Expanded high-throughput screening and chemotype-enrichment analysis of the phase II: e1k ToxCast library for human sodium-iodide symporter (NIS) inhibition.

The sodium-iodide symporter (NIS) mediates the uptake of iodide into the thyroid. Inhibition of NIS function by xenobiotics has been demonstrated to suppress circulating thyroid hormones and perturb related physiological functions. Until recently, few environmental chemicals had been screened for NIS inhibition activity. We previously screened over 1000 chemicals from the ToxCast Phase II (ph1v2 and ph2) libraries using an in vitro radioactive iodide uptake (RAIU) with the hNIS-HEK293T cell line to identify NIS inhibitors. Here, we broaden the chemical space by expanding screening to include the ToxCast e1k library (804 unique chemicals) with initial screening for RAIU at 1 × 10-4 M. Then 209 chemicals demonstrating > 20% RAIU inhibition were further tested in multiple-concentration, parallel RAIU and cell viability assays. This identified 55 chemicals as active, noncytotoxic RAIU inhibitors. Further cytotoxicity-adjusted potency scoring (with NaClO4 having a reference score of 200) revealed five chemicals with moderate to strong RAIU inhibition (scored > 100). These data were combined with our previous PhII screening data to produce binary hit-calls for ~ 1800 unique chemicals (PhII + e1k) with and without cytotoxicity filtering. Results were analyzed with a ToxPrint chemotype-enrichment workflow to identify substructural features significantly enriched in the NIS inhibition hit-call space. We assessed the applicability of enriched PhII chemotypes to prospectively predict NIS inhibition in the e1k dataset. Chemotype enrichments derived for the combined ~ 1800 dataset also identified additional enriched features, as well as chemotypes affiliated with cytotoxicity. These enriched chemotypes provide important new information that can support future data interpretation, structure-activity relationship, chemical use, and regulation.

High-Throughput Screening and Quantitative Chemical Ranking for Sodium-Iodide Symporter Inhibitors in ToxCast Phase I Chemical Library.

Thyroid uptake of iodide via the sodium-iodide symporter (NIS) is the first step in the biosynthesis of thyroid hormones that are critical for health and development in humans and wildlife. Despite having long been a known target of endocrine disrupting chemicals such as perchlorate, information regarding NIS inhibition activity is still unavailable for the vast majority of environmental chemicals. This study applied a previously validated high-throughput approach to screen for NIS inhibitors in the ToxCast phase I library, representing 293 important environmental chemicals. Here 310 blinded samples were screened in a tiered-approach using an initial single-concentration (100 µM) radioactive-iodide uptake (RAIU) assay, followed by 169 samples further evaluated in multi-concentration (0.001 µM-100 µM) testing in parallel RAIU and cell viability assays. A novel chemical ranking system that incorporates multi-concentration RAIU and cytotoxicity responses was also developed as a standardized method for chemical prioritization in current and future screenings. Representative chemical responses and thyroid effects of high-ranking chemicals are further discussed. This study significantly expands current knowledge of NIS inhibition potential in environmental chemicals and provides critical support to U.S. EPA's Endocrine Disruptor Screening Program (EDSP) initiative to expand coverage of thyroid molecular targets, as well as the development of thyroid adverse outcome pathways (AOPs).

Effects of chronic exposure to triclosan on reproductive and thyroid endpoints in the adult Wistar female rat.

Triclosan (TCS), an antibacterial, has been shown to be an endocrine disruptor in the rat. Previously, subchronic TCS treatment to female rats was found to advance puberty and potentiate the effect of ethinyl estradiol (EE) on uterine growth when EE and TCS were co-administered prior to weaning. In the pubertal study, a decrease in serum thyroxine (T4) concentrations with no significant change in serum thyroid-stimulating hormone (TSH) was also observed. The purpose of the present study was to further characterize the influence of TCS on the reproductive and thyroid axes of the female rat using a chronic exposure regimen. Female Wistar rats were exposed by oral gavage to vehicle control, EE (1 µg/kg), or TCS (2.35, 4.69, 9.375 or 37.5 mg/kg) for 8 months and estrous cyclicity monitored. Although a divergent pattern of reproductive senescence appeared to emerge from 5 to 11 months of age between controls and EE-treated females, no significant difference in cyclicity was noted between TCS-treated and control females. A higher % control females displayed persistent diestrus (PD) by the end of the study, whereas animals administered with positive control (EE) were predominately persistent estrus (PE). Thyroxine concentration was significantly decreased in TCS-administered 9.375 and 37.5 mg/kg groups, with no marked effects on TSH levels, thyroid tissue weight, or histology. Results demonstrate that a long-term exposure to TCS did not significantly alter estrous cyclicity or timing of reproductive senescence in females but suppressed T4 levels at a lower dose than previously observed.

Repetitive sequence variations in the promoter region of the adhesin-encoding gene sabA of Helicobacter pylori affect transcription.

The pathogenesis of diseases elicited by the gastric pathogen Helicobacter pylori is partially determined by the effectiveness of adaptation to the variably acidic environment of the host stomach. Adaptation includes appropriate adherence to the gastric epithelium via outer membrane protein adhesins such as SabA. The expression of sabA is subject to regulation via phase variation in the promoter and coding regions as well as repression by the two-component system ArsRS. In this study, we investigated the role of a homopolymeric thymine [poly(T)] tract -50 to -33 relative to the sabA transcriptional start site in H. pylori strain J99. We quantified sabA expression in H. pylori J99 by quantitative reverse transcription-PCR (RT-PCR), demonstrating significant changes in sabA expression associated with experimental manipulations of poly(T) tract length. Mimicking the length increase of this tract by adding adenines instead of thymines had similar effects, while the addition of other nucleotides failed to affect sabA expression in the same manner. We hypothesize that modification of the poly(T) tract changes DNA topology, affecting regulatory protein interaction(s) or RNA polymerase binding efficiency. Additionally, we characterized the interaction between the sabA promoter region and ArsR, a response regulator affecting sabA expression. Using recombinant ArsR in electrophoretic mobility shift assays (EMSA), we localized binding to a sequence with partial dyad symmetry -20 and +38 relative to the sabA +1 site. The control of sabA expression by both ArsRS and phase variation at two distinct repeat regions suggests the control of sabA expression is both complex and vital to H. pylori infection.

Association of proinflammatory cytokines and islet resident leucocytes with islet dysfunction in type 2 diabetes.

AIMS/HYPOTHESIS: Chronic inflammation in type 2 diabetes is proposed to affect islets as well as insulin target organs. However, the nature of islet inflammation and its effects on islet function in type 2 diabetes remain unclear. Moreover, the immune cell profiles of human islets in healthy and type 2 diabetic conditions are undefined. We aimed to investigate the correlation between proinflammatory cytokine expression, islet leucocyte composition and insulin secretion in type 2 diabetic human islets. METHODS: Human islets from organ donors with or without type 2 diabetes were studied. First and second phases of glucose-stimulated insulin secretion were determined by perifusion. The expression of inflammatory markers was obtained by quantitative PCR. Immune cells within human islets were analysed by FACS. RESULTS: Type 2 diabetic islets, especially those without first-phase insulin secretion, displayed higher CCL2 and TNFa expression than healthy islets. CD45(+) leucocytes were elevated in type 2 diabetic islets, to a greater extent in moderately functional type 2 diabetic islets compared with poorly functional ones, and corresponded with elevated ALOX12 but not with CCL2 or TNFa expression. T and B lymphocytes and CD11c(+) cells were detectable within both non-diabetic and type 2 diabetic islet leucocytes. Importantly, the proportion of B cells was significantly elevated within type 2 diabetic islets. CONCLUSIONS/INTERPRETATION: Elevated total islet leucocyte content and proinflammatory mediators correlated with islet dysfunction, suggesting that heterogeneous insulitis occurs during the development of islet dysfunction in type 2 diabetes. In addition, the altered B cell content highlights a potential role for the adaptive immune response in islet dysfunction.

Drinking water sources, quality, and associated health outcomes in Appalachian Virginia: A risk characterization study in two counties.

OBJECTIVES: In the US, violations of drinking water regulations are highest in lower-income rural areas overall, and particularly in Central Appalachia. However, data on drinking water use, quality, and associated health outcomes in rural Appalachia are limited. We sought to assess public and private drinking water sources and associated risk factors for waterborne pathogen exposures for individuals living in rural regions of Appalachian Virginia. METHODS: We administered surveys and collected tap water, bottled water, and saliva samples in lower-income households in two adjacent rural counties in southwest Virginia (bordering Kentucky and Tennessee). Water samples were tested for pH, temperature, conductivity, total coliforms, E. coli, free chlorine, nitrate, fluoride, heavy metals, and specific pathogen targets. Saliva samples were analyzed for antibody responses to potentially waterborne infections. We also shared water analysis results with households. RESULTS: We enrolled 33 households (83 individuals), 82% (n = 27) with utility-supplied water and 18% with private wells (n = 3) or springs (n = 3). 58% (n = 19) reported household incomes of <$20,000/year. Total coliforms were detected in water samples from 33% (n = 11) of homes, E. coli in 12%, all with wells or springs (n = 4), and Aeromonas, Campylobacter, and Enterobacter in 9%, all spring water (n = 3). Diarrhea was reported for 10% of individuals (n = 8), but was not associated with E. coli detection. 34% (n = 15) of saliva samples had detectable antibody responses for Cryptosporidium spp., C. jejuni, and Hepatitis E. After controlling for covariates and clustering, individuals in households with septic systems and straight pipes had significantly higher likelihoods of antibody detection (risk ratios = 3.28, 95%CI = 1.01-10.65). CONCLUSIONS: To our knowledge, this is the first study to collect and analyze drinking water samples, saliva samples, and reported health outcome data from low-income households in Central Appalachia. Our findings indicate that utility-supplied water in this region was generally safe, and individuals in low-income households without utility-supplied water or sewerage have higher exposures to waterborne pathogens.

Evaluation of hydroxyatrazine in the endocrine disruptor screening and testing program's male and female pubertal protocols.

Two critical components of the validation of any in vivo screening assay are to demonstrate sensitivity and specificity. Although the Endocrine Disruptor Screening Program's Tier 1 Male and Female Pubertal Protocols have been shown to be sensitive assays for the detection of weak endocrine disrupting chemicals (EDCs), there are concerns that the assays lack specificity for EDC effects when a chemical induces systemic toxicity. A lack of specificity, or the ability to correctly identify an inactive or "negative" chemical, would increase the probability of identifying false positives. Here, we orally exposed rats to hydroxyatrazine (OH-ATR), a biotransformation by-product of the chlorotriazine herbicides that produced nephrotoxicity following a 13-week dietary exposure. Based on a previous study in our laboratory, males were dosed with 11.4 to 183.4 mg/kg OH-ATR and females were dosed with 45.75 to 183.4 mg/kg OH-ATR. Following exposure in both sexes, there was a dose-response increase in mean kidney weights and the incidence and severity of kidney lesions. These lesions included the deposition of mineralized renal tubule concretions, hydronephrosis, renal tubule dilatation, and pyelonephritis. However, no differences in body weight, liver weight, or reproductive tissue weights, reproductive or thyroid histology, hormone concentrations or the age of pubertal onset were observed. Therefore, the results demonstrate that the endpoints included in the pubertal assay are useful for nonendocrine (systemic) effects that define an no observable effect level (NOEL) or lowest observable effect level (LOEL) and provide one example where an impact on kidney function does not alter any of the endocrine-specific endpoints of the assay.

High-throughput AR dimerization assay identifies androgen disrupting chemicals and metabolites.

Introduction: Analysis of streamlined computational models used to predict androgen disrupting chemicals revealed that assays measuring androgen receptor (AR) cofactor recruitment/dimerization were particularly indispensable to high predictivity, especially for AR antagonists. As the original dimerization assays used to develop the minimal assay models are no longer available, new assays must be established and evaluated as suitable alternatives to assess chemicals beyond the original 1,800+ supported by the current data. Here we present the AR2 assay, which is a stable, cell-based method that uses an enzyme complementation approach. Methods: Bipartite domains of the NanoLuc luciferase enzyme were fused to the human AR to quantitatively measure ligand-dependent AR homodimerization. 128 chemicals with known endocrine activity profiles including 43 AR reference chemicals were screened in agonist and antagonist modes and compared to the legacy assays. Test chemicals were rescreened in both modes using a retrofit method to incorporate robust cytochrome P450 (CYP) metabolism to assess CYP-mediated shifts in bioactivity. Results: The AR2 assay is amenable to high-throughput screening with excellent robust Z'-factors (rZ') for both agonist (0.94) and antagonist (0.85) modes. The AR2 assay successfully classified known agonists (balanced accuracy = 0.92) and antagonists (balanced accuracy = 0.79-0.88) as well as or better than the legacy assays with equal or higher estimated potencies. The subsequent reevaluation of the 128 chemicals tested in the presence of individual human CYP enzymes changed the activity calls for five compounds and shifted the estimated potencies for several others. Discussion: This study shows the AR2 assay is well suited to replace the previous AR dimerization assays in a revised computational model to predict AR bioactivity for parent chemicals and their metabolites.

Polymorphisms of the acid sensing histidine kinase gene arsS in Helicobacter pylori populations from anatomically distinct gastric sites.

Phase variation is frequently utilized by bacterial species to affect gene expression such that phenotypic variants are maintained within populations, ensuring survival as environmental or host conditions change. Unusual among Helicobacter pylori phase variable or contingency genes is arsS, encoding a sensory histidine kinase involved in the acid acclimation of the organism. The presence of a 3' homopolymeric cytosine tract of variable length in arsS among Helicobacter pylori strains allows for the expression of various functional ArsS isoforms, differing in carboxy-terminal protein domains. In this study, we analyzed this 3'arsS region via amplified fragment length polymorphism (AFLP) and sequencing analyses for H. pylori populations from 3 different gastric sites of 12 patients. Our data indicate the presence of multiple arsS alleles within each population of H. pylori derived from the gastric antrum, cardia, or corpus of these patients. We also show that H. pylori, derived from the same anatomical site and patient, are predicted to express multiple ArsS isoforms in each population investigated. Furthermore, we identify a polymorphic deletion within arsS that generates another alternate ArsS C-terminal end. These findings suggest that four C-terminal variations of ArsS adds to the complexity of the ArsRS acid adaptation mechanism as a whole and may influence the ability of H. pylori to persist in the gastric niche for decades.

A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail.

Noninvasive salivary antibody immunoassays can enable low-cost epidemiological surveillance of infections. This study involved developing and validating a multiplex suspension immunoassay on the Luminex platform to measure immunoglobulin G (IgG) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and spike (S) proteins, and the spike protein's S1 and S2 subunits and receptor binding domain. Multiple versions of these recombinant proteins acquired from commercial and noncommercial sources were evaluated. Assay development and validation utilized saliva and serum samples from coronavirus disease 2019 (COVID-19) cases procured from commercial sources and negative controls from a prepandemic survey. Saliva was also collected in a demonstration survey by mail involving adult individuals in the United States who were diagnosed with SARS-CoV-2 infection 15 to 80 days prior to sample collection. The survey had an 83% valid sample return rate (192 samples from 38 states). Most COVID-19 cases (93%) reported mildly symptomatic or asymptomatic infections. The final salivary assay based on the best-performing spike and nucleocapsid proteins had a sensitivity of 87.1% (95% bootstrap confidence interval, 82.1 to 91.7%) and specificity of 98.5% (95.0 to 100%) using 227 and 285 saliva samples, respectively. The same assay had 95.9% (92.8 to 98.9%) sensitivity and 100% (98.4 to 100%) specificity in serum (174 and 285 serum samples, respectively). Salivary and serum antibody responses to spike and nucleocapsid proteins were strongly correlated in 22 paired samples (r = 0.88 and r = 0.80, respectively). Antibody responses peaked at approximately 50 days postonset; greater illness severity was associated with stronger responses. This study demonstrated that a salivary antibody assay can be used in large-scale population surveys by mail to better characterize public health impacts of COVID-19. IMPORTANCE Given the enormous impacts of the COVID-19 pandemic, developing tools for population surveillance of infection is of paramount importance. This article describes the development of a multiplex immunoassay on a Luminex platform to measure salivary immunoglobulin G responses to the spike protein, its two subunits and receptor binding domain, and the nucleocapsid protein of SARS-CoV-2. The assay validation utilized serum and saliva samples from prepandemic controls and recent COVID-19 cases. A survey by mail targeting recent COVID-19 cases across the United States also demonstrated the utility of safe, at-home self-collection of saliva. By incorporating multiple SARS-CoV-2 proteins, this assay may differentiate responses to natural SARS-CoV-2 infections from responses to most vaccines. Application of this noninvasive immunoassay in COVID-19 surveillance can help provide estimates of cumulative incidence rates of symptomatic and asymptomatic infections in various communities and subpopulations, temporal patterns of antibody responses, and risk factors for infection.

Respirometric Screening and Characterization of Mitochondrial Toxicants Within the ToxCast Phase I and II Chemical Libraries.

Mitochondrial toxicity drives several adverse health outcomes. Current high-throughput screening assays for chemically induced mitochondrial toxicity typically measure changes to mitochondrial structure and may not detect known mitochondrial toxicants. We adapted a respirometric screening assay (RSA) measuring mitochondrial function to screen ToxCast chemicals in HepG2 cells using a tiered testing strategy. Of 1042 chemicals initially screened at a singlemaximal concentration, 243 actives were identified and rescreened at 7 concentrations. Concentration-response data for 3 respiration phases confirmed activity and indicated a mechanism for 193 mitochondrial toxicants: 149 electron transport chain inhibitors (ETCi), 15 uncouplers and 29 adenosine triphosphate synthase inhibitors. Subsequently, an electron flow assay was used to identify the target complex for 84 of the 149 ETCi. Sixty reference chemicals were used to compare the RSA to existing ToxCast and Tox21 mitochondrial toxicity assays. The RSA was most predictive (accuracy = 90%) of mitochondrial toxicity. The Tox21 mitochondrial membrane potential assay was also highly predictive (accuracy = 87%) of bioactivity but underestimated the potency of well-known ETCi and provided no mechanistic information. The tiered RSA approach accurately identifies and characterizes mitochondrial toxicants acting through diverse mechanisms and at a throughput sufficient to screen large chemical inventories. The electron flow assay provides additional confirmation and detailed mechanistic understanding for ETCi, the most common type of mitochondrial toxicants among ToxCast chemicals. The mitochondrial toxicity screening approach described herein may inform hazard assessment and the in vitro bioactive concentrations used to derive relevant doses for screening level chemical assessment using new approach methodologies.

High-throughput screening and chemotype-enrichment analysis of ToxCast phase II chemicals evaluated for human sodium-iodide symporter (NIS) inhibition.

In support of the Endocrine Disruptor Screening Program (EDSP), the U.S.EPA's Office of Research and Development (ORD) is developing high-throughput screening (HTS) approaches to identify chemicals that alter target sites in the thyroid hormone (TH) pathway. The sodium iodide symporter (NIS) is a transmembrane glycoprotein that mediates iodide uptake into the thyroid as the initial step of TH biosynthesis. Previously, we screened 293 ToxCast chemicals (ph1v2) using a HEK293T cell line expressing human NIS in parallel radioactive iodide uptake (RAIU) and cell viability assays to identify potential environmental NIS inhibitors. Here, we expanded NIS inhibitor screening for a set of 768 ToxCast Phase II (ph2) chemicals, and applied a novel computational toxicology approach based on the ToxPrint chemotype to identify chemical substructures associated with NIS inhibition. Following single-concentration screening (at 1 × 10-4 M with a 20% inhibition cutoff), 235 samples (228 chemicals) were further tested in multiple-concentration (1 × 10-9 - 1 × 10-4 M) format in both RAIU and cell viability assays. The 167 chemicals that exhibited significant RAIU inhibition were then prioritized using combined RAIU and cell viability responses that were normalized relative to the known NIS inhibitor sodium perchlorate. Some of the highest ranked chemicals, such as PFOS, tributyltin chloride, and triclocarban, have been previously reported to be thyroid disruptors. In addition, several novel chemicals were identified as potent NIS inhibitors. The present results were combined with the previous ph1v2 screening results to produce two sets of binary hit-calls for 1028 unique chemicals, consisting of 273 positives exhibiting significant RAIU inhibition, and 63 positives following application of a cell viability filter. A ToxPrint chemotype-enrichment analysis identified >20 distinct chemical substructural features, represented in >60% of the active chemicals, as significantly enriched in each NIS inhibition hit-call space. A shared set of 9 chemotypes enriched in both hit-call sets indicates stable chemotype signals (insensitive to cytotoxicity filters) that can help guide structure-activity relationship (SAR) investigations and inform future research.

Development of a screening approach to detect thyroid disrupting chemicals that inhibit the human sodium iodide symporter (NIS).

The U.S. EPA's Endocrine Disruptor Screening Program aims to use high-throughput assays and computational toxicology models to screen and prioritize chemicals that may disrupt the thyroid signaling pathway. Thyroid hormone biosynthesis requires active iodide uptake mediated by the sodium/iodide symporter (NIS). Monovalent anions, such as the environmental contaminant perchlorate, are competitive inhibitors of NIS, yet limited information exists for more structurally diverse chemicals. A novel cell line expressing human NIS, hNIS-HEK293T-EPA, was used in a radioactive iodide uptake (RAIU) assay to identify inhibitors of NIS-mediated iodide uptake. The RAIU assay was optimized and performance evaluated with 12 reference chemicals comprising known NIS inhibitors and inactive compounds. An additional 39 chemicals including environmental contaminants were evaluated, with 28 inhibiting RAIU over 20% of that observed for solvent controls. Cell viability assays were performed to assess any confounding effects of cytotoxicity. RAIU and cytotoxic responses were used to calculate selectivity scores to group chemicals based on their potential to affect NIS. RAIU IC50 values were also determined for chemicals that displayed concentration-dependent inhibition of RAIU (≥50%) without cytotoxicity. Strong assay performance and highly reproducible results support the utilization of this approach to screen large chemical libraries for inhibitors of NIS-mediated iodide uptake.

Perilipin 5 regulates islet lipid metabolism and insulin secretion in a cAMP-dependent manner: implication of its role in the postprandial insulin secretion.

Elevation of circulating fatty acids (FA) during fasting supports postprandial (PP) insulin secretion that is critical for glucose homeostasis and is impaired in diabetes. We tested our hypothesis that lipid droplet (LD) protein perilipin 5 (PLIN5) in ß-cells aids PP insulin secretion by regulating intracellular lipid metabolism. We demonstrated that PLIN5 serves as an LD protein in human islets. In vivo, Plin5 and triglycerides were increased by fasting in mouse islets. MIN6 cells expressing PLIN5 (adenovirus [Ad]-PLIN5) and those expressing perilipin 2 (PLIN2) (Ad-PLIN2) had higher [(3)H]FA incorporation into triglycerides than Ad-GFP control, which support their roles as LD proteins. However, Ad-PLIN5 cells had higher lipolysis than Ad-PLIN2 cells, which increased further by 8-Br-cAMP, indicating that PLIN5 facilitates FA mobilization upon cAMP stimulation as seen postprandially. Ad-PLIN5 in islets enhanced the augmentation of glucose-stimulated insulin secretion by FA and 8-Br-cAMP in G-protein-coupled receptor 40 (GPR40)- and cAMP-activated protein kinase-dependent manners, respectively. When PLIN5 was increased in mouse ß-cells in vivo, glucose tolerance after an acute exenatide challenge was improved. Therefore, the elevation of islet PLIN5 during fasting allows partitioning of FA into LD that is released upon refeeding to support PP insulin secretion in cAMP- and GPR40-dependent manners.

Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription.

Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA ) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct "cross-talk" between HpArsRS and HpNikR at neutral pH has been demonstrated.

Pancreatic islet neuropeptide Y overexpression has minimal effect on islet morphology and ß-cell adaptation to high-fat diet.

Neuropeptide Y (NPY) is highly expressed in the hypothalamus, where it regulates feeding and energy homeostasis. Interestingly, NPY and its receptors are also expressed in peripheral tissues with roles in metabolism, including pancreatic islets. In islets, NPY is known to suppress insulin secretion acutely. In addition, the role of NPY in ß-cell de-differentiation has been postulated recently. Therefore, we studied transgenic mice expressing NPY under rat insulin promoter (TG) to determine the effects of chronic up-regulation of NPY on islet morphology and function. NPY levels were 25 times higher in islets of TG mice compared with wild-type (WT) littermates, whereas no differences in NPY expression were noted in the brains of TG and WT mice. Islet NPY secretion was 2.3-fold higher in TG compared with WT mice. There were no significant changes in body weight, glucose tolerance, or insulin sensitivity in TG mice fed regular rodent diet or high-fat diet (HF). Islet ß-cell area was comparable between TG and WT mice both on regular rodent and HF diets, indicating that NPY overexpression is insufficient to alter ß-cell maturation or the compensatory increase of ß-cell area on HF. One abnormality noted was that the glucose-stimulated insulin secretion in islets isolated from TG was reduced compared with those from WT mice on HF diet. Overall, an increase in islet NPY level has little impact on islet function and is insufficient to affect glucose homeostasis in mice.

The effect of triclosan on the uterotrophic response to extended doses of ethinyl estradiol in the weanling rat.

Triclosan (TCS), an antibacterial, has been shown to be an endocrine disruptor in the rat. We reported previously that TCS potentiated the estrogenic effect of ethinyl estradiol (EE) on uterine growth in rats exposed to EE and TCS in the uterotrophic assay, whereas TCS alone had no effect. To further characterize this potentiation, we evaluated the effect of co-exposure with lower doses of EE that are comparable to the concentrations in hormone replacement regimens and began to assess the mechanisms by which this potentiation occurs. Changes in uterine weight, epithelial cell growth, and estrogen-sensitive gene expression were assessed. TCS expectedly enhanced the uterotrophic response to EE, however at significantly lower doses of EE. Similarly, TCS increased the EE-induced stimulation of epithelial cell height following cotreatment. Cotreatment also enhanced the estrogen-induced change in gene expression, which was reversed with an ER antagonist. Furthermore, the TCS-induced potentiation was independent of ER activation, as no effects were observed in the ER TA assay.