Gut metabolic changes during pregnancy reveal the importance of gastrointestinal region in sample collection.

INTRODUCTION: Studies of gastrointestinal physiology and the gut microbiome often consider the influence of intestinal region on experimental endpoints. However, this same consideration is not often applied to the gut metabolome. Understanding the contribution of gut regionality may be critically important to the rapidly changing metabolic environments, such as during pregnancy. OBJECTIVES: We sought to characterize the difference in the gut metabolome in pregnant mice stratified by region-comparing the small intestine, cecum, and feces. Pre-pregnancy feces were collected to understand the influence of pregnancy on the fecal metabolome. METHODS: Feces were collected from CD-1 female mice before breeding. On gestation day (GD) 18, gut contents were collected from the small intestine, cecum, and descending colon. Metabolites were analyzed with LC-MS/MS using the Biocrates MetaboINDICATOR™ MxP® Quant 500 kit. RESULTS: Of the 104 small molecule metabolites meeting analysis criteria, we found that 84 (81%) were differentially abundant based on gut region. The most significant regional comparison observed was between the cecum and small intestines, with 52 (50%) differentially abundant metabolites. Pregnancy itself altered 41 (39.4%) fecal small molecule metabolites. CONCLUSIONS: The regional variation observed in the gut metabolome are likely due to the microbial and physiological differences between the different parts of the intestines. Additionally, pregnancy impacts the fecal metabolome, which may be due to evolving needs of both the dam and fetus.

Bypassing the brain barriers: upregulation of serum miR-495 and miR-543-3p reflects thyroid-mediated developmental neurotoxicity in the rat.

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.

Ammonium perchlorate: serum dosimetry, neurotoxicity, and resilience of the neonatal rat thyroid system.

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.

Dose Response, Dosimetric, and Metabolic Evaluations of Replacement PFAS Perfluoro-(2,5,8-trimethyl-3,6,9-trioxadodecanoic) Acid (HFPO-TeA).

Few studies are available on the environmental and toxicological effects of perfluoroalkyl ether carboxylic acids (PFECAs), such as GenX, which are replacing legacy PFAS in manufacturing processes. To collect initial data on the toxicity and toxicokinetics of a longer-chain PFECA, male and female Sprague Dawley rats were exposed to perfluoro-(2,5,8-trimethyl-3,6,9-trioxadodecanoic) acid (HFPO-TeA) by oral gavage for five days over multiple dose levels (0.3-335.2 mg/kg/day). Clinically, we observed mortality at doses >17 mg/kg/day and body weight changes at doses ≤17 mg/kg/day. For the 17 mg/kg/day dose level, T3 and T4 thyroid hormone concentrations were significantly decreased (p < 0.05) from controls and HFPO-TeA plasma concentrations were significantly different between sexes. Non-targeted analysis of plasma and in vitro hepatocyte assay extractions revealed the presence of another GenX oligomer, perfluoro-(2,5-dimethyl-3,6-dioxanonanoic) acid (HFPO-TA). In vitro to in vivo extrapolation (IVIVE) parameterized with in vitro toxicokinetic data predicted steady-state blood concentrations that were within seven-fold of those observed in the in vivo study, demonstrating reasonable predictivity. The evidence of thyroid hormone dysregulation, sex-based differences in clinical results and dosimetry, and IVIVE predictions presented here suggest that the replacement PFECA HFPO-TeA induces a complex and toxic exposure response in rodents.

Structural Malformations in the Neonatal Rat Brain Accompany Developmental Exposure to Ammonium Perchlorate.

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.

Thyroid hormone action controls multiple components of cell junctions at the ventricular zone in the newborn rat brain.

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 αvß3 and isoforms of both thyroid receptors (TRα/TRß) 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.

Thyroid Disruptors: Extrathyroidal Sites of Chemical Action and Neurodevelopmental Outcome-An Examination Using Triclosan and Perfluorohexane Sulfonate.

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 (50 mg/kg/day) and the antimicrobial Triclosan (300 mg/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.

A transient window of hypothyroidism alters neural progenitor cells and results in abnormal brain development.

Cortical heterotopias are clusters of ectopic neurons in the brain and are associated with neurodevelopmental disorders like epilepsy and learning disabilities. We have previously characterized the robust penetrance of a heterotopia in a rat model, induced by thyroid hormone (TH) disruption during gestation. However, the specific mechanism by which maternal TH insufficiency results in this birth defect remains unknown. Here we first determined the developmental window susceptible to endocrine disruption and describe a cellular mechanism responsible for heterotopia formation. We show that five days of maternal goitrogen treatment (10 ppm propylthiouracil) during the perinatal period (GD19-PN2) induces a periventricular heterotopia in 100% of the offspring. Beginning in the early postnatal brain, neurons begin to aggregate near the ventricles of treated animals. In parallel, transcriptional and architectural changes of this region were observed including decreased Sonic hedgehog (Shh) expression, abnormal cell adhesion, and altered radial glia morphology. As the ventricular epithelium is juxtaposed to two sources of brain THs, the cerebrospinal fluid and vasculature, this progenitor niche may be especially susceptible to TH disruption. This work highlights the spatiotemporal vulnerabilities of the developing brain and demonstrates that a transient period of TH perturbation is sufficient to induce a congenital abnormality.

Developmental Thyroid Hormone Insufficiency Induces a Cortical Brain Malformation and Learning Impairments: A Cross-Fostering Study.

Thyroid hormones (THs) are essential for brain development, but few rodent models exist that link TH inefficiency to apical neurodevelopmental endpoints. We have previously described a structural anomaly, a heterotopia, in the brains of rats treated in utero with propylthiouracil (PTU). However, how the timing of an exposure relates to this birth defect is unknown. This study seeks to understand how various temporal treatments of the mother relates to TH insufficiency and adverse neurodevelopment of the offspring. Pregnant rats were exposed to PTU (0 or 3 ppm) through the drinking water from gestational day 6 until postnatal day (PN) 14. On PN2 a subset of pups was cross-fostered to a dam of the opposite treatment, to create 4 conditions: pups exposed to PTU prenatally, postnatally, during both periods, or not at all (control). Both PTU and TH concentrations were characterized in the mother and offspring over time, to capture the dynamics of a developmental xenobiotic exposure. Brains of offspring were examined for heterotopia presence and severity, and adult littermates were assessed for memory impairments. Heterotopia were observed under conditions of prenatal exposure, and its severity increased in animals in the most prolonged exposure group. This malformation was also permanent, but not sex biased. In contrast, behavioral impairments were limited to males, and only in animals exposed to PTU during both the gestational and postnatal periods. This suggests a distinct TH-dependent etiology for both phenotypes, and illustrates how timing of hypothyroxinemia can induce abnormal brain structure and function.