A high throughput screening assay for human Thyroperoxidase inhibitors.

Rapid, human relevant assays are needed to assess potential hazards of the many chemicals in commerce. An assay of thyroid peroxidase (TPO) inhibition, using the substrate Amplex Ultra Red, was recently adapted for human TPO (AUR-hTPO). We tested a large number (788) of chemicals through this AUR-hTPO assay and compared performance with published results from an assay using enzyme from rat thyroid microsomes (AUR-rTPO). Coded chemicals, from the US EPA ToxCast Inventory, were tested in a tiered approach: 1) Initial screening at a single concentration; 2) Potency estimation for active chemicals with multiple concentrations; 3) Screening active chemicals for the non-specific activity. The assay gave consistent results for positive chemical methimazole and several positive and negative reference chemicals. hTPO inhibition was observed for 190 chemicals reported as positive in rTPO. Of these, 158 showed no confounding activity (interference due to fluorescence or non-specific protein inhibition). Comparison of all result with rTPO data and with evidence of TPO inhibition found in the literature suggest that the current assay has a higher rate of false negative but a much lower rate of false positive compared with the rTPO screen. These findings underscore the effectiveness of the AUR assay, using hTPO enzyme from engineered cell lines, to identify moderate to strong inhibitors but some improvements may be needed to detect weak TPO inhibitors.

House dust-derived mixtures of organophosphate esters alter the phenotype, function, transcriptome, and lipidome of KGN human ovarian granulosa cells.

Organophosphate esters (OPEs), used as flame retardants and plasticizers, are present ubiquitously in the environment. Previous studies suggest that exposure to OPEs is detrimental to female fertility in humans. However, no experimental information is available on the effects of OPE mixtures on ovarian granulosa cells, which play essential roles in female reproduction. We used high-content imaging to investigate the effects of environmentally relevant OPE mixtures on KGN human granulosa cell phenotypes. Perturbations to steroidogenesis were assessed using ELISA and qRT-PCR. A high-throughput transcriptomic approach, TempO-Seq, was used to identify transcriptional changes in a targeted panel of genes. Effects on lipid homeostasis were explored using a cholesterol assay and global lipidomic profiling. OPE mixtures altered multiple phenotypic features of KGN cells, with triaryl OPEs in the mixture showing higher potencies than other mixture components. The mixtures increased basal production of steroid hormones; this was mediated by significant changes in the expression of critical transcripts involved in steroidogenesis. Further, the total-OPE mixture disrupted cholesterol homeostasis and the composition of intracellular lipid droplets. Exposure to complex mixtures of OPEs, similar to those found in house dust, may adversely affect female reproductive health by altering a multitude of phenotypic and functional endpoints in granulosa cells. This study provides novel insights into the mechanisms of actions underlying the toxicity induced by OPEs and highlights the need to examine the effects of human relevant chemical mixtures.

Effects of the pesticide deltamethrin on high fat diet-induced obesity and insulin resistance in male mice.

Worldwide, rates of metabolic diseases are rapidly increasing and environmental exposure to pesticides, pollutants and/or other chemicals may play a role. Reductions in Brown Adipose Tissue (BAT) thermogenesis, mediated in part by uncoupling protein 1 (Ucp1), are associated with metabolic diseases. In the current study, we investigated whether the pesticide deltamethrin (0.01-1 mg/kg bw/day) incorporated into a high-fat diet and fed to mice housed at either room temperature (21°C) or thermoneutrality (29°C) would suppress BAT activity and accelerate the development of metabolic disease. Importantly, thermoneutrality allows for more accurate modeling of human metabolic disease. We found that, 0.01 mg/kg bw/day of deltamethrin induced weight loss, improved insulin sensitivity and increased energy expenditure, effects that were associated with increases in physical activity. In contrast, exposure to 0.1 and 1 mg/kg bw/day deltamethrin had no effect on any of the parameters examined. Deltamethrin treatment in mice did not alter molecular markers of BAT thermogenesis, despite observing suppression of UCP1 expression in cultured brown adipocytes. These data indicate that while deltamethrin inhibits UCP1 expression in vitro, 16wks exposure does not alter BAT thermogenesis markers nor exacerbates the development of obesity and insulin resistance in mice.

The effects of cognitive impairment on the multi-scale dynamics of standing postural control during visual-search in older men.

Background: Cognitive impairment disrupts postural control, particularly when standing while performing an unrelated cognitive task (i.e., dual-tasking). The temporal dynamics of standing postural sway are "complex," and such complexity may reflect the capacity of the postural control system to adapt to task demands. We aimed to characterize the impact of cognitive impairment on such sway complexity in older adults. Methods: Forty-nine older adult males (Alzheimer's disease (AD): n = 21; mild cognitive impairment (MCI): n = 13; cognitively-intact: n = 15) completed two 60-s standing trials in each of single-task and visual-search dual-task conditions. In the dual-task condition, participants were instructed to count the frequency of a designated letter in a block of letters projected on screen. The sway complexity of center-of-pressure fluctuations in anterior-posterior (AP) and medial-lateral (ML) direction was quantified using multiscale entropy. The dual-task cost to complexity was obtained by calculating the percent change of complexity from single- to dual-task condition. Results: Repeated-measures ANOVAs revealed significant main effects of group (F > 4.8, p < 0.01) and condition (F = 7.7, p < 0.007) on both AP and ML sway complexity; and significant interaction between group and condition for ML sway complexity (F = 3.7, p = 0.03). The AD group had the lowest dual-task ML complexity, as well as greater dual-task cost to ML (p = 0.03) compared to the other two groups. Visual-search task accuracy was correlated with ML sway complexity in the dual-task condition (r = 0.42, p = 0.007), and the dual-task cost to ML sway complexity (r = 0.39, p = 0.01) across all participants. Conclusion: AD-related cognitive impairment was associated with a greater relative reduction in postural sway complexity from single- to dual-tasking. Sway complexity appears to be sensitive to the impact of cognitive impairment on standing postural control.

Microplastics exposure: implications for human fertility, pregnancy and child health.

Plastics found in our everyday environment are becoming an increasing concern for individual and population-level health, and the extent of exposure and potential toxic effects of these contaminants on numerous human organ systems are becoming clear. Microplastics (MPs), tiny plastic particles, appear to have many of the same biological effects as their plastic precursors and have the compounded effect of potential accumulation in different organs. Recently, microplastic accumulation was observed in the human placenta, raising important questions related to the biological effects of these contaminants on the health of pregnancies and offspring. These concerns are particularly heightened considering the developmental origins of health and disease (DOHaD) framework, which postulates that in utero exposure can programme the lifelong health of the offspring. The current review examines the state of knowledge on this topic and highlights important avenues for future investigation.

Chronic exposure to synthetic food colorant Allura Red AC promotes susceptibility to experimental colitis via intestinal serotonin in mice.

Chemicals in food are widely used leading to significant human exposure. Allura Red AC (AR) is a highly common synthetic colorant; however, little is known about its impact on colitis. Here, we show chronic exposure of AR at a dose found in commonly consumed dietary products exacerbates experimental models of colitis in mice. While intermittent exposure is more akin to a typical human exposure, intermittent exposure to AR in mice for 12 weeks, does not influence susceptibility to colitis. However, exposure to AR during early life primes mice to heightened susceptibility to colitis. In addition, chronic exposure to AR induces mild colitis, which is associated with elevated colonic serotonin (5-hydroxytryptamine; 5-HT) levels and impairment of the epithelial barrier function via myosin light chain kinase (MLCK). Importantly, chronic exposure to AR does not influence colitis susceptibility in mice lacking tryptophan hydroxylase 1 (TPH1), the rate limiting enzyme for 5-HT biosynthesis. Cecal transfer of the perturbed gut microbiota by AR exposure worsens colitis severity in the recipient germ-free (GF) mice. Furthermore, chronic AR exposure elevates colonic 5-HT levels in naïve GF mice. Though it remains unknown whether AR has similar effects in humans, our study reveals that chronic long-term exposure to a common synthetic colorant promotes experimental colitis via colonic 5-HT in gut microbiota-dependent and -independent pathway in mice.

Increased gut serotonin production in response to bisphenol A structural analogs may contribute to their obesogenic effects.

Obesogens are synthetic, environmental chemicals that can disrupt endocrine control of metabolism and contribute to the risk of obesity and metabolic disease. Bisphenol A (BPA) is one of the most studied obesogens. There is considerable evidence that BPA exposure is associated with weight gain, increased adiposity, poor blood glucose control, and nonalcoholic fatty liver disease in animal models and human populations. Increased usage of structural analogs of BPA has occurred in response to legislation banning their use in some commercial products. However, BPA analogs may also cause some of the same metabolic impairments because of common mechanisms of action. One key effector that is altered by BPA and its analogs is serotonin, however, it is unknown if BPA-induced changes in peripheral serotonin pathways underlie metabolic perturbations seen with BPA exposure. Upon ingestion, BPA and its analogs act as endocrine-disrupting chemicals in the gastrointestinal tract to influence serotonin production by the gut, where over 95% of serotonin is produced. The purpose of this review is to evaluate how BPA and its analogs alter gut serotonin regulation and then discuss how disruption of serotonergic networks influences host metabolism. We also provide evidence that BPA and its analogs enhance serotonin production in gut enterochromaffin cells. Taken together, we propose that BPA and many BPA analogs represent endocrine-disrupting chemicals that can influence host metabolism through the endogenous production of gut-derived factors, such as serotonin.

Disruption by stealth - Interference of endocrine disrupting chemicals on hormonal crosstalk with thyroid axis function in humans and other animals.

Thyroid hormones (THs) are important regulators of growth, development, and homeostasis of all vertebrates. There are many environmental contaminants that are known to disrupt TH action, yet their mechanisms are only partially understood. While the effects of Endocrine Disrupting Chemicals (EDCs) are mostly studied as "hormone system silos", the present critical review highlights the complexity of EDCs interfering with TH function through their interactions with other hormonal axes involved in reproduction, stress, and energy metabolism. The impact of EDCs on components that are shared between hormone signaling pathways or intersect between pathways can thus extend beyond the molecular ramifications to cellular, physiological, behavioral, and whole-body consequences for exposed organisms. The comparatively more extensive studies conducted in mammalian models provides encouraging support for expanded investigation and highlight the paucity of data generated in other non-mammalian vertebrate classes. As greater genomics-based resources become available across vertebrate classes, better identification and delineation of EDC effects, modes of action, and identification of effective biomarkers suitable for HPT disruption is possible. EDC-derived effects are likely to cascade into a plurality of physiological effects far more complex than the few variables tested within any research studies. The field should move towards understanding a system of hormonal systems' interactions rather than maintaining hormone system silos.

The pesticide chlorpyrifos promotes obesity by inhibiting diet-induced thermogenesis in brown adipose tissue.

Obesity results from a caloric imbalance between energy intake, absorption and expenditure. In both rodents and humans, diet-induced thermogenesis contributes to energy expenditure and involves the activation of brown adipose tissue (BAT). We hypothesize that environmental toxicants commonly used as food additives or pesticides might reduce BAT thermogenesis through suppression of uncoupling protein 1 (UCP1) and this may contribute to the development of obesity. Using a step-wise screening approach, we discover that the organophosphate insecticide chlorpyrifos suppresses UCP1 and mitochondrial respiration in BAT at concentrations as low as 1 pM. In mice housed at thermoneutrality and fed a high-fat diet, chlorpyrifos impairs BAT mitochondrial function and diet-induced thermogenesis, promoting greater obesity, non-alcoholic fatty liver disease (NAFLD) and insulin resistance. This is associated with reductions in cAMP; activation of p38MAPK and AMPK; protein kinases critical for maintaining UCP1 and mitophagy, respectively in BAT. These data indicate that the commonly used pesticide chlorpyrifos, suppresses diet-induced thermogenesis and the activation of BAT, suggesting its use may contribute to the obesity epidemic.

In Utero and Lactational Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants Induces a Premature Development of the Mammary Glands.

In utero and prepubertal development of the mammary glands occurs minimally in a hormone independent manner until puberty where maturation of the hypothalamic-pituitary-gonadal axis drives an extensive remodeling. Nevertheless, because the immature glands contain functional hormone receptors, they are especially vulnerable to the effects of endocrine disruptors, such as brominated flame retardants (BFRs). BFRs are widespread chemicals added to household objects to reduce their flammability, and to which humans are ubiquitously exposed. We previously reported that in utero and lactational exposure to BFRs resulted in an impaired mammary gland development in peripubertal animals. Here, we assessed whether BFR-induced disruption of mammary gland development could manifest earlier in life. Dams were exposed prior to mating until pups' weaning to a BFR mixture (0, 0.06, 20, or 60 mg/kg/day) formulated according to levels found in house dust. The mammary glands of female offspring were collected at weaning. Histo-morphological analyses showed that exposure to 0.06 mg/kg/day accelerates global epithelial development as demonstrated by a significant increase in total epithelial surface area, associated with a tendency to increase of the ductal area and thickness, and of lumen area. Significant increases of the Ki67 cell proliferation index and of the early apoptotic marker cleaved caspase-9 were also observed, as well as an upward trend in the number of thyroid hormone receptor α1 positive cells. These molecular, histologic, and morphometric changes are suggestive of accelerated pubertal development. Thus, our results suggest that exposure to an environmentally relevant mixture of BFRs induces precocious development of the mammary gland.

In Utero and Lactational Exposure to Flame Retardants Disrupts Rat Ovarian Follicular Development and Advances Puberty.

Brominated flame retardants (BFRs), including polybrominated diphenyl ethers and hexabromocyclododecane, leach out from consumer products into the environment. Exposure to BFRs has been associated with effects on endocrine homeostasis. To test the hypothesis that in utero and lactational exposure to BFRs may affect the reproductive system of female offspring, adult female Sprague Dawley rats were fed diets formulated to deliver nominal doses (0, 0.06, 20, or 60 mg/kg/day) of a BFR dietary mixture mimicking the relative congener levels in house dust from prior to mating until weaning. Vaginal opening and the day of first estrus occurred at a significantly earlier age among offspring from the 20 mg/kg/day BFR group, indicating that the onset of puberty was advanced. Histological analysis of ovaries from postnatal day 46 offspring revealed an increase in the incidence of abnormal follicles. A toxicogenomic analysis of ovarian gene expression identified upstream regulators, including HIF1A, CREB1, EGF, the ß-estradiol, and PPARA pathways, predicted to be downregulated in the 20 or 60 mg/kg/day group and to contribute to the gene expression patterns observed. Thus, perinatal exposure to BFRs dysregulated ovarian folliculogenesis and signaling pathways that are fundamental for ovarian function in the adult.

A rapid assay of human thyroid peroxidase activity.

Impaired synthesis or action of thyroid hormones (THs) during critically sensitive periods of development can have long term adverse effects on health. Development of rapid assays to identify chemicals that impair THs physiology is an important goal for reducing risks from chemical use. Thyroid peroxidase (TPO) is a key enzyme regulating THs synthesis in thyroid gland and a vulnerable target for chemicals that disrupt THs synthesis. To develop a human-relevant, rapid assay for TPO inhibition, we have engineered two cell lines (CHO and LentiX- 293) to express active human TPO (hTPO) enzyme and applied them in a recently-described assay using a stable fluorescent product (Amplex UltraRed). Assay performance was assessed by comparing activity of 19 reference chemicals with known strong, weak or no TPO inhibitory activity. The assay using hTPO from either cell line consistently identified the relative potency of strong to moderate inhibitors and chemicals known to be inactive. Results were less consistent for chemicals reported to be weak inhibitors of rodent TPO, possibly suggesting some species specificity. Our studies support the use of hTPO from stably transfected cell lines to substitute for animal-derived thyroid microsomes for rapid high throughput screening assays to identify and characterize TPO inhibitors.

Toxicity of Flame Retardant Isopropylated Triphenyl Phosphate: Liver, Adrenal, and Metabolic Effects.

The use of organophosphates phosphate flame retardants, particularly isopropylated triphenyl phosphate (IPTPP), has increased in recent years as replacements for polybrominated diphenyl ethers. This is despite limited understanding of the hazards of IPTPP. To examine the general and endocrine toxicity of IPTPP, adult Wistar rats were fed for 90 days on diets containing IPTPP estimated to deliver daily doses of 5 to 140 mg/kg/d. Exposure to IPTPP caused a dose-related increase in liver and adrenal gland weight in both sexes. Cells in the zona fasciculate (ZF) of the adrenal cortex were observed to be filled with droplets that stained with Nile red, suggesting they contained neutral lipid. Despite marked structural changes, there was no change in basal or stress-induced serum levels of their major secreted ZF product corticosterone (B), suggesting cell function was not altered. There were no effects on responses to glucose or insulin challenge, but serum levels of fructosamine were elevated by IPTPP exposure, suggesting a slight tendency of exposed animals to be hyperglycemic. Serum levels of total cholesterol and high-density lipoprotein cholesterol were significantly elevated in both sexes at the 2 highest doses. This study demonstrates that IPTPP exposure causes hypertrophy and neutral lipid accumulation in adrenal cortex ZF cells but does not result in impaired B production.

Development of a non-radioactive screening assay to detect chemicals disrupting the human sodium iodide symporter activity.

Adequate concentration of iodide ions within thyroid epithelial cells, which is mediated by the sodium iodide symporter (NIS), is essential for proper thyroid hormone synthesis. Inhibition of NIS activity represents a potential mechanism by which goitrogens/toxicants can disrupt thyroid hormone physiology. It is necessary to develop a rapid, simple, inexpensive and sensitive screening assay to identify chemicals affecting NIS function. The current study compares the sensitivities of non-radioactive Sandell-Kolthoff (SK) reaction and radioactive iodide uptake (RAIU) in a previously described NIS assay. The EPAhNIS cell line (HEK293T stably transfected to over-express the human NIS) was tested with the reference NIS inhibitor (sodium perchlorate) across multiple log concentration range. The results from SK reaction in EPAhNIS cells showed similar performance to published RAIU results from the same cell line, in terms of assay screening coefficient (Z') and variability (CV). Results from the reference chemicals tested in EPAhNIS cells revealed that SK reaction yielded IC50 and selectivity scores consistent with those observed for RAIU. However, RAIU seems marginally more sensitive than the SK reaction, as RAIU consistently detected weaker NIS inhibitors among the test chemicals. We developed a second hNIS assay based on the MCF-7 cell line. Applying reference anions and chemicals to MCF7hNIS cells, we found that in comparison with results from EPAhNIS cells, the SK reaction with MCF7hNIS: 1) yielded similar Z' and CV; 2) had similar IC50 and selectivity scores for reference chemicals; 3) identified more NIS inhibitors among reference chemicals than SK reaction, but less than the RAIU assay in EPAhNIS cells. In conclusion, the SK reaction can be used with both EPAhNIS and MCF7hNIS cells to measure iodide uptake and identify NIS inhibitors, except for those presenting an extremely weak potency.

Physical Growth, Body Scale, and Perceptual-Motor Development.

In this chapter we consider from the theoretical framework of the ecological approach to perception and action, the relations between physical growth and body scale in the context of children's perceptual-motor development. Body scale and the timescale of its change through growth are shown to relate to the emergence and dissolution of the fundamental skills in infancy, the perception of what an environment affords functionally for action, together with the emergent pattern of movement coordination. A central issue in typical and atypical motor development is the mapping of the timescale of adaptive change in the acquisition of perceptual-motor skill to the accompanying timescale of change in physical growth.

Developmental coordination disorder and its cause: The road less travelled.

We critically review the research literature that seeks to focus on the possible cause of children diagnosed with developmental coordination disorder (DCD). In so doing we contrast the traditional information processing (IP) approach as a model to explain the causal factors that account for the motor deficits present in children with DCD, with a dynamical systems (DS) account which argues that coordination deficits in children with DCD is less to do with problems of poor internal models (a cornerstone of IP theory) and more with a degrading of perception-action coupling. We review and comment on the extant empirical data and conclusions of both approaches. We conclude that the data for an IP explanation is weak and a reconsideration of DCD is in order with respect to the underlying cause of this issue.

From the Cover: Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants Decreased p-ß-Cateninser675 Expression and Its Interaction With E-Cadherin in the Mammary Glands of Lactating Rats.

Proper mammary gland development and function require precise hormonal regulation and bidirectional cross talk between cells provided by means of paracrine factors as well as intercellular junctions; exposure to environmental endocrine disruptors can disturb these processes. Exposure to one such family of chemicals, the brominated flame retardants (BFRs), is ubiquitous. Here, we tested the hypothesis that BFR exposures disrupt signaling pathways and intercellular junctions that control mammary gland development. Before mating, during pregnancy and throughout lactation, female Sprague-Dawley rats were fed diets containing that BFR mixture based on house dust, delivering nominal exposures of BFR of 0 (control), 0.06, 20, or 60 mg/kg/d. Dams were euthanized and mammary glands collected on postnatal day 21. BFR exposure had no significant effects on mammary gland/body weight ratios or the levels of proteins involved in milk synthesis, epithelial-mesenchymal transition, cell-cell interactions, or hormone signalling. However, BFR exposure (0.06 mg/kg/d) down-regulated phospho-ser675 ß-catenin (p-ß-catSer675) levels in the absence of any effect on total ß-catenin levels. Levels of p-CREB were also down-regulated, suggesting that PKA inhibition plays a role. p-ß-catSer675 co-localized with ß-catenin at the mammary epithelial cell membrane, and its expression was decreased in animals from the 0.06 and 20 mg/kg/d BFR treatment groups. Although ß-Catenin signaling was not affected by BFR exposure, the interaction between p-ß-catSer675 and E-cadherin was significantly reduced. Together, our results demonstrate that exposure to an environmentally relevant mixture of BFR during pregnancy and lactation decreases p-ß-catser675 at cell adhesion sites, likely in a PKA-dependant manner, altering mammary gland signaling.