it is a genuine concern that thyroid function can be altered by a very large number of chemicals routinely found in the environment and in samples of human and wildlife tissues. These chemicals range from natural to manufactured compounds. They can produce thyroid dysfunction when they are absent from the diet, as in the case of iodine, or when they are present in the diet, as in the case of thionamides. Recent clinical evidence strongly suggests that brain development is much more sensitive to thyroid hormone excess or deficit than previously believed. In addition, recent experimental research provides new insight into the developmental processes affected by thyroid hormone. Based on the authors' research focusing on the ability of polychlorinated biphenyls to alter the expression of thyroid hormone-responsive genes in the developing brain, this review provides background information supporting a new way of approaching risk analysis of thyroid disruptors.