Thyroid hormone (TH) availability is particularly critical for early brain development. TH transport across the blood-brain barrier is facilitated through two main transmembrane transporters: monocarboxylate transporter 8 (MCT8) and organic anion transporter 1C1 (OATP1C1). Inhibition of MCT8-mediated TH transport has been identified for a number of environmental chemicals using in vitro screening assays. Here we examined the in vivo effects of exposure to a potent in vitro inhibitor of MCT8, the flavonolignan silychristin, on several aspects of the TH system. Adult female rats were daily gavaged with 0, 250, or 500 mg/kg/day (n = 10/group) of silychristin for 7 days and euthanized on day 8. A smaller group (n = 5/group) of rats was administered the related flavonolignan, silybin (900 mg/kg), or the milk-thistle-derived flavonolignan mixture, silymarin (1,500 mg/kg). Serum TH concentrations were not changed in any treatment group. Mct8 and Oatp1c1 expression were upregulated in the choroid plexus upon silymarin exposure, without change in response to silychristin or silybin. Deiodinase 1 and dehalogenase activities, unchanged in the liver, were increased in the thyroid by the high dose of silychristin. These changes may have been triggered by increased thyroidal TH content, consequent to a reduction in MCT8-mediated TH efflux. Pharmacokinetic properties of silychristin and other flavonoids result in their low bioavailability and likely contributed to the largely negative findings. These observations demonstrate the challenges in extrapolating results from in vitro models to studies in intact organisms, showcasing the importance of selecting appropriate animal models and the best experimental design for assessing effects on human health.

Evaluating the neurodevelopmental effects of thyroid-disrupting chemicals is challenging. Although some standardized developmental and reproductive toxicity studies recommend serum thyroxine (T4) measures in developing rats, extrapolating between a serum T4 reduction and neurodevelopmental outcomes is not straightforward. Previously, we showed that the blood-brain and blood-cerebrospinal fluid barriers may be affected by developmental hypothyroidism in newborn rats. Here, we hypothesized that if the brain barriers were functionally disturbed by abnormal thyroid action, then small molecules may escape from the brain tissue and into general circulation. These small molecules could then be identified in blood samples, serving as a direct readout of thyroid-mediated developmental neurotoxicity. To address these hypotheses, pregnant rats were exposed to propylthiouracil (PTU, 0 or 3 ppm) to induce thyroid hormone insufficiency, and dams were permitted to give birth. PTU significantly reduced serum T4 in postnatal offspring. Consistent with our hypothesis, we show that tight junctions of the brain barriers were abnormal in PTU-exposed pups, and the blood-brain barrier exhibited increased permeability. Next, we performed serum microRNA Sequencing (miRNA-Seq) to identify noncoding RNAs that may reflect these neurodevelopmental disturbances. Of the differentially expressed miRNAs identified, 7 were upregulated in PTU-exposed pups. Validation by qRT-PCR shows that miR-495 and miR-543-3p were similarly upregulated in males and females. Interestingly, these miRNAs have been linked to cell junction dysfunction in other models, paralleling the identified abnormalities in the rat brain. Taken together, these data show that miR-495 and miR-543-3p may be novel in vivo biomarkers of thyroid-mediated developmental neurotoxicity.

The environmental contaminant perchlorate impairs the synthesis of thyroid hormones by reducing iodine uptake into the thyroid gland. Despite this known action, moderate doses of perchlorate do not significantly alter serum thyroid hormone in rat pups born to exposed dams. We examined perchlorate dosimetry and responsivity of the thyroid gland and brain in offspring following maternal exposure to perchlorate. Pregnant rat dams were delivered perchlorate in drinking water (0, 30, 100, 300, 1000 ppm) from gestational day 6 to postnatal day (PN) 21. Perchlorate was present in the placenta, milk, and serum, the latter declining in pups over the course of lactation. Serum and brain thyroid hormone were reduced in pups at birth but recovered to control levels by PN2. Dramatic upregulation of Nis was observed in the thyroid gland of the exposed pup. Despite the return of serum thyroid hormone to control levels by PN2, expression of several TH-responsive genes was altered in the PN14 pup brain. Contextual fear learning was unimpaired in the adults, supporting previous reports. Declining levels of serum perchlorate and a profound upregulation of Nis gene expression in the thyroid gland are consistent with the rapid return to the euthyroid state in the neonate. However, despite this recovery, thyroid hormone insufficiencies in serum and brain beginning in utero and present at birth appear sufficient to alter TH action in the fetus and subsequent trajectory of brain development. Biomarkers of that altered trajectory remain in the brain of the neonate, demonstrating that perchlorate is not devoid of effects on the developing brain.

Objective: To investigate the stability of magnesium isoglycyrrhizinate (Mg IG) injection solution when mixed with reduced glutathione (GSH) injection solution at room temperature. Method: High-performance liquid chromatography was used to determine the concentration changes of Mg IG injection solution mixed with GSH injection solution in 5% and 10% glucose solutions. Results: No significant changes in concentration were observed within 0, 0.5, 1, 1.5, 2, and 4 hours after mixing the Mg IG injection solution with the GSH injection solution. Conclusion: Mg IG injection solution can be mixed with GSH injection solution in 5% and 10% glucose solutions and should be used within 4 hours.

Known as "forever chemicals", per- and polyfluoroalkyl substances (PFAS) are synthetic compounds used in consumer goods but pose significant public health concerns, including disruption of the thyroid system. As thyroid hormones (THs) are required for normal brain development, PFAS may also be developmental neurotoxicants. However, this is not well understood. Here we examine the endocrine and neurodevelopmental consequences of perfluorohexane sulfonate (PFHxS) exposure in pregnant, lactating, and developing rats, and compare its effects to an anti-thyroid pharmaceutical (propylthiouracil, PTU) that induces thyroid-mediated developmental neurotoxicity. We show that PFHxS dramatically reduces maternal serum thyroxine (T4), nearly equivalently to PTU (-55 and -51%, respectively). However, only PTU increases thyroid stimulating hormone. The lactational transfer of PFHxS is significant and reduces pup serum T4 across the postnatal period. Surprisingly, brain THs are only minimally decreased by PFHxS, whereas PTU drastically diminishes them. Evaluation of brain TH action by phenotyping, RNA-Sequencing, and quantification of radial glia cell morphology supports that PTU interrupts TH signaling while PFHxS has limited to no effect. These data show that PFHxS induces abnormal serum TH profiles; however, there were no indications of hypothyroidism in the postnatal brain. We suggest the stark differences between the neurodevelopmental effects of PFHxS and a typical antithyroid agent may be due to its interaction with TH distributing proteins like transthyretin.

Thyroid hormones (THs) require iodine for biosynthesis and play critical roles in brain development. Perchlorate is an environmental contaminant that reduces serum THs by blocking the uptake of iodine from the blood to the thyroid gland. Using a pregnant rodent model, we examined the impact of maternal exposure to perchlorate under conditions of dietary iodine deficiency (ID) on the brain and behavior of offspring. We observed modest reductions in thyroxine (T4) in the serum of dams and no effect on T4 in pup serum in response to maternal exposure to 300 ppm of perchlorate in the drinking water. Likewise, serum T4 was reduced in ID dams, but, as with perchlorate, no effects were evident in the pup. However, when ID was coupled with perchlorate, reductions in pup serum THs and transcriptional alterations in the thyroid gland and pup brain were detected. These observations were accompanied by reductions in the number of cortical inhibitory interneurons containing the calcium-binding protein parvalbumin (Pvalb). Alterations in Pvalb expression in the neonatal brain were associated with deficits in the prepulse inhibition of acoustic startle in adult male offspring and enhanced fear conditioning in females. These findings support and extend structural defects in the brain previously reported in this model. Further, they underscore the critical need to consider additional non-chemical stressors in the determination of hazards and risks posed by environmental contaminants that affect the thyroid system.

Adolescents with intellectual and developmental disabilities (IDD) experience overweight and obesity (OW/OB) up to 1.8 times the rate of their typically developing peers. Parents may influence adolescent weight management behaviors in this population, but the association between parent factors and adolescent weight management behaviors is unclear.To examine the associations between parent BMI and sociodemographic characteristics with adolescents' BMI, diet quality, daily energy intake, moderate to vigorous physical activity (MVPA), and sedentary behavior.This study analyzed baseline data from an 18-month randomized controlled weight loss trial for adolescents with IDD. We assessed parent BMI (kg/m2) and sociodemographic factors, and adolescent BMI z-score, MVPA, sedentary time, daily energy intake, and diet quality. Associations between parent and adolescent factors were assessed with Pearson, Spearman or Kendall Tau-b correlations; mean differences for categorical outcomes were assessed with independent samples t-tests/Mann-Whitney U tests or ANOVA/Kruskall-Wallis tests.Ninety-five adolescent and parent dyads were included. Parent BMI was positively correlated with adolescent BMI z-score (n = 94: rs = 0.37, p < 0.01). Household income was inversely correlated with adolescent BMI z-score (n = 95: Tb = −0.18, p = 0.02). Parents with less than a bachelor's degree had adolescents with higher BMI z-scores than those with bachelor's or higher (2.1 ± 0.5 vs. 1.8 ± 0.5, p = 0.02) as well as higher sedentary behavior (n = 28, 515.2 ± 102.6 min/day vs. n = 40, 463.9 ± 148.1 min/day, p = 0.02).We found parent BMI, income, and education associated with adolescent BMI z-score. These findings contribute to the sparse literature on parental factors associated with OW/OB in this population.NCT02561754.

Environmental contaminants are often flagged as thyroid system disruptors due to their actions to reduce serum thyroxine (T4) in rodent models. The presence of a periventricular heterotopia (PVH), a brain malformation resulting from T4 insufficiency, has been described in response to T4 decrements induced by pharmaceuticals that reduce the hormone synthesis enzyme thyroperoxidase. In this report, we extend these observations to the environmental contaminant perchlorate, an agent that interferes with thyroid status by inhibiting iodine uptake into the thyroid gland. Pregnant rat dams were administered perchlorate in their drinking water (0, 30, 100, 300, 1000 ppm) from gestational day (GD) 6 until the weaning of pups on postnatal day (PN) 21. Serum T4 was reduced in dams and fetuses in late gestation and remained lower in lactating dams. Pup serum and brain T4, however, were not reduced beyond PN0, and small PVHs were evident in the brains of offspring when assessed on PN14. To emulate the developmental time window of the brain in humans, a second study was conducted in which pups from perchlorate-exposed dams were administered perchlorate orally from PN0 to PN6. This treatment reduced serum and brain T4 in the pup and resulted in large PVH. A third study extended the period of serum and brain TH suppression in pups by coupling maternal perchlorate exposure with maternal dietary iodine deficiency (ID). No PVHs were evident in the pups from ID dams, small PVHs were observed in the offspring of dams exposed to 300 ppm of perchlorate, and very large PVHs were present in the brains of pups born to dams receiving ID and perchlorate. These findings underscore the importance of the inclusion of serum hormone profiles in pregnant dams and fetuses in in vivo screens for thyroid-system-disrupting chemicals and indicate that chemical-induced decreases in fetal rat serum that resolve in the immediate postnatal period may still harbor considerable concern for neurodevelopment in humans.

Identifying xenobiotics that interrupt the thyroid axis has significant public health implications, given that thyroid hormones are required for brain development. As such, some developmental and reproductive toxicology (DART) studies now require or recommend serum total thyroxine (T4) measurements in pregnant, lactating, and developing rats. However, serum T4 concentrations are normally low in the fetus and pup which makes quan-tification difficult. These challenges can be circumvented by technologies like mass spectrometry, but these approaches are expensive and not always widely available. To demonstrate the feasibility of measuring T4 using a commercially available assay, we examine technical replicates of rat serum samples measured both by liquid chromatography mass spectrometry (LC/MS/MS) and radioimmunoassay (RIA). These samples were obtained from rats on gestational day 20 (dams and fetuses) or postnatal day 5 (pups), following maternal exposure to the goitrogen propylthiouracil (0-3 ppm) to incrementally decrease T4. We show that with assay modification, it is possible to measure serum T4 using low sample volumes (25-50 mu L) by an RIA, including in the GD20 fetus exposed to propylthiouracil. This proof-of-concept study demonstrates the technical feasibility of measuring serum T4 in DART studies.

A rare disease is classified as such if it affects less than 1 persons in 2,000. The Core Outcome Set STandards for Development (COS-STAD) is a set of standards that represent the minimum recommendations to be considered in the process of COS development. The aim of this study was to provide a baseline assessment of COS development standards for rare genetic diseases.Core Outcome Measures in Effectiveness Trials (COMET) database contains 447 published COS studies. Studies focusing on COS development for rare genetic diseases were eligible for inclusion and were assessed by two independent evaluators.Nine COS studies were included in the analysis. Eight different rare genetic diseases were investigated. None of the studies met all standards for development. The number of standards met ranged from 6 to 10 and the median was 7.This study is the first assessment of COS-STAD for rare genetic diseases COS studies and it highlights a great need for improvement. First in terms of numbers of rare diseases considered for COS developments, second in methodology, particularly regarding the consensus process and third in reporting of the COS development studies.

Metal additive manufacturing (AM) enables the production of complex and individualized designs. However, most AM parts require postprocessing with subtractive manufacturing processes, which can account for a significant percentage of the total manufacturing cost of an AM part. Positioning and clamping of complex AM parts within post-processing machines often lead to increased prestresses and reduced tool accessibility. One concept to address this problem is the integration of clamping interfaces in the part. But this leads to the new design challenge of optimal and material-saving placement of clamping interfaces on the part. To overcome this challenge new design tools are desired that facilitate this work and automatically generate the design of clamping interfaces.A recently developed clamping system uses bolts that are directly printed onto parts as clamping interfaces. These printed bolts and the clamping jaws of the system enable a unique spatial positioning and rigid clamping of AM parts for post-processing. This work introduces a design workflow that supports the positioning of bolts using a knowledge-based engineering (KBE) approach. The workflow thus allows the user to easily find a feasible clamping configuration and automatically generates the geometries of the bolt-shaped clamping interfaces. As input, the workflow uses the part geometry and an AM build direction. During the workflow, the user can modify the position of the clamping system relative to the part and find feasible positions for bolts. The bolt geometries are then generated automatically, and the part can be exported. This paper describes the workflow in detail and provides a vision for future developments of the tool and its potential for the AM process chain.

Despite the number of in vitro assays that have been recently developed to identify chemicals that interfere with the hypothalamic-pituitary-thyroid axis (HPT), the translation of those in vitro results into in vivo responses (in vitro to in vivo extrapolation, IVIVE) has received limited attention from the modeling community. To help advance this field a steady state biologically based dose response (BBDR) model for the HPT axis was constructed for the pregnant rat on gestation day (GD) 20. The BBDR HPT axis model predicts plasma levels of thyroid stimulating hormone (TSH) and the thyroid hormones, thyroxine (T4) and triiodothyronine (T3). Thyroid hormones are important for normal growth and development of the fetus. Perchlorate, a potent inhibitor of thyroidal uptake of iodide by the sodium iodide symporter (NIS) protein, was used as a case study for the BBDR HPT axis model. The inhibitory blocking of the NIS by perchlorate was associated with dose-dependent steady state decreases in thyroid hormone production in the thyroid gland. The BBDR HPT axis model predictions for TSH, T3, and T4 plasma concentrations in pregnant Sprague Dawley (SD) rats were within 2-fold of observations for drinking water perchlorate exposures ranging from 10 to 30,000 μg/kg/d. In Long Evans (LE) pregnant rats, for both control and perchlorate drinking water exposures, ranging from 85 to 82,000 μg/kg/d, plasma thyroid hormone and TSH concentrations were predicted within 2 to 3.4- fold of observations. This BBDR HPT axis model provides a successful IVIVE template for thyroid hormone disruption in pregnant rats.

A number of xenobiotics interfere with thyroid hormone (TH) signaling. Although adequate supplies of TH are necessary for normal brain development, regulatory reliance on serum TH as proxies for brain TH insufficiency is fraught with significant uncertainties. A more direct causal linkage to neurodevelopmental toxicity induced by TH-system disrupting chemicals is to measure TH in the target organ of most concern, the brain. However, the phospholipid-rich matrix of brain tissue presents challenges for TH extraction and measurement. We report optimized analytical procedures to extract TH in brain tissue of rats with recoveries >80% and low detection limits for T3, rT3, and T4 (0.013, 0.033, and 0.028 ng/g, respectively). Recovery of TH is augmented by enhancing phospholipid separation from TH using an anion exchange column coupled with a stringent column wash. Quality control measures incorporating a matrix-matched calibration procedure revealed excellent recovery and consistency across a large number of samples. Application of optimized procedures revealed age-dependent increases in neonatal brain T4, T3, and rT3 on the day of birth (postnatal day, PN0), PN2, PN6, and PN14. No sex-dependent differences in brain TH were observed at these ages, and similar TH levels were evident in perfused versus non-perfused brains. Implementation of a robust and reliable method to quantify TH in the fetal and neonatal rat brain will aid in the characterization of the thyroid-dependent chemical interference on neurodevelopment. A brain- in addition to a serum-based metric will reduce uncertainties in assessment of hazard and risk on the developing brain posed by thyroid system-disrupting chemicals.

Thyroid hormone (TH) action controls brain development in a spatiotemporal manner. Previously, we demonstrated that perinatal hypothyroidism led to formation of a periventricular heterotopia in developing rats. This heterotopia occurs in the posterior telencephalon, and its formation was preceded by loss of radial glia cell polarity. As radial glia mediate cell migration and originate in a progenitor cell niche called the ventricular zone (VZ), we hypothesized that TH action may control cell signaling in this region. Here we addressed this hypothesis by employing laser capture microdissection and RNA-Seq to evaluate the VZ during a known period of TH sensitivity. Pregnant rats were exposed to a low dose of propylthiouracil (PTU, 0.0003%) through the drinking water during pregnancy and lactation. Dam and pup THs were quantified postnatally and RNA-Seq of the VZ performed in neonates. The PTU exposure resulted in a modest increase in maternal thyroid stimulating hormone and reduced thyroxine (T4). Exposed neonates exhibited hypothyroidism and T4 and triiodothyronine (T3) were also reduced in the telencephalon. RNA-Seq identified 358 differentially expressed genes in microdissected VZ cells of hypothyroid neonates as compared to controls (q-values ≤0.05). Pathway analyses showed processes like maintenance of the extracellular matrix and cytoskeleton, cell adhesion, and cell migration were significantly affected by hypothyroidism. Immunofluorescence also demonstrated that collagen IV, F-actin, radial glia, and adhesion proteins were reduced in the VZ. Immunohistochemistry of integrin αvβ3 and isoforms of both thyroid receptors (TRα/TRβ) showed highly overlapping expression patterns, including enrichment in the VZ. Taken together, our results show that TH action targets multiple components of cell junctions in the VZ, and this may be mediated by both genomic and nongenomic mechanisms. Surprisingly, this work also suggests that the blood-brain and blood-cerebrospinal fluid barriers may also be affected in hypothyroid newborns.

To verify the analytical performance of cobas® HBV PCR and cobas® HCV PCR assays with Abbott m2000 RealTime System as the reference method.De-identified residual, archived patient specimens, and College of American Pathologists (CAP) proficiency testing samples were used. Analytical parameters verified were accuracy, precision, limit of detection (LOD), linear range, and cross-contamination. Experiments were designed in accordance with Clinical Laboratories Standards Institute (CLSI) guidelines and CAP standards. Analysis of accuracy was done through regression plots and Bland Altman analyses. Precision was analyzed through coefficient of variation and ANOVA; LOD through probit analysis; and linear range through polynomial fit analysis.The regression plots for accuracy showed a slope nearing 1, with a y-intercept close to zero, while Bland Altman analyses also showed no systematic evidence of bias, though concordance of results was not perfect near the lower limit of quantification. Coefficients of variation were all below 15%, while ANOVA returned p-values above 0.99, indicating no statistically significant imprecision. The LOD verified were an order of magnitude higher than the manufacturer reported ones for both assays, while the linear range verified was more limited. Within the verified range, polynomial fit analysis showed line to be the best fit for the data.cobas® HBV PCR and cobas® HCV PCR assays showed acceptable accuracy, acceptable precision, as well as no evidence of cross-contamination. The LOD verified were higher, and linear ranges more limited than those reported by the manufacturer. Verifications of these may be limited by availability of appropriate testing specimens.

Many xenobiotics are identified as potential thyroid disruptors due to their action to reduce circulating levels of thyroid hormone, most notably thyroxine (T4). Developmental neurotoxicity is a primary concern for thyroid disrupting chemicals yet correlating the impact of chemically induced changes in serum T4 to perturbed brain development remains elusive. A number of thyroid-specific neurodevelopmental assays have been proposed, based largely on the model thyroid hormone synthesis inhibitor propylthiouracil (PTU). This study examined whether thyroid disrupting chemicals acting distinct from synthesis inhibition would result in the same alterations in brain as expected with PTU. The perfluoroalkyl substance perfluorohexane sulfonate (50mg/kg/day) and the antimicrobial Triclosan (300mg/kg/day) were administered to pregnant rats from gestational day 6 to postnatal day (PN) 21, and a number of PTU-defined assays for neurotoxicity evaluated. Both chemicals reduced serum T4 but did not increase thyroid stimulating hormone. Both chemicals increased expression of hepatic metabolism genes, while thyroid hormone-responsive genes in the liver, thyroid gland, and brain were largely unchanged. Brain tissue T4 was reduced in newborns, but despite persistent T4 reductions in serum, had recovered in the PN6 pup brain. Neither treatment resulted in a low dose PTU-like phenotype in either brain morphology or neurobehavior, raising questions for the interpretation of serum biomarkers in regulatory toxicology. They further suggest that reliance on serum hormones as prescriptive of specific neurodevelopmental outcomes may be too simplistic and to understand thyroid-mediated neurotoxicity we must expand our thinking beyond that which follows thyroid hormone synthesis inhibition.

To date, approximately 200 chemicals have been tested in US Environmental Protection Agency (EPA) or Organization for Economic Co-operation and Development (OECD) developmental neurotoxicity (DNT) guideline studies, leaving thousands of chemicals without traditional animal information on DNT hazard potential. To address this data gap, a battery of in vitro DNT new approach methodologies (NAMs) has been proposed. Evaluation of the performance of this battery will increase the confidence in its use to determine DNT chemical hazards. One approach to evaluate DNT NAM performance is to use a set of chemicals to evaluate sensitivity and specificity. Since a list of chemicals with potential evidence of in vivo DNT has been established, this study aims to develop a curated list of "negative" chemicals for inclusion in a "DNT NAM evaluation set". A workflow, including a literature search followed by an expert-driven literature review, was used to systematically screen 39 chemicals for lack of DNT effect. Expert panel members evaluated the scientific robustness of relevant studies to inform chemical categorizations. Following review, the panel discussed each chemical and made categorical determinations of "Favorable", "Not Favorable", or "Indeterminate" reflecting acceptance, lack of suitability, or uncertainty given specific limitations and considerations, respectively. The panel determined that 10, 22, and 7 chemicals met the criteria for "Favorable", "Not Favorable", and "Indeterminate", for use as negatives in a DNT NAM evaluation set. Ultimately, this approach not only supports DNT NAM performance evaluation but also highlights challenges in identifying large numbers of negative DNT chemicals.