@article{starnes_jackson_rock_belcher_2024, title={Quantitative cross-species comparison of serum albumin binding of per- and polyfluoroalkyl substances from five structural classes}, url={https://doi.org/10.1093/toxsci/kfae028}, DOI={10.1093/toxsci/kfae028}, abstractNote={Per- and polyfluoroalkyl substances (PFAS) are a class of over 8,000 chemicals, many of which are persistent, bioaccumulative, and toxic to humans, livestock, and wildlife. Serum protein binding affinity is instrumental in understanding PFAS toxicity, yet experimental binding data is limited to only a few PFAS congeners. Previously, we demonstrated the usefulness of a high-throughput, in vitro differential scanning fluorimetry assay for determination of relative binding affinities of human serum albumin for 24 PFAS congeners from 6 chemical classes. In the current study, we used this assay to comparatively examine differences in human, bovine, porcine, and rat serum albumin binding of 8 structurally informative PFAS congeners from 5 chemical classes. With the exception of the fluorotelomer alcohol 1H, 1H, 2H, 2H-perfluorooctanol (6:2 FTOH), each PFAS congener bound by human serum albumin was also bound by bovine, porcine, and rat serum albumin. The critical role of the charged functional headgroup in albumin binding was supported by the inability of albumin of each species tested to bind 6:2 FTOH. Significant interspecies differences in serum albumin binding affinities were identified for each of the bound PFAS congeners. Relative to human albumin, perfluoroalkyl carboxylic and sulfonic acids were bound with greater affinity by porcine and rat serum albumin, and the perfluoroalkyl ether acid congener bound with lower affinity to porcine and bovine serum albumin. These comparative affinity data for PFAS binding by serum albumin from human, experimental model and livestock species reduce critical interspecies uncertainty and improve accuracy of predictive bioaccumulation and toxicity assessments for PFAS.}, journal={Toxicological Sciences}, author={Starnes, Hannah M and Jackson, Thomas W and Rock, Kylie D and Belcher, Scott M}, year={2024}, month={Mar} }
 @article{muncke_andersson_backhaus_belcher_boucher_almroth_collins_geueke_groh_heindel_et al._2023, title={A vision for safer food contact materials: Public health concerns as drivers for improved testing}, volume={180}, ISSN={["1873-6750"]}, url={https://doi.org/10.1016/j.envint.2023.108161}, DOI={10.1016/j.envint.2023.108161}, abstractNote={Food contact materials (FCMs) and food contact articles are ubiquitous in today's globalized food system. Chemicals migrate from FCMs into foodstuffs, so called food contact chemicals (FCCs), but current regulatory requirements do not sufficiently protect public health from hazardous FCCs because only individual substances used to make FCMs are tested and mostly only for genotoxicity while endocrine disruption and other hazard properties are disregarded. Indeed, FCMs are a known source of a wide range of hazardous chemicals, and they likely contribute to highly prevalent non-communicable diseases. FCMs can also include non-intentionally added substances (NIAS), which often are unknown and therefore not subject to risk assessment. To address these important shortcomings, we outline how the safety of FCMs may be improved by (1) testing the overall migrate, including (unknown) NIAS, of finished food contact articles, and (2) expanding toxicological testing beyond genotoxicity to multiple endpoints associated with non-communicable diseases relevant to human health. To identify mechanistic endpoints for testing, we group chronic health outcomes associated with chemical exposure into Six Clusters of Disease (SCOD) and we propose that finished food contact articles should be tested for their impacts on these SCOD. Research should focus on developing robust, relevant, and sensitive in-vitro assays based on mechanistic information linked to the SCOD, e.g., through Adverse Outcome Pathways (AOPs) or Key Characteristics of Toxicants. Implementing this vision will improve prevention of chronic diseases that are associated with hazardous chemical exposures, including from FCMs.}, journal={ENVIRONMENT INTERNATIONAL}, author={Muncke, Jane and Andersson, Anna-Maria and Backhaus, Thomas and Belcher, Scott M. and Boucher, Justin M. and Almroth, Bethanie Carney and Collins, Terrence J. and Geueke, Birgit and Groh, Ksenia J. and Heindel, Jerrold J. and et al.}, year={2023}, month={Oct} }
 @article{boatman_chappel_polera_dodds_belcher_baker_2023, title={Assessing Per- and Polyfluoroalkyl Substances (PFAS) in Fish Fillet Using Non-Targeted Analyses}, url={https://doi.org/10.1101/2023.09.01.555938}, DOI={10.1101/2023.09.01.555938}, abstractNote={Per- and polyfluoroalkyl substances (PFAS) are a class of thousands of man-made chemicals that are persistent and highly stable in the environment. The diverse structures of PFAS give them different chemical properties that influence their solubility in different environmental matrices and biological tissues. PFAS in drinking water have been extensively studied, but information on their presence in fish and other exposure routes is limited. To address this, a non-targeted analysis using liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) was performed to evaluate PFAS in fish fillets from in central North Carolina and compare with PFAS data from previously published water. A total of 22 different PFAS were detected in the fillets, including only 4 of the PFAS reported in water. Both more PFAS types and higher concentrations were observed in fish caught near a known PFAS point-source compared to those from a reservoir used for drinking water and recreation. Median fillet PFOS levels were 54 ppb in fish closest to the point source and 14-20 ppb in fish from the reservoir. Thus, future PFAS monitoring should include both targeted and non-targeted analyses of both water and fish to increase understanding of human exposure risks and ecosystem impacts. SYNOPSIS Fish fillet samples were collected from five sites in North Carolina. PFAS were detected in all samples and differences in analytes and abundances were observed at the different sites. GRAPHICAL ABSTRACT For use in table of contents only}, author={Boatman, Anna K. and Chappel, Jessie R. and Polera, Madison E. and Dodds, James N. and Belcher, Scott M. and Baker, Erin S.}, year={2023}, month={Sep} }
 @article{rock_polera_guillette_starnes_dean_watters_stevens-stewart_belcher_2023, title={Domestic Dogs and Horses as Sentinels of Per- and Polyfluoroalkyl Substance Exposure and Associated Health Biomarkers in Gray's Creek North Carolina}, volume={57}, ISSN={["1520-5851"]}, url={https://doi.org/10.1021/acs.est.3c01146}, DOI={10.1021/acs.est.3c01146}, abstractNote={Central North Carolina (NC) is highly contaminated with per- and polyfluoroalkyl substances (PFAS), in part due to local fluorochemical production. Little is known about the exposure profiles and long-term health impacts for humans and animals that live in nearby communities. In this study, serum PFAS concentrations were determined using liquid chromatography high-resolution mass spectrometry and diagnostic clinical chemistry endpoints were assessed for 31 dogs and 32 horses that reside in Gray's Creek NC at households with documented PFAS contamination in their drinking water. PFAS were detected in every sample, with 12 of the 20 PFAS detected in ≥50% of samples from each species. The average total PFAS concentrations in horses were lower compared to dogs who had higher concentrations of PFOS (dogs 2.9 ng/mL; horses 1.8 ng/mL), PFHxS (dogs 1.43 ng/mL, horses < LOD), and PFOA (dogs 0.37 ng/mL; horses 0.10 ng/mL). Regression analysis highlighted alkaline phosphatase, glucose, and globulin proteins in dogs and gamma glutamyl transferase in horses as potential biomarkers associated with PFAS exposure. Overall, the results of this study support the utility of companion animal and livestock species as sentinels of PFAS exposure differences inside and outside of the home. As in humans, renal and hepatic health in domestic animals may be sensitive to long-term PFAS exposures.}, number={26}, journal={ENVIRONMENTAL SCIENCE & TECHNOLOGY}, author={Rock, Kylie D. and Polera, Madison E. and Guillette, Theresa C. and Starnes, Hannah M. and Dean, Kentley and Watters, Mike and Stevens-Stewart, Debra and Belcher, Scott M.}, year={2023}, month={Jun}, pages={9567–9579} }
 @article{starnes_jackson_rock_belcher_2023, title={Quantitative Cross-Species Comparison of Serum Albumin Binding of Per- and Polyfluoroalkyl Substances from Five Structural Classes}, url={https://doi.org/10.1101/2023.11.10.566613}, DOI={10.1101/2023.11.10.566613}, abstractNote={Per- and polyfluoroalkyl substances (PFAS) are a class of over 8,000 chemicals that are persistent, bioaccumulative, and toxic to humans, livestock, and wildlife. Serum protein binding affinity is instrumental in understanding PFAS toxicity, yet experimental binding data is limited to only a few PFAS congeners. Previously, we demonstrated the usefulness of a high-throughput, in vitro differential scanning fluorimetry assay for determination of relative binding affinities of human serum albumin for 24 PFAS congeners from 6 chemical classes. In the current study, we used this differential scanning fluorimetry assay to comparatively examine differences in human, bovine, porcine, and rat serum albumin binding of 8 structurally informative PFAS congeners from 5 chemical classes. With the exception of the fluorotelomer alcohol 1H,1H,2H,2H-perfluorooctanol (6:2 FTOH), each PFAS congener bound by human serum albumin was also bound by bovine, porcine, and rat serum albumin. The critical role of the charged functional headgroup in albumin binding was supported by the inability of serum albumin of each species tested to bind 6:2 FTOH. Significant interspecies differences in serum albumin binding affinities were identified for each of the bound PFAS congeners. Relative to human albumin, perfluoroalkyl carboxylic and sulfonic acids were bound with greater affinity by porcine and rat serum albumin, and perfluoroalkyl ether congeners bound with lower affinity to porcine and bovine serum albumin. These comparative affinity data for PFAS binding by serum albumin from human, experimental model and livestock species reduce critical interspecies uncertainty and improve accuracy of predictive toxicity assessments for PFAS.}, author={Starnes, Hannah M. and Jackson, Thomas W. and Rock, Kylie D. and Belcher, Scott M.}, year={2023}, month={Nov} }
 @article{starnes_rock_jackson_belcher_2022, title={A Critical Review and Meta-Analysis of Impacts of Per- and Polyfluorinated Substances on the Brain and Behavior}, volume={4}, url={http://dx.doi.org/10.3389/ftox.2022.881584}, DOI={10.3389/ftox.2022.881584}, abstractNote={Per- and polyfluoroalkyl substances (PFAS) are a class of structurally diverse synthetic organic chemicals that are chemically stable, resistant to degradation, and persistent in terrestrial and aquatic environments. Widespread use of PFAS in industrial processing and manufacturing over the last 70 years has led to global contamination of built and natural environments. The brain is a lipid rich and highly vascularized organ composed of long-lived neurons and glial cells that are especially vulnerable to the impacts of persistent and lipophilic toxicants. Generally, PFAS partition to protein-rich tissues of the body, primarily the liver and blood, but are also detected in the brains of humans, wildlife, and laboratory animals. Here we review factors impacting the absorption, distribution, and accumulation of PFAS in the brain, and currently available evidence for neurotoxic impacts defined by disruption of neurochemical, neurophysiological, and behavioral endpoints. Emphasis is placed on the neurotoxic potential of exposures during critical periods of development and in sensitive populations, and factors that may exacerbate neurotoxicity of PFAS. While limitations and inconsistencies across studies exist, the available body of evidence suggests that the neurobehavioral impacts of long-chain PFAS exposures during development are more pronounced than impacts resulting from exposure during adulthood. There is a paucity of experimental studies evaluating neurobehavioral and molecular mechanisms of short-chain PFAS, and even greater data gaps in the analysis of neurotoxicity for PFAS outside of the perfluoroalkyl acids. Whereas most experimental studies were focused on acute and subchronic impacts resulting from high dose exposures to a single PFAS congener, more realistic exposures for humans and wildlife are mixtures exposures that are relatively chronic and low dose in nature. Our evaluation of the available human epidemiological, experimental, and wildlife data also indicates heightened accumulation of perfluoroalkyl acids in the brain after environmental exposure, in comparison to the experimental studies. These findings highlight the need for additional experimental analysis of neurodevelopmental impacts of environmentally relevant concentrations and complex mixtures of PFAS.}, journal={Frontiers in Toxicology}, publisher={Frontiers Media SA}, author={Starnes, Hannah M. and Rock, Kylie D. and Jackson, Thomas W. and Belcher, Scott M.}, year={2022}, month={Apr} }
 @article{guillette_jackson_guillette_mccord_belcher_2022, title={Blood concentrations of per- and polyfluoroalkyl substances are associated with autoimmune-like effects in American alligators from Wilmington, North Carolina}, volume={4}, ISSN={["2673-3080"]}, url={http://dx.doi.org/10.3389/ftox.2022.1010185}, DOI={10.3389/ftox.2022.1010185}, abstractNote={Surface and groundwater of the Cape Fear River basin in central and coastal North Carolina is contaminated with high levels of per- and polyfluoroalkyl substances (PFAS). Elevated levels of PFAS have also been found in blood of fish and wildlife from the Cape Fear River, and in the blood of human populations reliant on contaminated well or surface water from the Cape Fear River basin as a source of drinking water. While the public and environmental health impacts of long-term PFAS exposures are poorly understood, elevated blood concentrations of some PFAS are linked with immunotoxicity and increased incidence of some chronic autoimmune diseases in human populations. The goal of this One Environmental Health study was to evaluate PFAS exposure and biomarkers related to immune health in populations of American alligators (Alligator mississippiensis), a protected and predictive sentinel species of adverse effects caused by persistent toxic pollutants. We found that serum PFAS concentrations in alligator populations from the Cape Fear River were increased compared to a reference population of alligators from the adjoining Lumber River basin. The elevated serum PFAS concentrations in the Cape Fear River alligators were associated with increased innate immune activities, and autoimmune-like phenotypes in this population. In addition to evidence of significantly higher double stranded-DNA binding autoantibodies in adult Cape Fear River alligators, our qRT-PCR analysis found remarkably high induction of Interferon-α signature genes implicated in the pathology of human autoimmune disease. We interpret the association of increased PFAS exposure with disrupted immune functions to suggest that PFAS broadly alters immune activities resulting in autoimmune-like pathology in American alligators. This work substantiates and extends evidence from experimental models and human epidemiology studies showing that some PFAS are immune toxicants.}, journal={FRONTIERS IN TOXICOLOGY}, publisher={Frontiers Media SA}, author={Guillette, T. C. and Jackson, Thomas W. and Guillette, Matthew and McCord, James and Belcher, Scott M.}, year={2022}, month={Oct} }
 @article{belcher_guillette_robb_rock_2022, title={Comparative assessment of blood mercury in American alligators (Alligator mississippiensis) from Coastal North Carolina and Florida}, volume={8}, ISSN={["1573-3017"]}, url={https://doi.org/10.1007/s10646-022-02573-z}, DOI={10.1007/s10646-022-02573-z}, abstractNote={Mercury (Hg) is a widespread and harmful persistent pollutant of aquatic ecosystems. Except for the northern most populations of American alligators (Alligator Mississippiensis) found in North Carolina, the potential adverse health impacts of Hg on ecosystems and humans consuming alligator meat have been studied for over three decades. Now that alligators are being recreationally hunted and consumed across their range, it is especially important to monitor toxic contaminant levels to best understand possible adverse impacts of exposures on alligator populations and human health. In this study, we determined blood Hg concentrations in American alligators from an urbanized site in Wilmington, NC, a nearby site at Lake Waccamaw, NC, and a site on the St Johns River in Florida. Median blood total Hg (tHg) concentrations were particularly high at Lake Waccamaw (526 ng/g, range 152–946 ng/g), resulting in median muscle concentrations (0.48 mg/kg, range 0.13–0.88 mg/kg) well above US EPA screening values for fish consumption. Median concentrations at the Wilmington site (69 ng/g, range 22–336 ng/g) were generally low, and Hg concentrations from the St Johns River site (143 ng/g, range 54–244 ng/g) were comparable to those reported in previous studies. Analysis of relationships between tHg concentrations and a panel of blood chemistry biomarkers found only modest concentration-dependent impact on biomarkers of renal function. The results of this study reveal that local environmental factors greatly impact Hg bioaccumulation in alligators, findings that reaffirm local contaminant biomonitoring in alligator populations will be critical for affective management and determination of guidelines for safe consumption of harvested alligators.}, journal={ECOTOXICOLOGY}, publisher={Springer Science and Business Media LLC}, author={Belcher, Scott M. and Guillette, Matthew P. and Robb, Frank and Rock, Kylie D.}, year={2022}, month={Aug} }
 @article{jackson_baars_belcher_2022, title={Gestational Cd Exposure in the CD-1 Mouse Sex-Specifically Disrupts Essential Metal Ion Homeostasis}, volume={2}, ISSN={["1096-0929"]}, url={https://doi.org/10.1093/toxsci/kfac027}, DOI={10.1093/toxsci/kfac027}, abstractNote={Abstract In CD-1 mice, gestational-only exposure to cadmium (Cd) causes female-specific hepatic insulin resistance, metabolic disruption, and obesity. To evaluate whether sex differences in uptake and changes in essential metal concentrations contribute to metabolic outcomes, placental and liver Cd and essential metal concentrations were quantified in male and female offspring perinatally exposed to 500 ppb CdCl2. Exposure resulted in increased maternal liver Cd+2 concentrations (364 µg/kg) similar to concentrations found in non-occupationally exposed human liver. At gestational day (GD) 18, placental Cd and manganese concentrations were significantly increased in exposed males and females, and zinc was significantly decreased in females. Placental efficiency was significantly decreased in GD18-exposed males. Increases in hepatic Cd concentrations and a transient prenatal increase in zinc were observed in exposed female liver. Fetal and adult liver iron concentrations were decreased in both sexes, and decreases in hepatic zinc, iron, and manganese were observed in exposed females. Analysis of GD18 placental and liver metallothionein mRNA expression revealed significant Cd-induced upregulation of placental metallothionein in both sexes, and a significant decrease in fetal hepatic metallothionein in exposed females. In placenta, expression of metal ion transporters responsible for metal ion uptake was increased in exposed females. In liver of exposed adult female offspring, expression of the divalent cation importer (Slc39a14/Zip14) decreased, whereas expression of the primary exporter (Slc30a10/ZnT10) increased. These findings demonstrate that Cd can preferentially cross the female placenta, accumulate in the liver, and cause lifelong dysregulation of metal ion concentrations associated with metabolic disruption.}, journal={TOXICOLOGICAL SCIENCES}, publisher={Oxford University Press (OUP)}, author={Jackson, Thomas W. and Baars, Oliver and Belcher, Scott M.}, year={2022}, month={Feb} }
 @article{riegl_starnes_jima_baptissart_diehl_belcher_cowley_2022, title={The imprinted gene Zac1 regulates steatosis in developmental cadmium-induced nonalcoholic fatty liver disease}, volume={10}, ISSN={["1096-0929"]}, url={https://doi.org/10.1093/toxsci/kfac106}, DOI={10.1093/toxsci/kfac106}, abstractNote={Cadmium (Cd) exposure in adulthood is associated with non-alcoholic fatty liver disease (NAFLD), characterized by steatosis, inflammation, and fibrosis. The prevalence of NAFLD in children is increasing, suggesting a role for the developmental environment in programming susceptibility. However, the role of developmental Cd exposure in programming NAFLD and the underlying mechanisms remain unclear. We have proposed that imprinted genes are strong candidates for connecting the early life environment and later disease. In support of this, we previously identified roles for the Imprinted Gene Network (IGN) and its regulator Zac1 in programming NAFLD in response to maternal metabolic dysfunction. Here, we test the hypothesis that developmental Cd exposure is sufficient to program NAFLD, and further, that this process is mediated by Zac1 and the IGN. Using mice, we show that developmental CdCl2 exposure leads to histological, biochemical, and molecular signatures of steatosis and fibrosis in juveniles. Transcriptomic analyses comparing livers of CdCl2-exposed and control mice show up-regulation of Zac1 and the IGN coincident with disease presentation. Increased hepatic Zac1 expression is independent of promoter methylation and imprinting statuses. Finally, we show that over-expression of Zac1 in cultured hepatocytes is sufficient to induce lipid accumulation in a Pparγ-dependent manner, and demonstrate direct binding of Zac1 to the Pparγ promoter. Our findings demonstrate that developmental Cd exposure is sufficient to program NAFLD in later life, and with our previous work, establish Zac1 and the IGN as key regulators of prosteatotic and profibrotic pathways, two of the major pathological hallmarks of NAFLD.}, journal={TOXICOLOGICAL SCIENCES}, author={Riegl, Sierra D. and Starnes, Cassie and Jima, Dereje D. and Baptissart, Marine and Diehl, Anna Mae and Belcher, Scott M. and Cowley, Michael}, year={2022}, month={Oct} }
 @article{kirkwood_fleming_nguyen_reif_baker_belcher_2022, title={Utilizing Pine Needles to Temporally and Spatially Profile Per- and Polyfluoroalkyl Substances (PFAS)}, volume={56}, ISSN={["1520-5851"]}, url={https://doi.org/10.1021/acs.est.1c06483}, DOI={10.1021/acs.est.1c06483}, abstractNote={As concerns over exposure to per- and polyfluoroalkyl substances (PFAS) are continually increasing, novel methods to monitor their presence and modifications are greatly needed, as some have known toxic and bioaccumulative characteristics while most have unknown effects. This task however is not simple, as the Environmental Protection Agency (EPA) CompTox PFAS list contains more than 9000 substances as of September 2020 with additional substances added continually. Nontargeted analyses are therefore crucial to investigating the presence of this immense list of possible PFAS. Here, we utilized archived and field-sampled pine needles as widely available passive samplers and a novel nontargeted, multidimensional analytical method coupling liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) to evaluate the temporal and spatial presence of numerous PFAS. Over 70 PFAS were detected in the pine needles from this study, including both traditionally monitored legacy perfluoroalkyl acids (PFAAs) and their emerging replacements such as chlorinated derivatives, ultrashort chain PFAAs, perfluoroalkyl ether acids including hexafluoropropylene oxide dimer acid (HFPO-DA, "GenX") and Nafion byproduct 2, and a cyclic perfluorooctanesulfonic acid (PFOS) analog. Results from this study provide critical insight related to PFAS transport, contamination, and reduction efforts over the past six decades.}, number={6}, journal={ENVIRONMENTAL SCIENCE & TECHNOLOGY}, publisher={American Chemical Society (ACS)}, author={Kirkwood, Kaylie I and Fleming, Jonathon and Nguyen, Helen and Reif, David M. and Baker, Erin S. and Belcher, Scott M.}, year={2022}, month={Mar}, pages={3441–3451} }
 @article{jackson_baars_belcher_2021, title={Gestational Cd Exposure in the CD-1 Mouse Sex-Specifically Disrupts Essential Metal Ion Homeostasis}, volume={11}, url={https://doi.org/10.1101/2021.11.06.467551}, DOI={10.1101/2021.11.06.467551}, abstractNote={In CD-1 mice, gestational-only exposure to cadmium (Cd) causes female-specific hepatic insulin resistance, metabolic disruption, and obesity. To evaluate whether sex differences in cadmium uptake and changes in essential metal concentrations contribute to metabolic outcomes, placental and liver cadmium and essential metal concentrations were quantified in male and female offspring perinatally exposed to 500 ppb CdCl2. Exposure resulted in increased maternal liver Cd+2 concentrations (364 μg/kg) similar to concentrations found in non-occupationally exposed human liver. At gestational day (GD) 18, placental cadmium and manganese concentrations were significantly increased in exposed males and females, and zinc was significantly decreased in females. Placental efficiency was significantly decreased in GD18 exposed males. Increases in hepatic Cd concentrations and a transient prenatal increase in zinc were observed in exposed female liver. Fetal and adult liver iron concentrations were decreased in both sexes, and decreases in hepatic zinc, iron, and manganese were observed in exposed females. Analysis of GD18 placental and liver metallothionein mRNA expression revealed significant Cd-induced upregulation of placental metallothionein in both sexes, and a significant decrease in fetal hepatic metallothionein in exposed females. In placenta, expression of metal ion transporters responsible for metal ion uptake was increased in exposed females. In liver of exposed adult female offspring, expression of the divalent cation importer (Slc39a14/Zip14) decreased, whereas expression of the primary exporter (Slc30a10) increased. These findings demonstrate that Cd can preferentially cross the female placenta, accumulate in the liver, and cause lifelong dysregulation of metal ion concentrations associated with metabolic disruption.}, publisher={Cold Spring Harbor Laboratory}, author={Jackson, Thomas W. and Baars, Oliver and Belcher, Scott M.}, year={2021}, month={Nov} }
 @article{lind_araujo_barchowsky_belcher_berridge_chiamvimonvat_chiu_cogliano_elmore_farraj_et al._2021, title={Key Characteristics of Cardiovascular Toxicants}, volume={129}, ISSN={["1552-9924"]}, DOI={10.1289/EHP9321}, abstractNote={Background: The concept of chemical agents having properties that confer potential hazard called key characteristics (KCs) was first developed to identify carcinogenic hazards. Identification of KCs of cardiovascular (CV) toxicants could facilitate the systematic assessment of CV hazards and understanding of assay and data gaps associated with current approaches. Objectives: We sought to develop a consensus-based synthesis of scientific evidence on the KCs of chemical and nonchemical agents known to cause CV toxicity along with methods to measure them. Methods: An expert working group was convened to discuss mechanisms associated with CV toxicity. Results: The group identified 12 KCs of CV toxicants, defined as exogenous agents that adversely interfere with function of the CV system. The KCs were organized into those primarily affecting cardiac tissue (numbers 1–4 below), the vascular system (5–7), or both (8–12), as follows: 1) impairs regulation of cardiac excitability, 2) impairs cardiac contractility and relaxation, 3) induces cardiomyocyte injury and death, 4) induces proliferation of valve stroma, 5) impacts endothelial and vascular function, 6) alters hemostasis, 7) causes dyslipidemia, 8) impairs mitochondrial function, 9) modifies autonomic nervous system activity, 10) induces oxidative stress, 11) causes inflammation, and 12) alters hormone signaling. Discussion: These 12 KCs can be used to help identify pharmaceuticals and environmental pollutants as CV toxicants, as well as to better understand the mechanistic underpinnings of their toxicity. For example, evidence exists that fine particulate matter [PM ≤2.5μm in aerodynamic diameter (PM2.5)] air pollution, arsenic, anthracycline drugs, and other exogenous chemicals possess one or more of the described KCs. In conclusion, the KCs could be used to identify potential CV toxicants and to define a set of test methods to evaluate CV toxicity in a more comprehensive and standardized manner than current approaches. https://doi.org/10.1289/EHP9321}, number={9}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Lind, Lars and Araujo, Jesus A. and Barchowsky, Aaron and Belcher, Scott and Berridge, Brian R. and Chiamvimonvat, Nipavan and Chiu, Weihsueh A. and Cogliano, Vincent J. and Elmore, Sarah and Farraj, Aimen K. and et al.}, year={2021}, month={Sep} }
 @article{jackson_scheibly_polera_belcher_2021, title={Rapid Characterization of Human Serum Albumin Binding for Per- and Polyfluoroalkyl Substances Using Differential Scanning Fluorimetry}, volume={55}, ISSN={["1520-5851"]}, url={https://doi.org/10.1021/acs.est.1c01200}, DOI={10.1021/acs.est.1c01200}, abstractNote={Per- and polyfluoroalkyl substances (PFAS) are a diverse class of synthetic chemicals that accumulate in the environment. Many proteins, including the primary human serum transport protein albumin (HSA), bind PFAS. The predictive power of physiologically based pharmacokinetic modeling approaches is currently limited by a lack of experimental data defining albumin-binding properties for most PFAS. A novel thermal denaturation assay was optimized to evaluate changes in the thermal stability of HSA in the presence of increasing concentrations of known ligands and a structurally diverse set of PFAS. Assay performance was initially evaluated for fatty acids and HSA-binding drugs ibuprofen and warfarin. Concentration-response relationships were determined and dissociation constants (Kd) for each compound were calculated using regression analysis of the dose-dependent changes in HSA melting temperature. Estimated Kd values for HSA binding of octanoic acid, decanoic acid, hexadecenoic acid, ibuprofen, and warfarin agreed with established values. The binding affinities for 24 PFAS that included perfluoroalkyl carboxylic acids (C4-C12), perfluoroalkyl sulfonic acids (C4-C8), mono- and polyether perfluoroalkyl ether acids, and polyfluoroalkyl fluorotelomer substances were determined. These results demonstrate the utility of this differential scanning fluorimetry assay as a rapid high-throughput approach for determining the relative protein-binding properties and identification of chemical structures involved in binding for large numbers of structurally diverse PFAS.}, number={18}, journal={ENVIRONMENTAL SCIENCE & TECHNOLOGY}, publisher={American Chemical Society (ACS)}, author={Jackson, Thomas W. and Scheibly, Chris M. and Polera, M. E. and Belcher, Scott M.}, year={2021}, month={Sep}, pages={12291–12301} }
 @article{kirkwood_fleming_nguyen_reif_baker_belcher_2021, title={Utilizing Pine Needles to Temporally and Spatially Profile Per- and Polyfluoroalkyl Substances}, volume={8}, url={https://doi.org/10.1101/2021.08.24.457570}, DOI={10.1101/2021.08.24.457570}, abstractNote={As concerns continue to mount over exposure to per- and polyfluoroalkyl substances (PFAS), novel methods of profiling their presence and modifications are greatly needed as some have known toxic and bioaccumulative characteristics while others have unknown effects. This task however is not simple as over 5000 PFAS of interest have been named by the Environmental Protection Agency and this list continues to grow daily. In this work, we utilized widely available archived and field-sampled pine needles and a novel non-targeted analytical method to evaluate the temporal and spatial presence of numerous PFAS. Over 70 PFAS were detected in the pine needles from this study, providing information from the last six decades related to PFAS exposure, contamination, and reduction.}, publisher={Cold Spring Harbor Laboratory}, author={Kirkwood, Kaylie I. and Fleming, Jonathon and Nguyen, Helen and Reif, David M. and Baker, Erin S. and Belcher, Scott M.}, year={2021}, month={Aug} }
 @article{heindel_belcher_flaws_prins_ho_mao_patisaul_ricke_rosenfeld_soto_et al._2020, title={Data integration, analysis, and interpretation of eight academic CLARITY-BPA studies}, volume={98}, ISSN={["1873-1708"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85089071914&partnerID=MN8TOARS}, DOI={10.1016/j.reprotox.2020.05.014}, abstractNote={"Consortium Linking Academic and Regulatory Insights on BPA Toxicity" (CLARITY-BPA) was a comprehensive "industry-standard" Good Laboratory Practice (GLP)-compliant 2-year chronic exposure study of bisphenol A (BPA) toxicity that was supplemented by hypothesis-driven independent investigator-initiated studies. The investigator-initiated studies were focused on integrating disease-associated, molecular, and physiological endpoints previously found by academic scientists into an industry standard guideline-compliant toxicity study. Thus, the goal of this collaboration was to provide a more comprehensive dataset upon which to base safety standards and to determine whether industry-standard tests are as sensitive and predictive as molecular and disease-associated endpoints. The goal of this report is to integrate the findings from the investigator-initiated studies into a comprehensive overview of the observed impacts of BPA across the multiple organs and systems analyzed. For each organ system, we provide the rationale for the study, an overview of methodology, and summarize major findings. We then compare the results of the CLARITY-BPA studies across organ systems with the results of previous peer-reviewed studies from independent labs. Finally, we discuss potential influences that contributed to differences between studies. Developmental exposure to BPA can lead to adverse effects in multiple organs systems, including the brain, prostate gland, urinary tract, ovary, mammary gland, and heart. As published previously, many effects were at the lowest dose tested, 2.5μg/kg /day, and many of the responses were non-monotonic. Because the low dose of BPA affected endpoints in the same animals across organs evaluated in different labs, we conclude that these are biologically - and toxicologically - relevant.}, journal={REPRODUCTIVE TOXICOLOGY}, author={Heindel, Jerrold J. and Belcher, Scott and Flaws, Jodi A. and Prins, Gail S. and Ho, Shuk-Mei and Mao, Jiude and Patisaul, Heather B. and Ricke, William and Rosenfeld, Cheryl S. and Soto, Ana M. and et al.}, year={2020}, month={Dec}, pages={29–60} }
 @article{guillette_mccord_guillette_polera_rachels_morgeson_kotlarz_knappe_reading_strynar_et al._2020, title={Elevated levels of per- and polyfluoroalkyl substances in Cape Fear River Striped Bass (Morone saxatilis) are associated with biomarkers of altered immune and liver function}, volume={136}, ISSN={["1873-6750"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85079172705&partnerID=MN8TOARS}, DOI={10.1016/j.envint.2019.105358}, abstractNote={Per- and polyfluoroalkyl substances (PFAS) are anthropogenic chemicals of concern that persist in the environment. Environmental monitoring revealed high concentrations of hexafluoropropylene oxide dimer acid (HFPO-DA) and other novel PFAS in the lower Cape Fear River; however, there is limited information on PFAS exposures and effects of this contamination on aquatic biota. Serum concentrations of 23 PFAS in Striped Bass (Morone saxatilis) from the Cape Fear River (n = 58) and a reference population from an aquaculture laboratory on the Pamlico/Tar watershed (n = 29) were quantified using liquid chromatography and high-resolution mass spectrometry, and correlations between PFAS concentrations and health-related serum biomarkers were evaluated. Perfluorooctane sulfonate, the predominant PFAS in Cape Fear River Striped Bass serum, was detectable in every sample with serum concentrations reaching 977 ng/mL. Perfluorononanoic and perfluorodecanoic acid were also detected in all samples, with perfluorohexanesulfonic acid present in >98% of the samples. HFPO-DA (range <0.24-5.85 ng/mL) and Nafion byproduct 2 (range <0.2-1.03 ng/mL) were detected in 48% and 78% of samples, respectively. The mean total PFAS concentration found in domestic Striped Bass raised in well-water under controlled aquaculture conditions was 40 times lower, with HPFO-DA detected in 10% of the samples, and Nafion byproduct 2 was not detected. The elevated PFAS concentrations found in the Cape Fear River Striped Bass were associated with biomarkers of alterations in the liver and immune system.}, journal={ENVIRONMENT INTERNATIONAL}, author={Guillette, T. C. and McCord, James and Guillette, Matthew and Polera, M. E. and Rachels, Kyle T. and Morgeson, Clint and Kotlarz, Nadine and Knappe, Detlef R. U. and Reading, Benjamin J. and Strynar, Mark and et al.}, year={2020}, month={Mar} }
 @article{jackson_ryherd_scheibly_sasser_guillette_belcher_2020, title={Gestational Cd Exposure in the CD-1 Mouse Induces Sex-Specific Hepatic Insulin Insensitivity, Obesity, and Metabolic Syndrome in Adult Female Offspring}, volume={178}, ISSN={["1096-0929"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85097035964&partnerID=MN8TOARS}, DOI={10.1093/toxsci/kfaa154}, abstractNote={There is compelling evidence that developmental exposure to toxic metals increases risk for obesity and obesity-related morbidity including cardiovascular disease and type 2 diabetes. To explore the hypothesis that developmental Cd exposure increases risk of obesity later in life, male, and female CD-1 mice were maternally exposed to 500 ppb CdCl2 in drinking water during a human gestational equivalent period (gestational day 0-postnatal day 10 [GD0-PND10]). Hallmark indicators of metabolic disruption, hepatic steatosis, and metabolic syndrome were evaluated prior to birth through adulthood. Maternal blood Cd levels were similar to those observed in human pregnancy cohorts, and Cd was undetected in adult offspring. There were no observed impacts of exposure on dams or pregnancy-related outcomes. Results of glucose and insulin tolerance testing revealed that Cd exposure impaired offspring glucose homeostasis on PND42. Exposure-related increases in circulating triglycerides and hepatic steatosis were apparent only in females. By PND120, Cd-exposed females were 30% heavier with 700% more perigonadal fat than unexposed control females. There was no evidence of dyslipidemia, steatosis, increased weight gain, nor increased adiposity in Cd-exposed male offspring. Hepatic transcriptome analysis on PND1, PND21, and PND42 revealed evidence for female-specific increases in oxidative stress and mitochondrial dysfunction with significant early disruption of retinoic acid signaling and altered insulin receptor signaling consistent with hepatic insulin sensitivity in adult females. The observed steatosis and metabolic syndrome-like phenotypes resulting from exposure to 500 ppb CdCl2 during the pre- and perinatal period of development equivalent to human gestation indicate that Cd acts developmentally as a sex-specific delayed obesogen.}, number={2}, journal={TOXICOLOGICAL SCIENCES}, author={Jackson, Thomas W. and Ryherd, Garret L. and Scheibly, Chris M. and Sasser, Aubrey L. and Guillette, T. C. and Belcher, Scott M.}, year={2020}, month={Dec}, pages={264–280} }
 @article{jackson_bendfeldt_beam_rock_belcher_2020, title={Heterozygous mutation of Sonic Hedgehog receptor (Ptch) drives cerebellar overgrowth and sex-specifically alters hippocampal and cortical layer structure, activity, and social behavior in female mice}, volume={1}, url={https://doi.org/10.1101/2020.01.25.919506}, DOI={10.1101/2020.01.25.919506}, abstractNote={Sonic hedgehog (SHH) signaling is essential for the differentiation and migration of early stem cell populations during cerebellar development. Dysregulation of SHH-signaling can result in cerebellar overgrowth and the formation of the brain tumor medulloblastoma. Treatment for medulloblastoma is extremely aggressive and patients suffer life-long side effects including behavioral deficits. Considering that other behavioral disorders including autism spectrum disorders, holoprosencephaly, and basal cell nevus syndrome are known to present with cerebellar abnormalities, it is proposed that some behavioral abnormalities could be inherent to the medulloblastoma sequalae rather than treatment. Using a haploinsufficient SHH receptor knockout mouse model (Ptch1+/-), a partner preference task was used to explore activity, social behavior and neuroanatomical changes resulting from dysregulated SHH signaling. Compared to wild-type, Ptch1+/- females displayed increased activity by traveling a greater distance in both open-field and partner preference tasks. Social behavior was also sex-specifically modified in Ptch1+/- females that interacted more with both novel and familiar animals in the partner preference task compared to same-sex wild-type controls. Haploinsufficency of PTCH resulted in cerebellar overgrowth in lobules IV/V and IX of both sexes, and female-specific decreases in hippocampal size and isocortical layer thickness. Taken together, neuroanatomical changes related to deficient SHH signaling may alter social behavior.}, publisher={Cold Spring Harbor Laboratory}, author={Jackson, Thomas W. and Bendfeldt, Gabriel A. and Beam, Kelby A. and Rock, Kylie D. and Belcher, Scott M.}, year={2020}, month={Jan} }
 @article{jackson_bendfeldt_beam_rock_belcher_2020, title={Heterozygous mutation of sonic hedgehog receptor (Ptch1) drives cerebellar overgrowth and sex-specifically alters hippocampal and cortical layer structure, activity, and social behavior in female mice}, volume={78}, ISSN={["1872-9738"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85080064843&partnerID=MN8TOARS}, DOI={10.1016/j.ntt.2020.106866}, abstractNote={Sonic hedgehog (SHH) signaling is essential for the differentiation and migration of early stem cell populations during cerebellar development. Dysregulation of SHH-signaling can result in cerebellar overgrowth and the formation of the brain tumor medulloblastoma. Treatment for medulloblastoma is extremely aggressive and patients suffer life-long side effects including behavioral deficits. Considering that other behavioral disorders including autism spectrum disorders, holoprosencephaly, and basal cell nevus syndrome are known to present with cerebellar abnormalities, it is proposed that some behavioral abnormalities could be inherent to the medulloblastoma sequalae rather than treatment. Using a haploinsufficient SHH receptor knockout mouse model (Ptch1+/−), a partner preference task was used to explore activity, social behavior and neuroanatomical changes resulting from dysregulated SHH signaling. Compared to wild-type, Ptch1+/− females displayed increased activity by traveling a greater distance in both open-field and partner preference tasks. Social behavior was also sex-specifically modified in Ptch1+/− females that interacted more with both novel and familiar animals in the partner preference task compared to same-sex wild-type controls. Haploinsufficiency of PTCH1 resulted in cerebellar overgrowth in lobules IV/V and IX of both sexes, and female-specific decreases in hippocampal size and isocortical layer thickness. Taken together, neuroanatomical changes related to deficient SHH signaling may alter social behavior.}, journal={NEUROTOXICOLOGY AND TERATOLOGY}, author={Jackson, Thomas W. and Bendfeldt, Gabriel A. and Beam, Kelby A. and Rock, Kylie D. and Belcher, Scott M.}, year={2020} }
 @article{sorrow_maguire_murphy_belcher_hoyo_2019, title={Elevated metabolites of acetaminophen in cord blood of children with obesity}, volume={14}, ISSN={["2047-6302"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85053785805&partnerID=MN8TOARS}, DOI={10.1111/ijpo.12465}, abstractNote={High‐throughput metabolomics has been used cross‐sectionally to evaluate differential metabolic profiles associated with human obesity.}, number={1}, journal={PEDIATRIC OBESITY}, author={Sorrow, P. and Maguire, R. and Murphy, S. K. and Belcher, S. M. and Hoyo, C.}, year={2019}, month={Jan} }
 @article{belcher_cline_conley_groeters_jefferson_law_mackey_suen_williams_dixon_et al._2019, title={Endocrine Disruption and Reproductive Pathology}, volume={47}, ISSN={["1533-1601"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85076490433&partnerID=MN8TOARS}, DOI={10.1177/0192623319879903}, abstractNote={During the past 20 years, investigations involving endocrine active substances (EAS) and reproductive toxicity have dominated the landscape of ecotoxicological research. This has occurred in concert with heightened awareness in the scientific community, general public, and governmental entities of the potential consequences of chemical perturbation in humans and wildlife. The exponential growth of experimentation in this field is fueled by our expanding knowledge into the complex nature of endocrine systems and the intricacy of their interactions with xenobiotic agents. Complicating factors include the ever-increasing number of novel receptors and alternate mechanistic pathways that have come to light, effects of chemical mixtures in the environment versus those of single EAS laboratory exposures, the challenge of differentiating endocrine disruption from direct cytotoxicity, and the potential for transgenerational effects. Although initially concerned with EAS effects chiefly in the thyroid glands and reproductive organs, it is now recognized that anthropomorphic substances may also adversely affect the nervous and immune systems via hormonal mechanisms and play substantial roles in metabolic diseases, such as type 2 diabetes and obesity.}, number={8}, journal={TOXICOLOGIC PATHOLOGY}, author={Belcher, Scott M. and Cline, J. Mark and Conley, Justin and Groeters, Sibylle and Jefferson, Wendy N. and Law, Mac and Mackey, Emily and Suen, Alisa A. and Williams, Carmen J. and Dixon, Darlene and et al.}, year={2019}, month={Dec}, pages={1049–1071} }
 @article{hudson_belcher_cowley_2019, title={Maternal cadmium exposure in the mouse leads to increased heart weight at birth and programs susceptibility to hypertension in adulthood}, volume={9}, ISSN={["2045-2322"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85072393964&partnerID=MN8TOARS}, DOI={10.1038/s41598-019-49807-5}, abstractNote={Abstract Cadmium (Cd) is a toxic heavy metal ubiquitous in the environment. Maternal exposure to Cd is associated with fetal growth restriction, trace element deficiencies, and congenital malformations. Cd exposure during adulthood is associated with cardiovascular disease (CVD); however, the effects of maternal Cd exposure on offspring cardiovascular development and disease are not well-understood. Utilizing a mouse model of maternal Cd exposure, we show that offspring born to Cd-exposed mothers have increased heart weights at birth and susceptibility to hypertension during adulthood. Despite inefficient maternal-fetal transfer of Cd, maternal Cd alters fetal levels of essential trace elements including a deficiency in iron, which is required for cardiovascular system development, oxygen homeostasis, and cellular metabolism. RNA-seq on newborn hearts identifies differentially expressed genes associated with maternal Cd exposure that are enriched for functions in CVD, hypertension, enlarged hearts, cellular energy, and hypoxic stress. We propose that a maternal Cd exposure-induced iron deficiency leads to altered cellular metabolic pathways and hypoxic conditions during fetal development; this stress may contribute to increased heart weight at birth and the programming of susceptibility to hypertension in adulthood. These studies will give insights into potential mechanisms through which maternal Cd exposure impacts cardiovascular development and disease.}, number={1}, journal={SCIENTIFIC REPORTS}, publisher={Springer Science and Business Media LLC}, author={Hudson, Kathleen M. and Belcher, Scott M. and Cowley, Michael}, year={2019}, month={Sep} }
 @article{prins_patisaul_belcher_vandenberg_2018, title={CLARITY-BPA academic laboratory studies identify consistent low-dose Bisphenol A effects on multiple organ systems}, volume={125}, ISSN={1742-7835}, url={http://dx.doi.org/10.1111/bcpt.13125}, DOI={10.1111/bcpt.13125}, abstractNote={Bisphenol A (BPA) is a high-production chemical used in a variety of applications worldwide. While BPA has been documented as an endocrine-disrupting chemical (EDC) having adverse health-related outcomes in multiple studies, risk assessment for BPA has lagged due to reliance on guideline toxicology studies over academic ones with end-points considered more sensitive and appropriate. To address current controversies on BPA safety, the United States National Institute of Environmental Health Sciences (NIEHS), the National Toxicology Program (NTP) and the Food and Drug Administration (FDA) established the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA) using the NCTR Sprague-Dawley rats. The goal of CLARITY-BPA is to perform a traditional regulatory toxicology study (Core study) in conjunction with multiple behavioural, molecular and cellular studies by academic laboratories focused on previously identified BPA-sensitive organ systems (Academic studies). Combined analysis of the data from both study types will be undertaken by the NTP with the aim of resolving uncertainties on BPA toxicity. To date, the Core study has been completed and a draft report released. Most of the academic studies have also been finalized and published in peer-reviewed journals. In light of this important milestone, the PPTOX-VI meeting held in the Faroe Islands, 27-30 May 2018 devoted a plenary session to CLARITY-BPA with presentations by multiple investigators with the purpose of highlighting key outcome. This MiniReview synthesizes the results of three academic studies presented at this plenary session, evaluates recently published findings by other CLARITY-BPA academic studies to provide an early combined overview of this emerging data and places this in the context of the Core study findings. This co-ordinated effort revealed a plethora of significant BPA effects across multiple organ systems and BPA doses with non-monotonic responses across the dose range utilized. Remarkably consistent across most studies, including the Core study, are low-dose effects (2.5, 25 and 250 μg BPA/kg body-weight). Collectively, the findings highlighted herein corroborate a significant body of evidence that documents adverse effects of BPA at doses relevant to human exposures and emphasizes the need for updated risk assessment analysis.}, number={S3}, journal={Basic & Clinical Pharmacology & Toxicology}, publisher={Wiley}, author={Prins, Gail S. and Patisaul, Heather B. and Belcher, Scott M. and Vandenberg, Laura N.}, year={2018}, month={Oct}, pages={14–31} }
 @article{belcher_2018, title={CLARITY-BPA: Heart (Belcher)}, DOI={10.22427/ntp-data-018-00016-0001-000-7}, journal={Chemical Effects in Biological Systems (CEBS)}, publisher={NIEHS}, author={Belcher, Scott}, year={2018}, month={Aug} }
 @article{guillette_jackson_belcher_2018, title={Duality of estrogen receptor beta action in cancer progression}, volume={41}, ISSN={["1471-4973"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85047091078&partnerID=MN8TOARS}, DOI={10.1016/j.coph.2018.05.001}, abstractNote={The physiological actions of estrogens are primarily mediated by the nuclear hormone receptors estrogen receptor alpha (ERα) and beta (ERβ). Activities of these nuclear steroid hormone receptors in etiology and progression of many hormone-responsive cancers are well-established, yet the specific role of each receptor, and their various expressed isoforms, in estrogen-responsive cancers remains unclear. Recent advances in nuclear receptor profiling, characterization of expressed splice variants, and the availability of new experimental cancer models, has extended the understanding of the complex interplay between the differentially expressed nuclear estrogen receptors. In this review, we discuss proposed roles of ERβ in several subtypes of cancers that lack significant ERα expression and define current understanding of how different ERs collaborate to regulate cellular processes.}, journal={CURRENT OPINION IN PHARMACOLOGY}, author={Guillette, T. C. and Jackson, Thomas W. and Belcher, Scott M.}, year={2018}, month={Aug}, pages={66–73} }
 @article{gear_kendziorski_belcher_2017, title={Effects of bisphenol A on incidence and severity of cardiac lesions in the NCTR-Sprague-Dawley rat: A CLARITY-BPA study}, volume={275}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85019206640&partnerID=MN8TOARS}, DOI={10.1016/j.toxlet.2017.05.011}, abstractNote={The goal of this study was to determine whether bisphenol A (BPA) had adverse effects indicative of cardiac toxicity. As part of the "Consortium Linking Academic and Regulatory Insights on BPA Toxicity" (CLARITY-BPA), study dams and offspring were exposed by daily gavage to five doses of BPA ranging from 2.5 to 25000μg/kg/day, 0.05 or 0.5μg/kg/day 17α-ethinyl-estradiol (EE) or 0.3% carboxymethylcellulose vehicle. Exposure-related effects were analyzed in isolated hearts by quantitative morphometry and histopathology. No dose-related changes in body weight were detected. Across all exposure groups including vehicle controls, body weight of continuously dosed males was reduced compared to males dosed only until PND21. Heart weight was increased only in females exposed to EE, and consistent alterations in LV wall thickness were not observed. Exposure-related changes in collagen accumulation were minor and limited to highest EE exposure groups with increased collagen accumulation in PND21 males. Decreased collagen was observed in hearts of BPA or EE exposed females at PND90 and PND180. In BPA or EE treated females cardiomyopathy incidence and severity was significantly increased compared to control females at PND21 with myocardial degeneration observed in both males and females at PND21 and PND90.}, journal={Toxicology Letters}, author={Gear, R. and Kendziorski, J.A. and Belcher, Scott}, year={2017}, pages={123–135} }
 @book{patisaul_belcher_2017, title={Endocrine Disruptors, Brain, and Behavior}, DOI={10.1093/acprof:oso/9780199935734.001.0001}, abstractNote={Hormones play a foundational role in the sex-specific organization of the brain and, consequently, the complex behaviors they coordinate. Our world and bodies are becoming increasingly polluted with chemicals capable of interfering with hormone action and thus, possibly, our neural and mental health. If and how these endocrine-disrupting compounds (EDCs) affect the development and function of the brain, and may be contributing to neural disorders that are rapidly rising in prevalence, are the central concerns of this book. This work also examines why even the concept of endocrine disruption is controversial in some circles; how differing definitions of endocrine disruption and “adverse” outcomes shape public policy; and where the current capacity to evaluate chemicals for safety in a regulatory context begins and ends. Fundamental concepts of the EDC hypothesis, including critical windows of exposure and sexually dimorphic effects, are explained. A historical perspective on how the endocrine disruption hypothesis emerged and a summary of how and to what degree prototypical EDCs affect human brain health are provided as a prelude to a critical evaluation of the evidence linking EDC exposures to human neurobehavioral disorders. The book concludes with suggestions for future research needs and a summary of emerging technology that might prove more capable of effectively evaluating existing and new chemicals for endocrine-disrupting properties. The impossibility of disentangling the “science” of EDC action on the brain and behavior from its public health policy implications and economic influence is comprehensively addressed throughout.}, journal={Oxford Scholarship Online}, publisher={Oxford University Press}, author={Patisaul, Heather B. and Belcher, Scott M.}, year={2017}, month={May} }
 @article{belcher_burton_cookman_kirby_miranda_saeed_wray_2017, title={Estrogen and soy isoflavonoids decrease sensitivity of medulloblastoma and central nervous system primitive neuroectodermal tumor cells to chemotherapeutic cytotoxicity}, volume={18}, ISSN={["2050-6511"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85028957141&partnerID=MN8TOARS}, DOI={10.1186/s40360-017-0160-7}, abstractNote={Our previous studies demonstrated that growth and migration of medulloblastoma (MB), the most common malignant brain tumor in children, are stimulated by 17β-estradiol. The growth stimulating effects of estrogens are mediated through ERβ and insulin-like growth factor 1 signaling to inhibit caspase 3 activity and reduce tumor cell apoptosis. The objective of this study was to determine whether estrogens decreased sensitivity of MB cells to cytotoxic actions of chemotherapeutic drugs.Using in vitro cell viability and clonogenic survival assays, concentration response analysis was used to determine whether the cytoprotective effects of estradiol protected human D283 Med MB cells from the cytotoxic actions of the MB chemotherapeutic drugs cisplatin, vincristine, or lomustine. Additional experiments were done to determine whether the ER antagonist fulvestrant or the selective ER modulator tamoxifen blocked the cytoprotective actions of estradiol. ER-selective agonists and antagonists were used to define receptor specificity, and the impacts of the soy-derived phytoestrogens genistein, daidzein, and s-equol on chemosensitivity were evaluated.In D283 Med cells the presence of 10 nM estradiol increased the IC50 for cisplatin-induced inhibition of viability 2-fold from ~5 μM to >10 μM. In clonogenic survival assays estradiol decreased the chemosensitivity of D283 Med cells exposed to cisplatin, lomustine and vincristine. The ERβ selective agonist DPN and low physiological concentrations of the soy-derived phytoestrogens genistein, daidzein, and s-equol also decreased sensitivity of D283 Med cells to cisplatin. The protective effects of estradiol were blocked by the antiestrogens 4-hydroxytamoxifen, fulvestrant (ICI 182,780) and the ERβ selective antagonist PPHTP. Whereas estradiol also decreased chemosensitivity of PFSK-1 cells, estradiol increased sensitivity of Daoy cell to cisplatin, suggesting that ERβ mediated effects may vary in different MB celltypes.These findings demonstrate that E2 and environmental estrogens decrease sensitivity of MB to cytotoxic chemotherapeutics, and that ERβ selective and non-selective inhibition of estrogen receptor activity blocks these cytoprotective actions. These findings support the therapeutic potential of antiestrogen adjuvant therapies for MB, and findings that soy phytoestrogens also decrease sensitivity of MB cells to cytotoxic chemotherapeutics suggest that decreased exposure to environmental estrogens may benefit MB patient responses to chemotherapy.}, number={1}, journal={BMC PHARMACOLOGY & TOXICOLOGY}, author={Belcher, Scott M. and Burton, Caleb C. and Cookman, Clifford J. and Kirby, Michelle and Miranda, Gabriel L. and Saeed, Fatima O. and Wray, Kathleen E.}, year={2017}, month={Sep} }
 @article{leung_govindarajah_cheong_veevers_song_gear_zhu_ying_kendler_medvedovic_et al._2017, title={Gestational high-fat diet and bisphenol A exposure heightens mammary cancer risk}, volume={24}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85020741436&partnerID=MN8TOARS}, DOI={10.1530/ERC-17-0006}, abstractNote={In utero exposure to bisphenol A (BPA) increases mammary cancer susceptibility in offspring. High-fat diet is widely believed to be a risk factor of breast cancer. The objective of this study was to determine whether maternal exposure to BPA in addition to high-butterfat (HBF) intake during pregnancy further influences carcinogen-induced mammary cancer risk in offspring, and its dose–response curve. In this study, we found that gestational HBF intake in addition to a low-dose BPA (25 µg/kg BW/day) exposure increased mammary tumor incidence in a 50-day-of-age chemical carcinogen administration model and altered mammary gland morphology in offspring in a non-monotonic manner, while shortening tumor-free survival time compared with the HBF-alone group. In utero HBF and BPA exposure elicited differential effects at the gene level in PND21 mammary glands through DNA methylation, compared with HBF intake in the absence of BPA. Top HBF + BPA-dysregulated genes (ALDH1B1, ASTL, CA7, CPLX4, KCNV2, MAGEE2 and TUBA3E) are associated with poor overall survival in The Cancer Genomic Atlas (TCGA) human breast cancer cohort (n = 1082). Furthermore, the prognostic power of the identified genes was further enhanced in the survival analysis of Caucasian patients with estrogen receptor-positive tumors. In conclusion, concurrent HBF dietary and a low-dose BPA exposure during pregnancy increases mammary tumor incidence in offspring, accompanied by alterations in mammary gland development and gene expression, and possibly through epigenetic reprogramming.}, number={7}, journal={Endocrine-related cancer}, author={Leung, Y.-K. and Govindarajah, V. and Cheong, A. and Veevers, J. and Song, D. and Gear, R. and Zhu, X. and Ying, J. and Kendler, A. and Medvedovic, M. and et al.}, year={2017}, pages={365–378} }
 @article{gear_belcher_2017, title={Impacts of Bisphenol A and Ethinyl Estradiol on Male and Female CD-1 Mouse Spleen}, volume={7}, ISSN={["2045-2322"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85018428280&partnerID=MN8TOARS}, DOI={10.1038/s41598-017-00961-8}, abstractNote={Abstract The endocrine disruptor bisphenol A (BPA) and the pharmaceutical 17α-ethinyl estradiol (EE) are synthetic chemicals with estrogen-like activities. Despite ubiquitous human exposure to BPA, and the wide-spread clinical use of EE as oral contraceptive adjuvant, the impact of these estrogenic endocrine disrupting chemicals (EDCs) on the immune system is unclear. Here we report results of in vivo dose response studies that analyzed the histology and microstructural changes in the spleen of adult male and female CD-1 mice exposed to 4 to 40,000 μg/kg/day BPA or 0.02 to 2 μg/kg/day EE from conception until 12–14 weeks of age. Results of that analysis indicate that both BPA and EE have dose- and sex-specific impacts on the cellular and microanatomical structures of the spleens that reveal minor alterations in immunomodulatory and hematopoietic functions. These findings support previous studies demonstrating the murine immune system as a sensitive target for estrogens, and that oral exposures to BPA and EE can have estrogen-like immunomodulatory affects in both sexes.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Gear, Robin B. and Belcher, Scott M.}, year={2017}, month={Apr} }
 @article{arambula_belcher_planchart_turner_patisaul_2016, title={Impact of Low Dose Oral Exposure to Bisphenol A (BPA) on the Neonatal Rat Hypothalamic and Hippocampal Transcriptome: A CLARITY-BPA Consortium Study}, volume={157}, ISSN={0013-7227 1945-7170}, url={http://dx.doi.org/10.1210/en.2016-1339}, DOI={10.1210/en.2016-1339}, abstractNote={Bisphenol A (BPA) is an endocrine disrupting, high volume production chemical found in a variety of products. Evidence of prenatal exposure has raised concerns that developmental BPA may disrupt sex-specific brain organization and, consequently, induce lasting changes on neurophysiology and behavior. We and others have shown that exposure to BPA at doses below the no-observed-adverse-effect level can disrupt the sex-specific expression of estrogen-responsive genes in the neonatal rat brain including estrogen receptors (ERs). The present studies, conducted as part of the Consortium Linking Academic and Regulatory Insights of BPA Toxicity program, expanded this work by examining the hippocampal and hypothalamic transcriptome on postnatal day 1 with the hypothesis that genes sensitive to estrogen and/or sexually dimorphic in expression would be altered by prenatal BPA exposure. NCTR Sprague-Dawley dams were gavaged from gestational day 6 until parturition with BPA (0-, 2.5-, 25-, 250-, 2500-, or 25 000-μg/kg body weight [bw]/d). Ethinyl estradiol was used as a reference estrogen (0.05- or 0.5-μg/kg bw/d). Postnatal day 1 brains were microdissected and gene expression was assessed with RNA-sequencing (0-, 2.5-, and 2500-μg/kg bw BPA groups only) and/or quantitative real-time PCR (all exposure groups). BPA-related transcriptional changes were mainly confined to the hypothalamus. Consistent with prior observations, BPA induced sex-specific effects on hypothalamic ERα and ERβ (Esr1 and Esr2) expression and hippocampal and hypothalamic oxytocin (Oxt) expression. These data demonstrate prenatal BPA exposure, even at doses below the current no-observed-adverse-effect level, can alter gene expression in the developing brain.}, number={10}, journal={Endocrinology}, publisher={The Endocrine Society}, author={Arambula, Sheryl E. and Belcher, Scott M. and Planchart, Antonio and Turner, Stephen D. and Patisaul, Heather B.}, year={2016}, month={Aug}, pages={3856–3872} }
 @article{belcher_gear_kendig_2015, title={Bisphenol a alters autonomic tone and extracellular matrix structure and induces sex-specific effects on cardiovascular function in male and female CD-1 mice}, volume={156}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84923884313&partnerID=MN8TOARS}, DOI={10.1210/en.2014-1847}, abstractNote={The aim of this study was to determine whether bisphenol A (BPA) has adverse effects on cardiovascular functions in CD-1 mice and define sex-specific modes of BPA action in the heart. Dams and analyzed progeny were maintained on a defined diet containing BPA (0.03, 0.3, 3, 30, or 300 ppm) that resulted in BPA exposures from 4-5 to approximately 5000 μg/kg · d or a diet containing 17α-ethinyl estradiol (EE; ∼0.02, 0.2, and 0.15 μg/kg · d) as an oral bioavailable estrogen control. Assessment of electrocardiogram parameters using noninvasive methods found that ventricular functions in both male and female mice were not altered by either BPA or EE. However, exposure-related changes in the rates of ventricular contraction, suggestive of a shift in sympathovagal balance of heart rate control toward increased parasympathetic activity, were detected in males. Decreased systolic blood pressure was observed in males exposed to BPA above 5 μg/kg · d and in females from the highest BPA exposure group. Morphometric histological measures revealed sexually dimorphic changes in the composition of the cardiac collagen extracellular matrix, increases in fibrosis, and evidence of modest exposure-related remodeling. Experiments using the α-selective adrenergic agonist phenylephrine found that BPA enhanced reflex bradycardia in females, but not males, revealed that BPA and EE exposure sex specifically altered the sympathetic regulation of the baroreflex circuits. Increased sensitivity to the cardiotoxic effects of the β-adrenergic agonist isoproterenol was observed in BPA- and EE-exposed females. This effect was not observed in males, in which BPA or EE exposures were protective of isoproterenol-induced ischemic damage and hypertrophy. The results of RNA sequence analysis identified significant sex-specific changes in gene expression in response to BPA that were consistent with the observed exposure-related phenotypic changes in the collagenous and noncollagenous extracellular matrix, cardiac remodeling, altered autonomic responses, changes in ion channel and transporter functions, and altered glycolytic and lipid metabolism.}, number={3}, journal={Endocrinology}, author={Belcher, S.M. and Gear, R.B. and Kendig, E.L.}, year={2015}, pages={882–895} }
 @article{kendziorski_belcher_2015, title={Effects of whole life exposure to Bisphenol A or 17α-ethinyl estradiol in uterus of nulligravida CD1 mice}, volume={5}, ISSN={2352-3409}, url={http://dx.doi.org/10.1016/J.DIB.2015.10.034}, DOI={10.1016/J.DIB.2015.10.034}, abstractNote={Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) with known estrogenic activity. Exposure to BPA in adult mice was shown previously to increase uterine pathology with associated alterations in the immune response and fibrosis. Reported here are uterine histopathology findings from CD1 mice exposed to BPA or 17α-ethinyl estradiol at multiple doses from conception through postnatal day 90. Along with uterine pathology, impacts of exposure on collagen accumulation and F4/80 positive macrophage numbers, as an indicator of immune response in the endometrium and myometrium, are presented. These companion data are from offspring (F1) of the dams analyzed for effects of adult exposures published in the Reproductive Toxicology manuscript titled “Strain-Specific Induction of Endometrial Periglandular Fibrosis in Mice Exposed during Adulthood to the Endocrine Disrupting Chemical Bisphenol A” (doi: 10.1016/j.reprotox.2015.08.001).}, journal={Data in Brief}, publisher={Elsevier BV}, author={Kendziorski, Jessica A. and Belcher, Scott M.}, year={2015}, month={Dec}, pages={948–953} }
 @article{cookman_belcher_2015, title={Estrogen receptor-β up-regulates IGF1R expression and activity to inhibit apoptosis and increase growth of medulloblastoma}, volume={156}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84937605783&partnerID=MN8TOARS}, DOI={10.1210/en.2015-1141}, abstractNote={Medulloblastoma (Med) is the most common malignant brain tumor in children. The role of ESR2 [estrogen receptor (ER)-β] in promoting Med growth was comprehensively examined in three in vivo models and human cell lines. In a novel Med ERβ-null knockout model developed by crossing Esr2(-/-) mice with cerebellar granule cell precursor specific Ptch1 conditional knockout mice, the tumor growth rate was significantly decreased in males and females. The absence of Esr2 resulted in increased apoptosis, decreased B-cell lymphoma 2 (BCL2), and IGF-1 receptor (IGF1R) expression, and decreased levels of active MAPKs (ERK1/2) and protein kinase B (AKT). Treatment of Med in Ptch1(+/-) Trp53(-/-) mice with the antiestrogen chemotherapeutic drug Faslodex significantly increased symptom-free survival, which was associated with increased apoptosis and decreased BCL2 and IGF1R expression and signaling. Similar effects were also observed in nude mice bearing D283Med xenografts. In vitro studies in human D283Med cells metabolically stressed by glutamine withdrawal found that 17β-estradiol and the ERβ selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile dose dependently protected Med cells from caspase-3-dependent cell death. Those effects were associated with increased phosphorylation of IGF1R, long-term increases in ERK1/2 and AKT signaling, and increased expression of IGF-1, IGF1R, and BCL2. Results of pharmacological experiments revealed that the cytoprotective actions of estradiol were dependent on ERβ and IGF1R receptor tyrosine kinase activity and independent of ERα and G protein-coupled estrogen receptor 1 (G protein coupled receptor 30). The presented results demonstrate that estrogen promotes Med growth through ERβ-mediated increases in IGF1R expression and activity, which induce cytoprotective mechanisms that decrease apoptosis.}, number={7}, journal={Endocrinology}, author={Cookman, C.J. and Belcher, S.M.}, year={2015}, pages={2395–2408} }
 @article{matlib_belcher_rapoport_millard_wang_maggio_2015, title={New Master of Science program emphasizing Safety Pharmacology—Results to date}, volume={75}, ISSN={1056-8719}, url={http://dx.doi.org/10.1016/J.VASCN.2015.08.114}, DOI={10.1016/J.VASCN.2015.08.114}, journal={Journal of Pharmacological and Toxicological Methods}, publisher={Elsevier BV}, author={Matlib, Abdul and Belcher, Scott and Rapoport, Robert and Millard, Ron and Wang, Hong-Sheng and Maggio, John}, year={2015}, month={Sep}, pages={191–192} }
 @article{lagarde_beausoleil_belcher_belzunces_emond_guerbet_rousselle_2015, title={Non-monotonic dose-response relationships and endocrine disruptors: A qualitative method of assessment -No section-}, volume={14}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84929008164&partnerID=MN8TOARS}, DOI={10.1186/1476-069X-14-13}, abstractNote={Experimental studies investigating the effects of endocrine disruptors frequently identify potential unconventional dose-response relationships called non-monotonic dose-response (NMDR) relationships. Standardized approaches for investigating NMDR relationships in a risk assessment context are missing. The aim of this work was to develop criteria for assessing the strength of NMDR relationships. A literature search was conducted to identify published studies that report NMDR relationships with endocrine disruptors. Fifty-one experimental studies that investigated various effects associated with endocrine disruption elicited by many substances were selected. Scoring criteria were applied by adaptation of an approach previously used for identification of hormesis-type dose-response relationships. Out of the 148 NMDR relationships analyzed, 82 were categorized with this method as having a “moderate” to “high” level of plausibility for various effects. Numerous modes of action described in the literature can explain such phenomena. NMDR can arise from numerous molecular mechanisms such as opposing effects induced by multiple receptors differing by their affinity, receptor desensitization, negative feedback with increasing dose, or dose-dependent metabolism modulation. A stepwise decision tree was developed as a tool to standardize the analysis of NMDR relationships observed in the literature with the final aim to use these results in a Risk Assessment purpose. This decision tree was finally applied to studies focused on the effects of bisphenol A.}, number={1}, journal={Environmental Health: A Global Access Science Source}, author={Lagarde, F. and Beausoleil, C. and Belcher, S.M. and Belzunces, L.P. and Emond, C. and Guerbet, M. and Rousselle, C.}, year={2015} }
 @article{belcher_2015, title={Response to the letter by Gallo D., et al}, volume={156}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84938684568&partnerID=MN8TOARS}, DOI={10.1210/en.2015-1484}, number={8}, journal={Endocrinology}, author={Belcher, S.M.}, year={2015}, pages={L8–L9} }
 @article{kendziorski_belcher_2015, title={Strain-specific induction of endometrial periglandular fibrosis in mice exposed during adulthood to the endocrine disrupting chemical bisphenol A}, volume={58}, ISSN={0890-6238}, url={http://dx.doi.org/10.1016/J.REPROTOX.2015.08.001}, DOI={10.1016/J.REPROTOX.2015.08.001}, abstractNote={The aim of this study was to compare effects of bisphenol A (BPA) on collagen accumulation in uteri of two mouse strains. Adult C57Bl/6N and CD-1 mice were exposed to dietary BPA (0.004-40mg/kg/day) or 17α-ethinyl estradiol (0.00002-0.001mg/kg/day) as effect control. An equine endometrosis-like phenotype with increased gland nesting and periglandular collagen accumulation was characteristic of unexposed C57Bl/6N, but not CD-1, endometrium. BPA non-monotonically increased gland nest density and periglandular collagen accumulation in both strains. Increased collagen I and III expression, decreased matrix metalloproteinase 2 (MMP2) and MMP14 expression, and increased immune response were associated with the endometrosis phenotype in the C57Bl/6N strain and the 30ppm BPA CD-1 group. The association between the pro-collagen shift in increased collagen expression and decreased MMP2 expression and activity implies that strain differences and BPA exposure alter regulation of endometrial remodeling and contribute to increased fibrosis, a component of several human uterine diseases.}, journal={Reproductive Toxicology}, publisher={Elsevier BV}, author={Kendziorski, Jessica A. and Belcher, Scott M.}, year={2015}, month={Dec}, pages={119–130} }
 @article{kopras_potluri_bermudez_williams_belcher_kasper_2014, title={Actions of endocrine-disrupting chemicals on stem/progenitor cells during development and disease}, volume={21}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84899804987&partnerID=MN8TOARS}, DOI={10.1530/ERC-13-0360}, abstractNote={Development and fate of the stem cell are regulated by extrinsic signals from the environment. Endocrine-disrupting chemicals which perturb hormonal signaling in utero and during early childhood may cause deregulation of multiple developmental processes, ranging from breakdown of stem cell niche architecture, developmental reprograming and altered stem cell fate to impaired organ and gonad development and sexual differentiation. Therefore, study of the environmental effects on stem cell integrity and normal development is a new and emerging focus for developmental biologists and cell toxicologists. When combined with new human and mouse stem cell-based models, stem cell differentiation dynamics can be studied in more biologically relevant ways. In this study, we review the current status of our understanding of the molecular mechanisms by which endocrine disruptors alter embryonic stem cell and adult stem/progenitor cell fate, organ development, cancer stem cell activity, and tumorigenesis.}, number={2}, journal={Endocrine-Related Cancer}, author={Kopras, E. and Potluri, V. and Bermudez, M.-L. and Williams, K. and Belcher, S. and Kasper, S.}, year={2014} }
 @article{khalil_ebert_wang_belcher_lee_czerwinski_kannan_2014, title={Bisphenol A and cardiometabolic risk factors in obese children}, volume={470-471}, ISSN={0048-9697}, url={http://dx.doi.org/10.1016/J.SCITOTENV.2013.09.088}, DOI={10.1016/J.SCITOTENV.2013.09.088}, abstractNote={Bisphenol-A (BPA) is an endocrine disruptor (ED) that has been associated with obesity and metabolic changes in liver in humans. Non-alcoholic fatty liver disease (NAFLD) affects 40% of all obese children in the United States. Association of BPA with NAFLD in children is poorly understood. We investigated if BPA might play a role.In a cross sectional study of 39 obese and overweight children aged 3-8 years enrolled from the Children Medical Center of Dayton, Ohio, anthropometric, clinical and biochemical assessment of serum samples were conducted. Urinary BPA was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was adjusted for urinary creatinine BPA (creatinine) using linear regression and spline analyses.Higher urinary BPA (creatinine) concentration in overweight and obese children was associated with increasing free thyroxine. In male children BPA (creatinine) decreased with age, and was associated with elevated liver enzyme aspartate aminotransferase and diastolic blood pressure. The association of BPA (creatinine) persisted even after adjusting for age and ethnicity. Also in males, BPA concentration unadjusted for creatinine was significantly associated with serum fasting insulin and homeostasis model assessment for insulin resistance (HOMA-IR) showing non-monotonic exposure-response relationship.Urinary BPA in obese children, at least in males is associated with adverse liver and metabolic effects, and high diastolic blood pressure.}, journal={Science of The Total Environment}, publisher={Elsevier BV}, author={Khalil, Naila and Ebert, James R. and Wang, Lei and Belcher, Scott and Lee, Miryoung and Czerwinski, Stefan A. and Kannan, Kurunthachalam}, year={2014}, month={Feb}, pages={726–732} }
 @article{cookman_belcher_2014, title={Classical nuclear hormone receptor activity as a mediator of complex concentration response relationships for endocrine active compounds}, volume={19}, ISSN={1471-4892}, url={http://dx.doi.org/10.1016/J.COPH.2014.09.013}, DOI={10.1016/J.COPH.2014.09.013}, abstractNote={Nonmonotonic concentration response relationships are frequently observed for endocrine active ligands that act via nuclear receptors. The curve of best fit for nonmonotonic concentration response relationships are often inverted U-shaped with effects at intermediate concentrations that are different from effects at higher or lower concentrations. Cytotoxicity is a major mode of action responsible for inverted U-shaped concentration response relationships. However, evidence suggests that ligand selectivity, activation of multiple molecular targets, concerted regulation of multiple opposing endpoints, and multiple ligand binding sites within nuclear receptors also contribute to nonmonotonic concentration response relationships of endocrine active ligands. This review reports the current understanding of mechanisms involved in classical nuclear receptor mediated nonmonotonic concentration response relationships with a focus on studies published between 2012 and 2014.}, journal={Current Opinion in Pharmacology}, publisher={Elsevier BV}, author={Cookman, Clifford J and Belcher, Scott M}, year={2014}, month={Dec}, pages={112–119} }
 @article{belcher_2014, title={Editorial overview: Endocrine and metabolic diseases: Conversations on endocrine disruptors — rising above the din}, volume={19}, ISSN={1471-4892}, url={http://dx.doi.org/10.1016/J.COPH.2014.10.003}, DOI={10.1016/J.COPH.2014.10.003}, abstractNote={Nonmonotonic concentration response relationships are frequently observed for endocrine active ligands that act via nuclear receptors. The curve of best fit for nonmonotonic concentration response relationships are often inverted U-shaped with effects at intermediate concentrations that are different from effects at higher or lower concentrations. Cytotoxicity is a major mode of action responsible for inverted U-shaped concentration response relationships. However, evidence suggests that ligand selectivity, activation of multiple molecular targets, concerted regulation of multiple opposing endpoints, and multiple ligand binding sites within nuclear receptors also contribute to nonmonotonic concentration response relationships of endocrine active ligands. This review reports the current understanding of mechanisms involved in classical nuclear receptor mediated nonmonotonic concentration response relationships with a focus on studies published between 2012 and 2014.}, journal={Current Opinion in Pharmacology}, publisher={Elsevier BV}, author={Belcher, Scott M}, year={2014}, month={Dec}, pages={vi-vii} }
 @article{belcher_cookman_patisaul_stapleton_2014, title={In vitro assessment of human nuclear hormone receptor activity and cytotoxicity of the flame retardant mixture FM 550 and its triarylphosphate and brominated components}, volume={228}, ISSN={0378-4274}, url={http://dx.doi.org/10.1016/J.TOXLET.2014.04.017}, DOI={10.1016/J.TOXLET.2014.04.017}, abstractNote={Firemaster® 550 (FM 550) is a mixture of brominated and triarylphosphate flame retardants used in polyurethane foam-based products. The primary components are also used in numerous other applications and are thus common household and industrial contaminants. Our previous animal studies suggested that FM 550 exposure may alter metabolism and cause weight gain. Employing human nuclear receptor (NR) luciferase reporter assays, the goal of this study was to evaluate the agonist actions of FM 550 and its constituent compounds at NRs with known roles in establishing or regulating energy balance. FM 550 was found to have significant agonist activity only at the master regulator of adipocyte differentiation PPARγ. As a result, the concentration response relationships and relative activities of FM 550 at PPARγ were investigated in more detail with the contribution of each chemical component defined and compared to the activities of the prototypical PPARγ environmental ligands triphenyltin and tributyltin. The resulting data indicated that the primary metabolic disruptive effects of FM 550 were likely mediated by the activity of the triarylphosphates at PPARγ, and have identified TPP as a candidate metabolic disruptor that also acts as a cytotoxicant.}, number={2}, journal={Toxicology Letters}, publisher={Elsevier BV}, author={Belcher, Scott M. and Cookman, Clifford J. and Patisaul, Heather B. and Stapleton, Heather M.}, year={2014}, month={Jul}, pages={93–102} }
 @article{patisaul_roberts_mabrey_mccaffrey_gear_braun_belcher_stapleton_2013, title={Accumulation and Endocrine Disrupting Effects of the Flame Retardant Mixture Firemaster® 550 in Rats: An Exploratory Assessment}, volume={27}, ISSN={1095-6670}, url={http://dx.doi.org/10.1002/jbt.21439}, DOI={10.1002/jbt.21439}, abstractNote={Firemaster® 550 (FM 550), a fire‐retardant mixture used in foam‐based products, was recently identified as a common contaminant in household dust. The chemical structures of its principle components suggest they have endocrine disrupting activity, but nothing is known about their physiological effects at environmentally relevant exposure levels. The goal of this exploratory study was to evaluate accumulation, metabolism and endocrine disrupting effects of FM 550 in rats exposed to 100 or 1000 µg/day across gestation and lactation. FM 550 components accumulated in tissues of exposed dams and offspring and induced phenotypic hallmarks associated with metabolic syndrome in the offspring. Effects included increased serum thyroxine levels and reduced hepatic carboxylesterease activity in dams, and advanced female puberty, weight gain, male cardiac hypertrophy, and altered exploratory behaviors in offspring. Results of this study are the first to implicate FM 550 as an endocrine disruptor and an obesogen at environmentally relevant levels. #x000A9; 2012 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:124‐136, 2013; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21439}, number={2}, journal={Journal of Biochemical and Molecular Toxicology}, publisher={Wiley}, author={Patisaul, Heather B. and Roberts, Simon C. and Mabrey, Natalie and McCaffrey, Katherine A. and Gear, Robin B. and Braun, Joe and Belcher, Scott M. and Stapleton, Heather M.}, year={2013}, month={Feb}, pages={124–136} }
 @article{thigpen_setchell_kissling_locklear_caviness_whiteside_belcher_brown_collins_lih_et al._2013, title={The estrogenic content of rodent diets, bedding, cages, and water bottles and its effect on bisphenol a studies}, volume={52}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84875526300&partnerID=MN8TOARS}, number={2}, journal={Journal of the American Association for Laboratory Animal Science}, author={Thigpen, J.E. and Setchell, K.D.R. and Kissling, G.E. and Locklear, J. and Caviness, G.F. and Whiteside, T. and Belcher, S.M. and Brown, N.M. and Collins, B.J. and Lih, F.B. and et al.}, year={2013}, pages={130–141} }
 @article{kendig_buesing_christie_cookman_gear_hugo_kasper_kendziorski_ungi_williams_et al._2012, title={Estrogen-like disruptive effects of dietary exposure to bisphenol A or 17α-ethinyl estradiol in CD1 mice}, volume={31}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84872079878&partnerID=MN8TOARS}, DOI={10.1177/1091581812463254}, abstractNote={Bisphenol A (BPA) is an endocrine disrupting chemical that is ubiquitous in wild and built environments. Due to variability in study design, the disruptive effects of BPA have proven difficult to experimentally replicate. This study was designed to assess the disruptive actions of dietary BPA exposure, while carefully controlling for known confounders. Parental CD1 mice were acclimated to defined diet containing BPA (0.03, 0.3, 3, 30, or 300 ppm) or 17α-ethinyl estradiol (EE; 0.0001, 0.001, and 0.01 ppm) and bred to produce progeny (F1) that were maintained through adulthood on the same diet as the parents. In F1 females, uterine weights were increased in all EE and the 30-ppm BPA-exposure groups, demonstrating model sensitivity and estrogen-like actions of BPA. In BPA-exposed females, no treatment-related differences were observed in parental reproductive function, or in the timing of puberty and metabolic function in female offspring. In F1 males, modest changes in body weight, adiposity and glucose tolerance, consistent with improved metabolic function, were observed. Associated with increased prolactin and increased circulating testosterone levels, balanopreputial separation was accelerated by 0.03 and 3.0 ppm BPA and anogenital distance at postnatal day 21 was increased in males by 0.03 ppm BPA. Sperm counts were also increased with 3.0 ppm BPA exposures. Overall, BPA was found to have modest, sex specific endocrine disruptive effects on a variety of end points below the established no observed adverse effect level. The dose response characteristics for many of the effects were nonmonotonic and not predictable from high-dose extrapolations.}, number={6}, journal={International Journal of Toxicology}, author={Kendig, E.L. and Buesing, D.R. and Christie, S.M. and Cookman, C.J. and Gear, R.B. and Hugo, E.R. and Kasper, S.N. and Kendziorski, J.A. and Ungi, K.R. and Williams, K. and et al.}, year={2012}, pages={537–550} }
 @article{williams_ghosh_giridhar_gu_case_belcher_kasper_2012, title={Inhibition of stathmin1 accelerates the metastatic process}, volume={72}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84867500471&partnerID=MN8TOARS}, DOI={10.1158/0008-5472.CAN-12-1158}, abstractNote={The oncoprotein stathmin 1 (STMN1) is upregulated in most, if not all, cancers of epithelial cell origin; therefore STMN1 is considered a target for cancer therapy. However, its role during metastasis has not been investigated. Here, we report for the first time that STMN1 strongly inhibits metastatic behavior in both normal epithelial and cancerous epithelial cells. Initially, loss-of-STMN1 compromises cell-cell adhesion. This is followed by epithelial-to-mesenchymal transition (EMT), increased cell migration, and metastasis via cooperative activation of p38 and through TGF-β-independent and -dependent mechanisms. In contrast, expressing STMN1 restores cell-cell adhesion and reverses the metastatic cascade. Primary prostate epithelial cell cultures from benign to undifferentiated adenocarcinoma (UA) clinical biopsies show that EMT-like cells arise while the cancer is still organ-confined and that their emergence is tumor-stage specific. Furthermore, primary EMT-like cells exhibit metastatic behavior both in vitro and in vivo as compared with their non-EMT counterpart. These observations predict that using STMN1 as a generic therapeutic target might accelerate metastasis. Instead, there may be a tumor stage-specific window-of-opportunity in which conserving STMN1 expression is required to inhibit emergence of metastatic disease.}, number={20}, journal={Cancer Research}, author={Williams, K. and Ghosh, R. and Giridhar, P.V. and Gu, G. and Case, T. and Belcher, S.M. and Kasper, S.}, year={2012}, pages={5407–5417} }
 @article{belcher_chen_yan_wang_2012, title={Rapid estrogen receptor-mediated mechanisms determine the sexually dimorphic sensitivity of ventricular myocytes to 17β-estradiol and the environmental endocrine disruptor bispheno A}, volume={153}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84862923332&partnerID=MN8TOARS}, DOI={10.1210/en.2011-1772}, abstractNote={Previously we showed that 17β-estradiol (E(2)) and/or the xenoestrogen bisphenol A (BPA) alter ventricular myocyte Ca(2+) handing, resulting in increased cardiac arrhythmias in a female-specific manner. In the present study, the roles of estrogen receptors (ER) in mediating the rapid contractile and arrhythmogenic effects of estrogens were examined. Contractility was used as an index to assess the impact of E(2) or BPA on Ca(2+) handling in rodent ventricular myocytes. The concentration-response curve for the stimulatory effects of BPA and E(2) on female myocyte was inverted-U shaped. Detectable effects for each compound were observed at 10(-12) M, and the most efficacious concentrations for each were at 10(-9) M. Sensitivity to E(2) and BPA was not observed in male myocytes and was abolished in myocytes from ovariectomized females. Analysis using protein-conjugated E(2) suggests that these rapid actions are induced by membrane-associated receptors. Analysis using selective ER agonists and antagonists and a genetic ERβ knockout mouse model showed that ERα and ERβ have opposing actions in myocytes and that the balance between ERβ and ERα signaling is the prime regulator of the sex-specific sensitivity toward estrogens. The response of female myocytes to E(2) and BPA is dominated by the stimulatory ERβ-mediated signaling, and the absence of BPA and E(2) responsiveness in males is due to a counterbalancing-suppressive action of ERα. We conclude that the sex-specific sensitivity of myocytes to estrogens and the rapid arrhythmogenic effects of BPA and estradiol in the female heart are regulated by the balance between ERα and ERβ signaling.}, number={2}, journal={Endocrinology}, author={Belcher, S.M. and Chen, Y. and Yan, S. and Wang, H.-S.}, year={2012}, pages={712–720} }
 @article{kendziorski_kendig_gear_belcher_2012, title={Strain specific induction of pyometra and differences in immune responsiveness in mice exposed to 17α-ethinyl estradiol or the endocrine disrupting chemical bisphenol A}, volume={34}, ISSN={0890-6238}, url={http://dx.doi.org/10.1016/j.reprotox.2012.03.001}, DOI={10.1016/j.reprotox.2012.03.001}, abstractNote={Pyometra is an inflammatory disease of the uterus that can be caused by chronic exposure to estrogens. It is unknown whether weakly estrogenic endocrine disruptors can cause pyometra. We investigated whether dietary exposures to the estrogenic endocrine disruptor bisphenol A (BPA) induced pyometra. Pyometra did not occur in CD1 mice exposed to different dietary doses of BPA ranging from 4.1 to >4000 μg/kg-d or 17α-ethinyl estradiol (EE; 1.2 to >150 μg/kg-d). In the C57BL/6 strain, pyometra occurred in the 15 μg/kg-d EE and 33 μg/kg-d BPA treatment groups. At the effective concentration of BPA, histological analysis revealed pathological alterations of uterine morphology associated with a >5.3-fold increase in macrophage numbers in non-pyometra uteri of C57BL/6 mice exposed to BPA. These results suggest that BPA enhances immune responsiveness of the uterus and that heightened responsiveness in C57BL/6 females is related to increased susceptibility to pyometra.}, number={1}, journal={Reproductive Toxicology}, publisher={Elsevier BV}, author={Kendziorski, Jessica A. and Kendig, Eric L. and Gear, Robin B. and Belcher, Scott M.}, year={2012}, month={Aug}, pages={22–30} }
 @article{cooper_kendig_belcher_2011, title={Assessment of bisphenol A released from reusable plastic, aluminium and stainless steel water bottles}, volume={85}, ISSN={0045-6535}, url={http://dx.doi.org/10.1016/j.chemosphere.2011.06.060}, DOI={10.1016/j.chemosphere.2011.06.060}, abstractNote={Bisphenol A (BPA) is a ubiquitous high volume industrial chemical that is an estrogen and an environmental endocrine disrupting chemical. Bisphenol A is used extensively in the production of consumer goods, polycarbonate plastics, epoxy resins and coatings used to line metallic food and beverage cans. There is great concern regarding the possible harmful effects from exposures that result from BPA leaching into foods and beverages from packaging or storage containers. The objective of this study was to independently assess whether BPA contamination of water was occurring from different types of reusable drinking bottles marketed as alternatives to BPA-containing polycarbonate plastics. Using a sensitive and quantitative BPA-specific competitive enzyme-linked immunosorbent assay we evaluated whether BPA migrated into water stored in polycarbonate or copolyester plastic bottles, and different lined or unlined metallic reusable water bottles. At room temperature the concentration of BPA migrating from polycarbonate bottles ranged from 0.2 to 0.3 mg L⁻¹. Under identical conditions BPA migration from aluminium bottles lined with epoxy-based resins was variable depending on manufacturer ranging from 0.08 to 1.9 mg L⁻¹. Boiling water significantly increased migration of BPA from the epoxy lined bottles. No detectable BPA contamination was observed in water stored in bottles made from Tritan™ copolyester plastic, uncoated stainless steel, or aluminium lined with EcoCare™. The results from this study demonstrate that when used according to manufacturers' recommendations reusable water bottles constructed from "BPA-free" alternative materials are suitable for consumption of beverages free of BPA contamination.}, number={6}, journal={Chemosphere}, publisher={Elsevier BV}, author={Cooper, James E. and Kendig, Eric L. and Belcher, Scott M.}, year={2011}, month={Oct}, pages={943–947} }
 @article{yan_chen_dong_song_belcher_wang_2011, title={Bisphenol A and 17β-estradiol promote arrhythmia in the female heart via alteration of calcium handling}, volume={6}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-80053201979&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0025455}, abstractNote={Background There is wide-spread human exposure to bisphenol A (BPA), a ubiquitous estrogenic endocrine disruptor that has been implicated as having potentially harmful effects on human heart health. Higher urine BPA concentrations have been shown to be associated with cardiovascular diseases in humans. However, neither the nature nor the mechanism(s) of BPA action on the heart are understood. Methodology/Principal Findings The rapid (<7 min) effects of BPA and 17β-estradiol (E2) in the heart and ventricular myocytes from rodents were investigated in the present study. In isolated ventricular myocytes from young adult females, but not males, physiological concentrations of BPA or E2 (10−9 M) rapidly induced arrhythmogenic triggered activities. The effects of BPA were particularly pronounced when combined with estradiol. Under conditions of catecholamine stimulation, E2 and BPA promoted ventricular arrhythmias in female, but not male, hearts. The cellular mechanism of the female-specific pro-arrhythmic effects of BPA and E2 were investigated. Exposure to E2 and/or BPA rapidly altered myocyte Ca2+ handling; in particular, estrogens markedly increased sarcoplasmic reticulum (SR) Ca2+ leak, and increased SR Ca2+ load. Ryanodine (10−7 M) inhibition of SR Ca2+ leak suppressed estrogen-induced triggered activities. The rapid response of female myocytes to estrogens was abolished in an estrogen receptor (ER) β knockout mouse model. Conclusions/Significance Physiologically-relevant concentrations of BPA and E2 promote arrhythmias in a female-specific manner in rat hearts; the pro-arrhythmic actions of estrogens are mediated by ERβ-signaling through alterations of myocyte Ca2+ handling, particularly increases in SR Ca2+ leak. Our study provides the first experimental evidence suggesting that exposure to estrogenic endocrine disrupting chemicals and the unique sensitivity of female hearts to estrogens may play a role in arrhythmogenesis in the female heart.}, number={9}, journal={PLoS ONE}, author={Yan, S. and Chen, Y. and Dong, M. and Song, W. and Belcher, S.M. and Wang, H.-S.}, year={2011} }
 @article{kendig_le_belcher_2010, title={Defining hormesis: Evaluation of a complex concentration response phenomenon}, volume={29}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77953860378&partnerID=MN8TOARS}, DOI={10.1177/1091581810363012}, abstractNote={Hormesis describes dose-response relationships characterized by a reversal of response between low and high doses of chemicals, biological molecules, physical stressors, or other initiators of a response. Acceptance of hormesis as a viable dose-response theory has been limited until recently, in part, because of poor conceptual understanding, ad hoc and inappropriate use, and lack of a defined mechanism. By examining the history of this dose-response theory, it is clear that both pharmacological and toxicological studies provide evidence for hormetic dose responses, but retrospective examination of studies can be problematic at best. Limited scientific evidence and lack of a common lexicon with which to describe these responses have left hormesis open to inappropriate application to unrelated dose-response relationships. Future studies should examine low-dose effects using unbiased, descriptive criteria to further the scientific understanding of this dose response. A clear, concise definition is required to further the limited scientific evidence for hormetic dose responses.}, number={3}, journal={International Journal of Toxicology}, author={Kendig, E.L. and Le, H.H. and Belcher, S.M.}, year={2010}, pages={235–246} }
 @article{saal_akingbemi_belcher_crain_crews_guidice_hunt_leranth_myers_nadal_et al._2010, title={Flawed experimental design reveals the need for guidelines requiring appropriate positive controls in endocrine disruption research}, volume={115}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77954075822&partnerID=MN8TOARS}, DOI={10.1093/toxsci/kfq048}, abstractNote={Frederick S. vom Saal,* Benson T. Akingbemi,† Scott M. Belcher,‡ David A. Crain,§ David Crews,{ Linda C. Guidice,jj Patricia A. Hunt,jjj Csaba Leranth,jjjj John Peterson Myers,# Angel Nadal,** Nicholas Olea,†† Vasantha Padmanabhan, Cheryl S. Rosenfeld, Alan Schneyer, Gilbert Schoenfelder, Carlos Sonnenschein, Ana M. Soto, Richard W. Stahlhut, Shanna H. Swan, Laura N. Vandenberg, Hong-Sheng Wang, Cheryl S. Watson, Wade V. Welshons, and Robert T. Zoeller}, number={2}, journal={Toxicological Sciences}, author={Saal, F.S. and Akingbemi, B.T. and Belcher, S.M. and Crain, D.A. and Crews, D. and Guidice, L.C. and Hunt, P.A. and Leranth, C. and Myers, J.P. and Nadal, A. and et al.}, year={2010}, pages={612–613} }
 @article{le_belcher_2010, title={Rapid signaling actions of environmental estrogens in developing granule cell neurons are mediated by estrogen receptor β}, volume={151}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-78649850881&partnerID=MN8TOARS}, DOI={10.1210/en.2010-0710}, abstractNote={Estrogenic endocrine disrupting chemicals (EDCs) constitute a diverse group of man-made chemicals and natural compounds derived from plants and microbial metabolism. Estrogen-like actions are mediated via the nuclear hormone receptor activity of estrogen receptor (ER)α and ERβ and rapid regulation of intracellular signaling cascades. Previous study defined cerebellar granule cell neurons as estrogen responsive and that granule cell precursor viability was developmentally sensitive to estrogens. In this study experiments using Western blot analysis and pharmacological approaches have characterized the receptor and signaling modes of action of selective and nonselective estrogen ligands in developing cerebellar granule cells. Estrogen treatments were found to briefly increase ERK1/2-phosphorylation and then cause prolonged depression of ERK1/2 activity. The sensitivity of granule cell precursors to estrogen-induced cell death was found to require the integrated activation of membrane and intracellular ER signaling pathways. The sensitivity of granule cells to selective and nonselective ER agonists and a variety of estrogenic and nonestrogenic EDCs was also examined. The ERβ selective agonist DPN, but not the ERα selective agonist 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol or other ERα-specific ligands, stimulated cell death. Only EDCs with selective or nonselective ERβ activities like daidzein, equol, diethylstilbestrol, and bisphenol A were observed to induce E2-like neurotoxicity supporting the conclusion that estrogen sensitivity in granule cells is mediated via ERβ. The presented results also demonstrate the utility of estrogen sensitive developing granule cells as an in vitro assay for elucidating rapid estrogen-signaling mechanisms and to detect EDCs that act at ERβ to rapidly regulate intracellular signaling.}, number={12}, journal={Endocrinology}, author={Le, H.H. and Belcher, S.M.}, year={2010}, pages={5689–5699} }
 @article{belcher_ma_le_2009, title={Blockade of estrogen receptor signaling inhibits growth and migration of medulloblastoma}, volume={150}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-67649644713&partnerID=MN8TOARS}, DOI={10.1210/en.2008-1363}, abstractNote={Medulloblastoma (MD) is the most common malignant brain tumor in children. These invasive neuroectodermal tumors arise from cerebellar granule cell-like precursors. In the developing cerebellum, estrogen influences growth and viability of granule cell precursors that transiently express elevated levels estrogen receptor-beta (ERbeta) during differentiation. Immunoanalysis revealed that ERbeta was expressed in the maturing human cerebellum, in all 22 primary MD tumors analyzed, and in two MD-derived cell lines (D283Med and Daoy). Very low levels of ERalpha-like proteins were detected in each cell line and 41% of tumor samples. Physiological concentrations of the 17beta-estradiol- or the ERbeta-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile diarylpropionitrile dose-dependently increased MD growth and cellular migration. In contrast, the ERalpha-selective agonist (4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol did not influence MD growth. Similar to previous studies in normal cerebellar granule cell precursors, these studies demonstrate that the physiological actions of estrogens in MD are mediated by ERbeta. Preclinical studies assessing the therapeutic efficacy of antiestrogen chemotherapeutics for treating human MD were performed. It was found that pharmacological inhibition of ER-mediated signaling with the ER antagonist drug Faslodex (ICI182,780) blocked all estrogen-mediated effects in both cell culture and xenograft models of human MD. These studies have revealed that functional ERbeta expression is a fundamental aspect of MD biology and has defined antiestrogen therapy as a potentially efficacious clinical approach to improve the long-term outcomes for MD patients.}, number={3}, journal={Endocrinology}, author={Belcher, S.M. and Ma, X. and Le, H.H.}, year={2009}, pages={1112–1121} }
 @article{belcher_2009, title={Blockade of estrogen receptor signaling to improve outlook for medulloblastoma sufferers}, volume={5}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-70349209368&partnerID=MN8TOARS}, DOI={10.2217/fon.09.69}, abstractNote={Future OncologyVol. 5, No. 6 EditorialFree AccessBlockade of estrogen receptor signaling to improve outlook for medulloblastoma sufferersScott M BelcherScott M Belcher† Author for correspondenceDepartment of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, P.O. Box 670575, Cincinnati, OH 45267–0575, USA. Search for more papers by this authorEmail the corresponding author at scott.belcher@uc.eduPublished Online:11 Aug 2009https://doi.org/10.2217/fon.09.69AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit For children and adolescents less than 20 years old, neoplasms of the CNS account for more than 16% of all childhood malignancies and are the second most frequent childhood cancer [1]. Medulloblastoma are the most common malignant brain tumors in children. These invasive primitive neuroectodermal tumors (PNET) arise from granule cell-like precursors that have escaped terminal differentiation. As a histological class, medulloblastoma are the most common pediatric brain tumor. These embryonal neural tumors have a disproportionately worse outcome, and thus represent nearly 10% of cancer deaths in children younger than 15 years of age [2,3]. Despite recent advances in understanding of the molecular and cellular mechanisms involved in the etiology of some types of medulloblastoma, the 5-year survival rate for medulloblastoma patients varies between 20 and 80%. For young children and infants, the survival rate is especially poor [1,4].A cornerstone of medulloblastoma management is aggressive craniospinal radiotherapy that is unfortunately associated with deleterious neurocognitive and endocrine impairments in survivors. Continued refinement of aggressive multimodal therapy has increased survival rates for children with medulloblastoma. However, those benefits come with the price of life-long therapy-induced side-effects [5,6]. The addition of chemotherapeutic approaches to radiotherapy has improved disease management, and in some cases has allowed a reduction in the dose of craniospinal irradiation while maintaining disease control. The aggressive therapeutic approaches used to treat medulloblastoma still result in an array of debilitating complications and increased risks for adverse endocrine and cardiovascular effects in adulthood, and well-define neurological and cognitive sequelae in survivors [5–8]. Clearly there is a pressing need for new approaches that improve management of medulloblastoma while decreasing negative side effects of therapy.In the brain, 17β-estradiol (E2) acts as a mitogen and trophic factor that influences many facets of brain function and development [9]. As in other estrogen-responsive tissues, the estrogen-sensitive cells of the nervous system bind estradiol at membrane-associated and intracellular estrogen receptors to regulate the activity of various growth factor-like signaling pathways, and transcription of estrogen-responsive genes [10–12].Initial indications for a possible role of estrogen receptors in medulloblastoma came from our studies showing that estrogen receptor β (ERβ) was transiently expressed at high levels in differentiating granule cell precursors of the developing rat cerebellum, and that low concentrations of estrogens regulate granule cell precursor migration, proliferation and viability [13–16]. With a detailed understanding of cerebellar development, the obvious parallels between normal granule cell ontogeny and the etiology of medulloblastoma led us to investigate whether estrogens and estrogen receptor-mediated signaling mechanisms played a role in medulloblastoma pathology. The results of studies published by Belcher et al.[17] reveal for the first time that ERβ is widely expressed in primary medulloblastoma tumors and in medulloblastoma-derived cell lines. Very low levels of nuclear-localized ERα could also be observed in a minority of the tumors analyzed. While detectable, the expression of ERα was very limited and localized in small focal cell clusters representing much less than 1% of the total number of tumor cells. Those results suggest at most a limited role for ERα in medulloblastoma. While underpowered to demonstrate specific associations between tumor grade/outcome and ER expression patterns, it was notable that tumors anticipated to have better clinical outcomes (classical or desmoplastic medulloblastoma) expressed relatively lower levels of ERβ. The more aggressive anaplastic tumors were characterized as having the highest levels of ERβ staining and lacked detectable ERα expression [17].Cell culture and xenograft models of human medulloblastoma were developed using the ERβ-positive D283Med cell line. In those preclinical models, physiological concentrations of the endogenous nonselective estrogen 17β-estradiol caused dose-dependent increases in tumor cell proliferation and greatly stimulated tumor growth rates. Additional in vivo and in vitro experiments using ER-selective agonists and the ER antagonist fluvesterent (Faslodex®) revealed that the growth-stimulating actions of estradiol were mediated by ERβ-dependent mechanisms [17]. Using an in vitro model of tumor cell invasion, estradiol was also shown to stimulate medulloblastoma cell migration via ERβ-mediated growth factor-like signaling mechanisms. Collectively, the reported findings closely mirrored the actions anticipated from previous findings in normal developing granule cell precursors, and strongly support the conclusion that medulloblastoma are estrogen-responsive tumors in which tumor growth and tumor cell invasion are enhanced by estrogens via multiple ER-mediated mechanisms. The generality of those results are supported by previous studies that demonstrated that cerebrocortical-derived PNET tumor cells express active ERs [18]. There it was shown that estrogen, via ERβ activation of ERK mitogen-activated signaling, increased ER- and ERK-dependent cell migration, suggesting that rapid estrogen signaling (i.e., nongenomic actions) and ER-mediated regulation of estrogen-responsive gene expression combined to increase the invasive nature of PNETs [18].Our characterization of medulloblastoma as an estrogen-responsive cancer prompted us to draw upon the extensive wealth of basic and clinical science surrounding successful anti-estrogen treatment of estrogen-responsive (ER-positive) breast cancer. Chemotherapeutic agents, such as the selective estrogen receptor modulator (SERM) tamoxifen and the ER transcriptional antagonist fluvesterent, are used clinically as first-line and adjuvant pharmacotherapy to block ER activity and decrease growth of estrogen-responsive tumors of the breast. We hypothesized that pharmacologic blockade of ER-mediated signaling could be used to block growth of human medulloblastoma. Preclinical studies assessing the therapeutic efficacy of anti-estrogen chemotherapeutics for treating human medulloblastoma revealed that pharmacological inhibition of ER-mediated signaling with the ER antagonist fluvesterent blocked all estrogen-mediated effects in cell culture. In vivo, Faslodex (fluvesterent drug preparation) fully blocked estrogen-stimulated growth of tumors in xenograft models of human medulloblastoma [17].Additional evidence for functional roles of ERs in CNS tumors is limited and comes mainly from studies that report expression of functional ERs in various cell lines initially derived from CNS or SNS tumors [18–20]. While limited in scope, those observations suggest that estrogens and ERs might have various roles in many different types of nervous system tumors (e.g., neuroblastoma, glioma and so on). Associative evidence has begun to accumulate from retrospective clinical studies comparing differential ER expression in astrocytic carcinomas. Based on the observation that ERβ is expressed in some low-grade astrocytic tumors, ERβ was suggested to be an indicator of less aggressive tumors and a potential marker of favorable treatment response and clinical outcome [21]. The significance of this observation remains unknown.Results of in vitro studies in ER-positive glioma-derived cell lines showed that high concentrations of tamoxifen increased the cytotoxic sensitivity of tumor cells to chemotherapeutic drugs and radiation. Rather than disruption of ER-mediated signaling mechanisms, the rationale for high-dose tamoxifen therapy for treating those CNS malignancies is based on its ability to sensitize these glial-derived tumor cells in vitro to other chemotherapeutic agents and radiation via inhibition of protein kinase C (PKC). While tamoxifen therapy has revolutionized breast cancer treatment, the use of high-dose tamoxifen for treatment of astrocytic brain tumors in adults and children was found to be without benefit [22–24]. Not only have recent clinical studies employing tamoxifen as adjuvant therapy failed to prove beneficial, some studies have demonstrated that high-dose tamoxifen increases recurrence of multifocal tumors in glioblastoma patients [25]. In light of the current understanding of ER-mediated mechanism and specific receptor expression patterns, the lack of efficacy of high-dose tamoxifen therapy is not surprising.The revelation that functional ERβ expression is a fundamental aspect of cerebellar development and medulloblastoma biology defines anti-estrogen therapy as a potentially efficacious clinical approach to improve the long-term outcome for medulloblastoma patients. In contrast to the use of tamoxifen as a chemo-sensitizing agent, our results suggest a rational application of ER-signaling inhibitors for treatment of medulloblastoma. From the human medulloblastoma xenograft experiments, it is clear that the lack of estrogen, or blockade of ER activity, arrests medulloblastoma tumor growth [17]. While the effects of estrogen on medulloblastoma growth are distinctive from the typical estrogen dependence of ER-positive breast cancers, medulloblastoma growth is stimulated by, but not dependent on the presence of estrogens. The elimination of the estrogen growth-stimulating actions could conceivably aid medulloblastoma management and potentially lead to a decreased dose of radiotherapy to achieve cure.Because peak medulloblastoma incidence is around age five, blockade of estrogen signaling as a temporary adjuvant treatment would be expected to have minimal long-term side effects in the majority of patients. In most patients, anti-estrogen treatments are anticipated to be efficacious, while avoiding critical periods of most estrogen-mediated effects including those involved with development of secondary sexual characteristics during puberty, and the much earlier critical periods of estrogen-sensitive CNS development controlling sexual development and specialization of the brain.The use of nonselective anti-estrogens, and especially SERMs, for treating medulloblastoma is considered inappropriate and likely to result in numerous side effects from unpredictable disruption of normal endocrine functions in various estrogen-responsive tissues. Agents that selectively inhibit ERβ signaling may prove the most effective anti-estrogen treatments for medulloblastoma by limiting the specificity of the anti-estrogen effects to those mediated via the tumor-specific forms of ERβ. Numerous ERβ-selective antagonists are currently being developed and are in clinical trials. However, well-characterized ERβ-selective drugs are clinically unavailable. The development of highly selective ERβ-antagonists specific for treating medulloblastoma is a logical extension of our studies. To limit systemic side effects of estrogen signaling blockade, any anti-estrogen signaling treatment modalities developed for medulloblastoma must keep the potential impact that blockade will have on other estrogen-responsive processes (e.g., bone growth) squarely in focus.Appreciating the poorly understood and changing developmental and cell-specific activities of ERs, it is clear that development of highly tumor-selective anti-estrogen drugs with limited side effects will be an especially challenging endeavor. However, immediate benefit to patient outcome can be realized by limiting the exposure of medulloblastoma patients to dietary phytoestrogens and estrogenic endocrine-disrupting chemicals migrating from medical devices. It is noteworthy that the youngest patients, those that have the worst outcomes, are likely exposed to the highest levels of estrogenic endocrine-disrupting chemicals. Owing to their lower body weights and decreased abilities to metabolize some endocrine-disrupting compounds, it is likely that infants, who are generally most sensitive to the disruptive actions of exogenous estrogens, are exposed to the highest doses of these estrogens [26]. Careful attention must be paid to the exposure of medulloblastoma patients to compounds with estrogen-like activities, not only those derived from medical devices (e.g., phthalates and bisphenol A), but also those derived from plant-based dietary sources (e.g., soy-based infant formula). Additional important sources of estrogenic chemical exposures of concern include infant formula packaging, canned foods and beverages, all of which are well-known dietary sources of human exposure to bisphenol A. Interventions that limit exogenous estrogen exposures are considered especially exciting because their immediate translation into medulloblastoma patient management may improve outcome with no risk of increasing treatment-related side effects.Financial & competing interests disclosureThe research studies described were supported by NIH/NIEHS grant R01-ES015145, the University of Cincinnati Center for Environmental Genetics (P30-ES06096), Pediatric Brain Tumor Foundation, and a Translational Research Initiative grant from Cincinnati Children’s Hospital Research Foundation. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Bibliography1 Ries LAG, Smith MA, Gurney JG et al.: Cancer Incidence and Survival among Children and Adolescents: United States SEER Program 1975–1995. National Cancer Institute, SEER Program. NIH Pub. No. 99–4649. Bethesda, MD, USA (1999).Google Scholar2 Rubin JB, Rowitch DH: Medulloblastoma: a problem of developmental biology. Cancer Cell.2,7–8 (2002).Crossref, Medline, CAS, Google Scholar3 Wechsler-Reya R, Scott MP: The developmental biology of brain tumors. Annu. Rev. Neurosci.24,385–428 (2001).Crossref, Medline, CAS, Google Scholar4 Gatta G, Capocaccia R, Coleman MP, Ries LA, Berrino F: Childhood cancer survival in Europe and the United States. 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Oncol.130,405–410 (2004).Crossref, Medline, CAS, Google Scholar22 Broniscer A, Leite CdC, Lanchote VL, Machado TMS, Cristofani LM: Radiation therapy and high-dose tamoxifen in the treatment of patients with diffuse brainstem gliomas: results of a Brazilian cooperative study. J. Clin. Oncol.18,1246–1253 (2000)Crossref, Medline, CAS, Google Scholar23 Robins HI, Won M, Seiferheld WF et al.: Phase II trial of radiation plus high-dose tamoxifen for glioblastoma multiforme: RTOG protocol BR-0021. Neuro. Oncol.8,47–52 (2006).Crossref, Medline, CAS, Google Scholar24 Spence AM, Peterson RA, Scharnhorst JD, Silbergeld DL, Rostomily RC: Phase II study of concurrent continuous temozolomide (TMZ) and tamoxifen (TMX) for recurrent malignant astrocytic gliomas. J. Neurooncol.70,91–95 (2004).Crossref, Medline, Google Scholar25 Puchner MJ, Giese A, Lohmann F, Cristante L: High-dose tamoxifen treatment increases the incidence of multifocal tumor recurrences in glioblastoma patients. Anticancer Res.24,4195–4203 (2004).Medline, CAS, Google Scholar26 vom Saal FS, Akingbemi BT, Belcher SM et al.: Chapel Hill bisphenol A expert panel consensus statement: Integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure. Reprod. Toxicol.24,131–138 (2007).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByDuality of estrogen receptor β action in cancer progressionCurrent Opinion in Pharmacology, Vol. 41Estrogen and soy isoflavonoids decrease sensitivity of medulloblastoma and central nervous system primitive neuroectodermal tumor cells to chemotherapeutic cytotoxicity6 September 2017 | BMC Pharmacology and Toxicology, Vol. 18, No. 1Estrogen Receptor-β Up-Regulates IGF1R Expression and Activity to Inhibit Apoptosis and Increase Growth of Medulloblastoma17 April 2015 | Endocrinology, Vol. 156, No. 7Calmodulin-kinases regulate basal and estrogen stimulated medulloblastoma migration via Rac124 November 2010 | Journal of Neuro-Oncology, Vol. 104, No. 1 Vol. 5, No. 6 eToC Sign up Follow us on social media for the latest updates Metrics History Published online 11 August 2009 Published in print August 2009 Information© Future Medicine LtdFinancial & competing interests disclosureThe research studies described were supported by NIH/NIEHS grant R01-ES015145, the University of Cincinnati Center for Environmental Genetics (P30-ES06096), Pediatric Brain Tumor Foundation, and a Translational Research Initiative grant from Cincinnati Children’s Hospital Research Foundation. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download}, number={6}, journal={Future Oncology}, author={Belcher, S.M.}, year={2009}, pages={751–754} }
 @article{myers_vom saal_taylor_akingbemi_arizono_belcher_colborn_chahoud_2009, title={Good laboratory practices: Myers et al. respond}, volume={117}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-74949095138&partnerID=MN8TOARS}, DOI={10.1289/ehp.0900884R}, abstractNote={We are in complete agreement with the statement by Becker et al. that “having confidence in scientific procedures and data is the sine qua non for determining the safety of chemicals and chemical products.” Our aim in writing the commentary (Myers et al. 2009) was not to challenge the original intent of Good Laboratory Practices (GLP) requirements, which was to establish standards of record keeping in contract laboratory research so as to reduce the likelihood of fraud. Our goal instead was to show—through an analysis of the application of GLP data on bisphenol A (BPA) in regulatory proceedings—that GLP by itself is insufficient to guarantee valid and reliable science. Becker et al. appear to have missed the point of our commentary entirely. 
 
In the case of BPA, three GLP studies have been offered by industry-sponsored laboratories as proof of the chemical’s safety (Cagen et al. 1999; Tyl et al. 2002, 2008). Each has errors in study design and/or data interpretation that are sufficiently serious as to invalidate the conclusions of these studies (Myers et al. 2009). Nevertheless, because the studies were conducted using GLP guidelines, they were judged by regulators as being more reliable than the many National Institutes of Health (NIH)-funded and peer-reviewed studies that have reported adverse effects (Richter et al. 2007;vom Saal et al. 2007). 
 
As our commentary (Myers et al. 2009) clearly establishes, GLP did not guarantee the scientific validity of these three studies. Because previous analyses had identified serious flaws in the first two of those GLP studies, we focused critical attention on the most recent (Tyl et al. 2008), which both the European Food Safety Authority (EFSA 2006) and the U.S. Food and Drug Administration (FDA) had identified as key in their BPA risk assessments (FDA 2008). We found three main flaws: a) the animals were inexplicably insensitive to estrogen; b) the assays were outdated and insensitive compared with methods used in NIH-funded research showing adverse effects; and c ) validity of the findings was challenged. For example, the prostate weights of control animals reported by Tyl et al. (2008) were > 70% larger (mean, > 72 mg) than those reported by numerous laboratories, including a previously published study using CD-1 mice [conducted at RTI, where the study by Tyl et al. (2008) was conducted] that reported mean prostate weights of 46 mg in CD-1 males that were examined at a similar age (Heindel et al. 1995). 
 
Since we published our commentary (Myers et al. 2009), a possible contributor to both the estrogen insensitivity and the enlarged control prostates has been suggested: Approximately 3 years before the experiments that formed the basis of the study by Tyl et al. (2008), there was a polycarbonate fire that released BPA into the RTI laboratory where the research was conducted (Kissinger and Rust 2009). An investigation revealed that animals in the laboratory were exposed to low doses of BPA that government-funded science (Richter et al. 2007) indicates could affect research animals. 
 
Additional uncertainties about Tyl et al.’s study (Tyl et al. 2008) have now been identified by the lead author. Whereas the published paper reports that the animals were examined at approximately 14 weeks of age, Tyl testified at an FDA hearing in September 2008 that they were 6 months of age, and then at a German Environmental Protection Agency hearing in March 2009 that they were 5 months of age (Kissinger and Rust 2009). There she confirmed that the information in the original article was inaccurate. Because an animal’s physiology changes as it ages, these contradictory statements are problematic for all reported outcomes; even at 5–6 months of age, normal, healthy CD-1 male mice would not have the grossly enlarged prostates reported by Tyl et al. (2008). 
 
The use of flawed science, however, is not the only concern. The type of multigeneration testing approach used in these studies is, quite simply, insufficient for the testing of endocrine-disrupting chemicals. This is not a new concept. The need for more specific tests for endocrine-active compounds led in 1998 to the establishment at the U.S. Environmental Protection Agency (U.S. EPA) of the Endocrine Disruptor Screening Program, mandated by Congress (U.S. EPA 1998). After virtually no progress for over a decade, in 2009 the U.S. EPA finally announced a set of testing procedures that will be examined. The proposed “new” methodology, heavily dependent upon traditional toxicologic methods used in multigenerational GLP studies, is still woefully inadequate (Colborn 2009). 
 
The letter by Becker et al. provides a striking example of the reluctance of industry lobbyists to hear this message. In the eyes of the 36 scientific colleagues who coauthored our commentary (Myers et al. 2009), the BPA studies that Becker et al. attempt to defend are so seriously flawed as to be indefensible. Rather than continue to defend a dead issue, we encourage industry representatives to come into the 21st century and help us devise new paradigms for testing endocrine-disrupting chemicals that will safeguard human health.}, number={11}, journal={Environmental Health Perspectives}, author={Myers, J.P. and Vom Saal, F.S. and Taylor, J.A. and Akingbemi, B.T. and Arizono, K. and Belcher, S. and Colborn, T. and Chahoud, I.}, year={2009} }
 @article{why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: the case of bisphenol a_2009, volume={117}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-64049090987&partnerID=MN8TOARS}, DOI={10.1289/ehp.0800173}, abstractNote={Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.}, number={3}, journal={Environmental Health Perspectives}, year={2009}, pages={309–315} }
 @article{le_carlson_chua_belcher_2008, title={Bisphenol A is released from polycarbonate drinking bottles and mimics the neurotoxic actions of estrogen in developing cerebellar neurons}, volume={176}, ISSN={0378-4274}, url={http://dx.doi.org/10.1016/j.toxlet.2007.11.001}, DOI={10.1016/j.toxlet.2007.11.001}, abstractNote={The impact of endocrine disrupting chemical (EDC) exposure on human health is receiving increasingly focused attention. The prototypical EDC bisphenol A (BPA) is an estrogenic high-production chemical used primarily as a monomer for the production of polycarbonate and epoxy resins. It is now well established that there is ubiquitous human exposure to BPA. In the general population, exposure to BPA occurs mainly by consumption of contaminated foods and beverages that have contacted epoxy resins or polycarbonate plastics. To test the hypothesis that bioactive BPA was released from polycarbonate bottles used for consumption of water and other beverages, we evaluated whether BPA migrated into water stored in new or used high-quality polycarbonate bottles used by consumers. Using a sensitive and quantitative competitive enzyme-linked immunosorbent assay, BPA was found to migrate from polycarbonate water bottles at rates ranging from 0.20 ng/h to 0.79 ng/h. At room temperature the migration of BPA was independent of whether or not the bottle had been previously used. Exposure to boiling water (100 °C) increased the rate of BPA migration by up to 55-fold. The estrogenic bioactivity of the BPA-like immunoreactivity released into the water samples was confirmed using an in vitro assay of rapid estrogen signaling and neurotoxicity in developing cerebellar neurons. The amounts of BPA found to migrate from polycarbonate drinking bottles should be considered as a contributing source to the total “EDC-burden” to which some individuals are exposed.}, number={2}, journal={Toxicology Letters}, publisher={Elsevier BV}, author={Le, Hoa H. and Carlson, Emily M. and Chua, Jason P. and Belcher, Scott M.}, year={2008}, month={Jan}, pages={149–156} }
 @book{melchert_belcher_kennedy_2008, title={Cardiovascular effects of steroidal agents}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85057367081&partnerID=MN8TOARS}, journal={Cardiovascular Toxicology, Fourth Edition}, author={Melchert, R.B. and Belcher, S.M. and Kennedy, R.H.}, year={2008}, pages={367–428} }
 @article{belcher_2008, title={Rapid signaling mechanisms of estrogens in the developing cerebellum}, volume={57}, ISSN={0165-0173}, url={http://dx.doi.org/10.1016/j.brainresrev.2007.07.020}, DOI={10.1016/j.brainresrev.2007.07.020}, abstractNote={The steroid hormone 17β-estradiol regulates the normal function and development of the mammalian nervous system. Many of estradiol's effects are mediated via the nuclear hormone estrogen receptors ERα and ERβ. In addition to regulating estrogen-responsive gene expression, estradiol also acts in an immediate and cell-specific fashion to regulate various intracellular signal transduction pathways. The goal of this review is to develop a contextual framework to understand the generalized function of estrogen during development of brain regions not known to be sexually specialized. However, it is first important to build this framework on the more well-developed foundation of estrogen's gonad-driven sex-specific actions. As a result, a discussion of known and proposed mechanisms of estrogen actions in reproductive and other tissues will be presented. Building upon this information, a review of our research group's recent in vitro and in vivo studies that have focused on elucidating the mechanisms of estrogen actions in neurons of the non-sexually specialized cerebellum will be presented. While the full spectrum of estrogen action during normal cerebellar development remains unresolved, results of recent studies have revealed a pathologic role for estrogen and estrogen receptors in medulloblastoma, common pediatric brain tumors that arise from cerebellar granule cell-like precursors. The potential use of anti-estrogen signaling agents as adjuvant therapy for medulloblastoma is proposed based on those finding.}, number={2}, journal={Brain Research Reviews}, publisher={Elsevier BV}, author={Belcher, Scott M.}, year={2008}, month={Mar}, pages={481–492} }
 @article{vom saal_akingbemi_belcher_birnbaum_crain_eriksen_farabollini_guillette_hauser_heindel_et al._2007, title={Chapel Hill bisphenol A expert panel consensus statement: Integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure}, volume={24}, ISSN={0890-6238}, url={http://dx.doi.org/10.1016/j.reprotox.2007.07.005}, DOI={10.1016/j.reprotox.2007.07.005}, abstractNote={Existing cross-sectional studies indicated a positive association of bisphenol A (BPA) with overweight and obesity. However, the relationship and potential mechanisms underlying this association remain to be elucidated in prospective studies.This study was designed to investigate whether serum BPA is associated with incident overweight and obesity risk, and to further explore whether adiponectin plays a mediating role in the association.We measured blood BPA and adiponectin in Chinese populations. The association of serum BPA with overweight and obesity risk was evaluated using multivariable logistic regression models. We further examined the mediating effect of adiponectin by causal mediation analysis.Among 796 participants free of overweight and obesity at baseline, 133 individuals developed overweight and obesity during the follow-up period. Compared with those in the lowest quartile of serum BPA, those in the second and third quartiles were positively associated with incident overweight and obesity risk adjusting for covariates (all P-values < 0.05), whereas this association was not observed in the fourth quartile. Further spline analysis showed an inverted U-shaped dose-response relationship (Pnon-linear = 0.04). Furthermore, each unit of serum log10-transformed BPA levels was associated with higher changes in waist-to-height ratio and body roundness index (all P-values < 0.05). Mediation analysis indicated significant indirect effects of adiponectin on the associations of BPA with overweight and obesity prevalence (mediation proportion: 46.08%; P = 0.02), and BMI levels (mediation proportion: 30.32%; P = 0.03).Serum BPA displayed a positive association with incident overweight and obesity risk in a non-monotonic pattern, and adiponectin might mediate the association. Further mechanistic studies are warranted.}, number={2}, journal={Reproductive Toxicology}, publisher={Elsevier BV}, author={vom Saal, Frederick S. and Akingbemi, Benson T. and Belcher, Scott M. and Birnbaum, Linda S. and Crain, D. Andrew and Eriksen, Marcus and Farabollini, Francesca and Guillette, Louis J., Jr. and Hauser, Russ and Heindel, Jerrold J. and et al.}, year={2007}, month={Aug}, pages={131–138} }
 @article{wetherill_akingbemi_kanno_mclachlan_nadal_sonnenschein_watson_zoeller_belcher_2007, title={In vitro molecular mechanisms of bisphenol A action}, volume={24}, ISSN={0890-6238}, url={http://dx.doi.org/10.1016/j.reprotox.2007.05.010}, DOI={10.1016/j.reprotox.2007.05.010}, abstractNote={Bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane; CAS# 80-05-7) is a chemical used primarily in the manufacture of polycarbonate plastic, epoxy resins and as a non-polymer additive to other plastics. Recent evidence has demonstrated that human and wildlife populations are exposed to levels of BPA which cause adverse reproductive and developmental effects in a number of different wildlife species and laboratory animal models. However, there are major uncertainties surrounding the spectrum of BPA's mechanisms of action, the tissue-specific impacts of exposures, and the critical windows of susceptibility during which target tissues are sensitive to BPA exposures. As a foundation to address some of those uncertainties, this review was prepared by the "In vitro" expert sub-panel assembled during the "Bisphenol A: An Examination of the Relevance of Ecological, In vitro and Laboratory Animal Studies for Assessing Risks to Human Health" workshop held in Chapel Hill, NC, Nov 28-29, 2006. The specific charge of this expert panel was to review and assess the strength of the published literature pertaining to the mechanisms of BPA action. The resulting document is a detailed review of published studies that have focused on the mechanistic basis of BPA action in diverse experimental models and an assessment of the strength of the evidence regarding the published BPA research.}, number={2}, journal={Reproductive Toxicology}, publisher={Elsevier BV}, author={Wetherill, Yelena B. and Akingbemi, Benson T. and Kanno, Jun and McLachlan, John A. and Nadal, Angel and Sonnenschein, Carlos and Watson, Cheryl S. and Zoeller, R. Thomas and Belcher, Scott M.}, year={2007}, month={Aug}, pages={178–198} }
 @article{belcher_zsarnovszky_crawford_hemani_spurling_kirley_2006, title={Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: Implications for modulation of multiple homeostatic systems including feeding and sleep–wake behaviors}, volume={137}, ISSN={0306-4522}, url={http://dx.doi.org/10.1016/j.neuroscience.2005.08.086}, DOI={10.1016/j.neuroscience.2005.08.086}, abstractNote={Three anti-peptide antisera were raised against three distinct amino acid sequences of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), characterized by Western blot analyses, and used to determine the distribution of NTPDase3 protein in adult rat brain. The three antisera all yielded similar immunolocalization data, leading to increased reliability of the results obtained. Unlike NTPDase1 and NTPDase2, NTPDase3 immunoreactivity was detected exclusively in neurons. Immunoreactivity was localized primarily to axon-like structures with prominent staining of presynaptic elements. Specific perikaryal immunostaining was detected primarily in scattered neurons near the lateral hypothalamic area and the perifornical nucleus. High densities of immunoreactive axon-like fibers were present in midline regions of the forebrain and midbrain. Highly scattered NTPDase3 positive fibers were observed in the cerebral cortex, the hippocampal formation, and the basal ganglia. Moreover, very high densities of immunostained fibers were detected in the mediobasal hypothalamus, with the overall mesencephalic pattern of staining associated closely with hormone responsive nuclei. High densities of NTPDase3 positive terminals were also associated with noradrenergic neurons. However, co-immunolocalization studies revealed clearly that NTPDase3 immunoreactivity was not localized within the noradrenaline cells or terminals. In contrast, nearly all of the NTPDase3 immunopositive hypothalamic cells, and most fibers in the mid- and hindbrain, also expressed hypocretin-1/orexin-A. The overall pattern of expression and co-localization with hypocretin-1/orexin-A suggests that NTPDase3, by regulating the extracellular turnover of ATP, may modulate feeding, sleep–wake, and other behaviors through diverse homeostatic systems.}, number={4}, journal={Neuroscience}, publisher={Elsevier BV}, author={Belcher, S.M. and Zsarnovszky, A. and Crawford, P.A. and Hemani, H. and Spurling, L. and Kirley, T.L.}, year={2006}, pages={1331–1346} }
 @article{zsarnovszky_le_wang_belcher_2005, title={Ontogeny of rapid estrogen-mediated extracellular signal-regulated kinase signaling in the rat cerebellar cortex: Potent nongenomic agonist and endocrine disrupting activity of the xenoestrogen bisphenol A}, volume={146}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-27844570800&partnerID=MN8TOARS}, DOI={10.1210/en.2005-0565}, abstractNote={In addition to regulating estrogen receptor-dependent gene expression, 17beta-estradiol (E(2)) can directly influence intracellular signaling. In primary cultured cerebellar neurons, E(2) was previously shown to regulate growth and oncotic cell death via rapid stimulation of ERK1/2 signaling. Here we show that ERK1/2 signaling in the cerebellum of neonatal and mature rats was rapidly responsive to E(2) and during development to the environmental estrogen bisphenol A (BPA). In vivo dose-response analysis for each estrogenic compound was performed by brief (6-min) intracerebellar injection, followed by rapid fixation and phosphorylation-state-specific immunohistochemistry to quantitatively characterize changes in activated ERK1/2 (pERK) immunopositive cell numbers. Beginning on postnatal d 8, E(2) significantly influenced the number of pERK-positive cells in a cell-specific manner that was dependent on concentration and age but not sex. In cerebellar granule cells on postnatal d 10, E(2) or BPA increased pERK-positive cell numbers at low doses (10(-12) to 10(-10) M) and at higher (10(-7) to 10(-6) M) concentrations. Intermediate concentrations of either estrogenic compound did not modify basal ERK signaling. Rapid E(2)-induced increases in pERK immunoreactivity were specific to the ERK1/2 pathway as demonstrated by coinjection of the mitogen-activated, ERK-activating kinase (MEK)1/2 inhibitor U0126. Coadministration of BPA (10(-12) to 10(-10) M) with 10(-10) M E(2) dose-dependently inhibited rapid E(2)-induced ERK1/2 activation in developing cerebellar neurons. The ability of BPA to act as a highly potent E(2) mimetic and to also disrupt the rapid actions of E(2) at very low concentrations during cerebellar development highlights the potential low-dose impact of xenoestrogens on the developing brain.}, number={12}, journal={Endocrinology}, author={Zsarnovszky, A. and Le, H.H. and Wang, H.-S. and Belcher, S.M.}, year={2005}, pages={5388–5396} }
 @article{belcher_le_spurling_wong_2005, title={Rapid estrogenic regulation of extracellular signal-regulated kinase 1/2 signaling in cerebellar granule cells involves a G protein- and protein kinase A-dependent mechanism and intracellular activation of protein phosphatase 2A}, volume={146}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-27844489524&partnerID=MN8TOARS}, DOI={10.1210/en.2005-0564}, abstractNote={In neonatal rat cerebellar neurons, 17beta-estradiol (E(2)) rapidly stimulates ERK1/2 phosphorylation through a membrane-associated receptor. Here the mechanism of rapid E(2)-induced ERK1/2 signaling in primary cultured granule cells was investigated in more detail. The results of these studies show that E(2) and ICI182,780, a steroidal antagonist of estrogen receptor transactivation, rapidly increased ERK signaling with a time course similar to the transient activation induced by epidermal growth factor (EGF). However, EGF receptor (EGFR) autophosphorylation was not increased by E(2), and blockade of EGFR tyrosine kinase activity did not abrogate the rapid actions of E(2). The involvement of Src-tyrosine kinase activity was demonstrated by detection of increased c-Src phosphorylation in response to E(2) and by blockade of E(2)-induced ERK1/2 activation by inhibition of Src-family tyrosine kinase activity. Inhibition of Galphai signaling or protein kinase A (PKA) activity blocked the ability of ICI182,780 to rapidly stimulate ERK signaling. Under those conditions, E(2) treatment induced a rapid and transient suppression of basal ERK1/2 phosphorylation. Protein phosphatase 2A (PP2A) activity was rapidly increased by E(2) but not by E(2) covalently linked to BSA. Rapid E(2)-induced increases in PP2A activity were insensitive to pertussis toxin. The presented evidence indicates that the rapid effects of estrogens on ERK signaling in cerebellar granule cells are induced through a novel G protein-coupled receptor mechanism that requires PKA and Src-kinase activity to link E(2) to the ERK/MAPK signaling module. Along with stimulating ERK signaling, E(2) rapidly activates PP2A via an independent signaling mechanism that may serve as a cell-specific regulator of signal duration.}, number={12}, journal={Endocrinology}, author={Belcher, S.M. and Le, H.H. and Spurling, L. and Wong, J.K.}, year={2005}, pages={5397–5406} }
 @article{ge_belcher_light_2004, title={Alterations of cerebellar mRNA specific for BDNF, p75NTR, and TrkB receptor isoforms occur within hours of ethanol administration to 4-day-old rat pups}, volume={151}, ISSN={0165-3806}, url={http://dx.doi.org/10.1016/j.devbrainres.2004.04.002}, DOI={10.1016/j.devbrainres.2004.04.002}, abstractNote={Developing cerebellar Purkinje cells of the rat are extremely sensitive to ethanol during postnatal days (PN) 4–6, but not at later times during development. Ethanol exposure during this vulnerable window induces rapid apoptotic Purkinje cell death that is hypothesized to result from ethanol inhibition in brain-derived nerve growth factor (BDNF)–TrkB neurotrophic signaling that results in loss of apoptotic suppression. In this study, the effect that different concentrations of ethanol (1.5, 3.0, 4.5 and 6.0 g/kg) have on steady-state mRNA expression of BDNF and different TrkB receptor isoforms in the cerebellum on PN4 was determined at 1, 4, 6, and 8 h after treatment. Significant decreases in mRNA specific for BDNF and TrkB isoforms were detected within 1 h after ethanol administration. No significant alterations in expression of mRNA specific to the low affinity p75NTR receptor were identified. These alterations are concurrent with the PN4 vulnerable period for Purkinje cells since equivalent treatment of PN9 rat pups does not produce significant alterations in mRNA specific to BDNF or TrkB at 4 h after exposure. These results support the hypothesis that ethanol induces a disruption of BDNF–TrkB signaling that results in loss of apoptotic suppression in vulnerable Purkinje cells by growth factor withdrawal.}, number={1-2}, journal={Developmental Brain Research}, publisher={Elsevier BV}, author={Ge, Yun and Belcher, Scott M and Light, Kim E}, year={2004}, month={Jul}, pages={99–109} }
 @article{ge_belcher_pierce_light_2004, title={Altered expression of Bcl2, Bad and Bax mRNA occurs in the rat cerebellum within hours after ethanol exposure on postnatal day 4 but not on postnatal day 9}, volume={129}, ISSN={0169-328X}, url={http://dx.doi.org/10.1016/j.molbrainres.2004.06.034}, DOI={10.1016/j.molbrainres.2004.06.034}, abstractNote={Previous studies have demonstrated that ethanol exposure during the vulnerable postnatal (PN) day 4–6 period results in a dose-dependent loss of Purkinje neurons in rats by apoptosis. Although the mechanism of ethanol action and the reasons for Purkinje cell vulnerability are unknown, we hypothesize that during the PN4–6 vulnerable period Purkinje cells are dependent on active trophic factor suppression of apoptosis. Furthermore, ethanol acts to prevent the reception of this trophic signaling resulting in the execution of the apoptotic pathway that includes specific alterations of proteins in the Bcl2 gene family. Ethanol exposure that occurs after this vulnerable period (i.e. PN9) would not be expected to demonstrate alterations in these apoptotic proteins since the Purkinje cells no longer demonstrate vulnerability to ethanol. The current study was undertaken to identify the alterations in mRNA expression for members of the Bcl2-family within the initial hours following ethanol administration on PN4 or PN9. Semi-quantitative reverse transcriptase with polymerase chain reaction (PCR) techniques were used to determine the expression levels of pro-apoptotic factors Bad and Bax, and anti-apoptotic Bcl2 mRNA. Ethanol was administered at four different doses (1.5, 3.0, 4.5, and 6.0 g/kg) on PN4 and analyses of whole cerebellar mRNA was conducted at 1, 4, 6, and 8 h after treatment. Doses greater than 1.5 g/kg produced significant decreases in Bcl2 and significant increases in Bad and Bax mRNA during the 8-h period after treatment. In stark contrast, when ethanol was administered at 3.0 or 6.0 g/kg to PN9 pups, no significant alterations of these apoptotic factors were identified at either 1 or 4 h after treatment. These results are in agreement with and provide further support for our hypothesis that ethanol interrupts the active suppression of apoptosis that is a crucial feature of Purkinje cell vulnerability during this time period.}, number={1-2}, journal={Molecular Brain Research}, publisher={Elsevier BV}, author={Ge, Yun and Belcher, Scott M. and Pierce, Dwight R. and Light, Kim E.}, year={2004}, month={Oct}, pages={124–134} }
 @article{ge_belcher_pierce_light_2004, title={Detection of Purkinje cell loss following drug exposures to developing rat pups using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for calbindin-D28k mRNA expression}, volume={150}, ISSN={0378-4274}, url={http://dx.doi.org/10.1016/j.toxlet.2004.02.003}, DOI={10.1016/j.toxlet.2004.02.003}, abstractNote={A technique is described that allows for the identification and quantification of Purkinje cell loss in cerebellum subsequent to developmental toxic exposures. This technique relies upon the extensively validated findings that the Purkinje cell is the only site of expression in the cerebellum of the calcium binding protein calbindin-D28k. Thus, analysis of mRNA expression specific to this protein by comparison to matched controls provides a reliable means of determining whether cell loss has occurred. Purkinje cell loss was induced in rat pups by ethanol exposure on postnatal day (PN) 4 or valproic acid administration to pregnant dams on gestational day 13. Analysis was conducted on PN5 or PN10 and the results compared to parallel groups of pups where the Purkinje cells were counted by traditional means. When compared to matched control rat pups the decrease in calbindin-D28k mRNA expression indicates Purkinje cell loss regardless of whether the cell loss was induced by prenatal valproic acid or postnatal ethanol exposure. The availability of a biochemical alternative to histological cell counting allows for more detailed analyses of the mechanisms of Purkinje cell death induced by these two toxicants, including analyses of the early alterations in signal transduction proteins.}, number={3}, journal={Toxicology Letters}, publisher={Elsevier BV}, author={Ge, Yun and Belcher, Scott M. and Pierce, Dwight R. and Light, Kim E.}, year={2004}, month={May}, pages={325–334} }
 @article{kirby_zsarnovszky_belcher_2004, title={Estrogen receptor expression in a human primitive neuroectodermal tumor cell line from the cerebral cortex: estrogen stimulates rapid ERK1/2 activation and receptor-dependent cell migration}, volume={319}, ISSN={0006-291X}, url={http://dx.doi.org/10.1016/j.bbrc.2004.05.049}, DOI={10.1016/j.bbrc.2004.05.049}, abstractNote={Primitive neuroectodermal tumors (PNETs) are the most common form of pediatric brain tumor. Most often these malignant childhood brain tumors arise from neuroepithelial precursor cells in the cerebellum, and less frequently in the cerebral cortex. Because the normal PNET precursor cells from the cerebrum and cerebellum transiently express high levels of estrogen receptors (ERs), we hypothesized that the PNET cells of the cerebrocortical-derived cell line PFSK1 may also express ERs and would be responsive to estrogen. Results of immunoblot studies using ER-specific antiserum indicate that both ERα and ERβ are expressed in PFSK1 cells. The ability of estrogen to rapidly activate MAPK signaling was tested; low physiological concentrations of E2 stimulated ERK1/2 phosphorylation and nuclear translocation within 15 min of exposure. Exogenously added 17β-estradiol (E2) could not stimulate PFSK1 growth, however E2 significantly increased PFSK1 cell migration, suggesting that rapid actions of E2 and ER-mediated processes might contribute to the metastatic phenotype of some PNETs.}, number={3}, journal={Biochemical and Biophysical Research Communications}, publisher={Elsevier BV}, author={Kirby, Michelle and Zsarnovszky, Attila and Belcher, Scott M}, year={2004}, month={Jul}, pages={753–758} }
 @article{zsarnovszky_belcher_2004, title={Spatial, temporal, and cellular distribution of the activated extracellular signal regulated kinases 1 and 2 in the developing and mature rat cerebellum}, volume={150}, ISSN={0165-3806}, url={http://dx.doi.org/10.1016/j.devbrainres.2004.03.012}, DOI={10.1016/j.devbrainres.2004.03.012}, abstractNote={The extracellular signal regulated kinases 1 and 2 (ERK1/2) are important members of an intracellular signaling cascade that is involved in many aspects of the cellular physiology and development of neurons and glia. ERK1/2 are expressed in many brain regions including the cerebellum; however, their role during cerebellar development is poorly understood. Immunohistochemical approaches using phosphorylation-state specific antiserum that recognizes only the activated-ERK1/2 (pERK) were used to characterize the spatial and temporal patterns of activated-ERK in the developing and adult rat cerebellum. The distribution and cell type-specificity of pERK-immunoreactivity (IR) followed an age-related pattern, with the density of pERK-IR Purkinje cells decreasing between P6 and P15 and increasing at later times. Immunopositive granule cell neurons increased from P6 to P12, became decreased during much of late postnatal cerebellar development, and absent in adults. Co-localization of pERK with glial fibrillary acidic protein or the neuronal marker β-tubulin revealed that activated ERK is present in maturing Purkinje and granule cells, and the soma of Bergmann glia on P4, P10 and P15; pERK was detected in astrocytes on P10 and P15. Associated with weaning, there was a general increase in activated-ERK in all cell types on P22. In adults, pERK-IR was confined to the Purkinje cell layer and scattered cells in the corpus medullare. In summary, a high degree of developmental plasticity was observed in the spatiotemporal distribution of cerebellar pERK-IR suggesting that the ERK-pathway plays a dynamic role in regulating neuronal and glial migration, proliferation and differentiation in the developing cerebellum. In the mature cerebellum, ERK signaling may also mediate postsynaptic information processing.}, number={2}, journal={Developmental Brain Research}, publisher={Elsevier BV}, author={Zsarnovszky, Attila and Belcher, Scott M}, year={2004}, month={Jun}, pages={199–209} }
 @article{wong_le_zsarnovszky_belcher_2003, title={Estrogens and ICI182,780 (Faslodex) modulate mitosis and cell death in immature cerebellar neurons via rapid activation of p44/p42 mitogen-activated protein kinase}, volume={23}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0038790428&partnerID=MN8TOARS}, number={12}, journal={Journal of Neuroscience}, author={Wong, J.K. and Le, H.H. and Zsarnovszky, A. and Belcher, S.M.}, year={2003}, pages={4984–4995} }
 @article{zsarnovszky_smith_hajos_belcher_2002, title={Estrogen regulates GFAP-expression in specific subnuclei of the female rat interpeduncular nucleus: A potential role for estrogen receptor β}, volume={958}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037184712&partnerID=MN8TOARS}, DOI={10.1016/S0006-8993(02)03771-X}, abstractNote={We previously demonstrated that in rat, astrocytic glial fibrillary acidic protein- (GFAP) expression in the interpeduncular nucleus (IPN) was responsive to testosterone and in females the intensity of GFAP-immunoreactivity (IR) followed the periodic hormonal changes of the estrous cycle. The aim of this study was to test whether 17beta-estradiol (E(2)), in the absence of other ovarian hormones, can influence GFAP-expression within individual subnuclei of the IPN and to determine the cellular distribution of estrogen receptor beta (ERbeta) in the IPN. Quantitative surface-density analysis was used to compare the intensity of GFAP-IR at different anterio-posterior (AP) levels of the IPN in ovariectomized female rats 24 h after treatment with E(2) or vehicle. Estrogen-treatment resulted in a significant increase in GFAP-IR in the rostrolateral subnucleus of the IPN at AP: -5.60, in the lateral-, dorsolateral-, dorsomedial- and central subnuclei at -6.04 and in the lateral subnucleus at -6.72. No significant differences were observed at -5.80 and -6.30. These results indicate that E(2), in the absence of other ovarian hormones, modulates GFAP-expression within select IPN subnuclei and that these affects are dependent on position along the AP axis. To determine whether ERbeta was a possible mediator of the observed estrogenic effects, adjacent section pairs of the IPN were immunostained for ERbeta or GFAP. Using the 'mirror' method, ERbeta-IR was detected in the cytoplasm of GFAP-immunopositive astroglia and in the nuclei of GFAP-immunonegative neurons. These findings suggest that in the IPN, E(2) may directly modulate GFAP-expression through ERbeta-mediated mechanisms.}, number={2}, journal={Brain Research}, author={Zsarnovszky, A. and Smith, T. and Hajos, F. and Belcher, S.M.}, year={2002}, pages={488–496} }
 @article{light_brown_newton_belcher_kane_2002, title={Ethanol-induced alterations of neurotrophin receptor expression on Purkinje cells in the neonatal rat cerebellum}, volume={924}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037016323&partnerID=MN8TOARS}, DOI={10.1016/S0006-8993(01)03224-3}, abstractNote={Ethanol causes loss of Purkinje cells in the cerebellum during the early stages of differentiation and maturation by a presently unknown mechanism. Neuronal vulnerability in the cerebellum parallels the prominent temporal and anatomical gradients of development (i.e. early to late interlobular and posterior to anterior, respectively). Development of Purkinje cells is known to require binding of the neurotrophins, including brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), to the tyrosine-kinase (Trk) receptors TrkB and TrkC, respectively. In addition, Purkinje cells are reported to experience a critical switch between BDNF dependence and NT3 dependence during the period of highest ethanol sensitivity between postnatal days (PN) 4-6. To test the hypothesis that ethanol alters neurotrophin signaling leading to Purkinje neuronal death, the immunohistochemical expression of TrkB and TrkC receptors on Purkinje cells of rat pups following a moderate dose of ethanol was determined at various times surrounding the period of postnatal ethanol vulnerability. Ethanol selectively decreased Purkinje cell expression of TrkB and TrkC receptors following exposures within the vulnerable period (PN4-6). These results suggest that ethanol may induce loss of Purkinje cells by alteration of neurotrophic regulation at this critical stage.}, number={1}, journal={Brain Research}, author={Light, K.E. and Brown, D.P. and Newton, B.W. and Belcher, S.M. and Kane, C.J.M}, year={2002}, pages={71–81} }
 @article{light_belcher_pierce_2002, title={Time course and manner of Purkinje neuron death following a single ethanol exposure on postnatal day 4 in the developing rat}, volume={114}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0036775655&partnerID=MN8TOARS}, DOI={10.1016/S0306-4522(02)00344-5}, abstractNote={The present study was designed to evaluate the time course and manner of Purkinje cell death following a single ethanol dose delivered intragastrically on postnatal day (PN) 4 to rat pups. Analysis included immunolabeling of Purkinje cells with antibody specific for calbindin D28k and counting of Purkinje cells in each lobule of a mid-vermal slice. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis and immunodetection for cleaved (activated) caspase-3 enzyme was used to identify apoptosis, with calbindin D28k co-immunolabeling to identify apoptotic Purkinje cells. Finally, immunodetection for cytochrome c, again with co-labeling using calbindin D28k antibody, identified intracellular release of cytochrome c from the mitochondria into the cytoplasm of Purkinje cells. The data demonstrate that a single dose of ethanol results in a significant and extensive, lobular dependent loss of Purkinje cells within 24 h after administration. Extensive loss in the early developing lobules (I-III, VIII-X) and less to no loss in the later developing lobules (IV-VII) is consistent with prior literature reports on the ethanol-induced effects on Purkinje cells at this age. Clear and consistent evidence of apoptotic Purkinje cells was identified and the pattern was transient in nature. Finally, cytochrome c is released from the mitochondria of Purkinje cells in a time course consistent with the activation of the mitochondrial pathway of apoptosis. These data support the hypothesis that ethanol-induced loss of Purkinje cells involves apoptotic mechanisms. Furthermore, the initiation of apoptosis by ethanol is consistent with ethanol-induced interruptions of Purkinje cell neurotrophic support leading to activation of the mitochondrial pathway of apoptosis.}, number={2}, journal={Neuroscience}, author={Light, K.E. and Belcher, S.M. and Pierce, D.R.}, year={2002}, pages={327–337} }
 @article{light_ge_belcher_2001, title={Early postnatal ethanol exposure selectively decreases BDNF and truncated TrkB-T2 receptor mRNA expression in the rat cerebellum}, volume={93}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035840303&partnerID=MN8TOARS}, DOI={10.1016/S0169-328X(01)00182-6}, abstractNote={Binge-like ethanol exposure on postnatal day (PN) 4 induces a concentration dependent loss of Purkinje cells in the rat cerebellum. The mechanism of this ethanol-induced Purkinje cell vulnerability is not presently understood. Nevertheless, the specific timing of this vulnerability leads us to consider the neurotrophin system crucial to the regulation of neuronal development. Differentiation, maturation, and survival of Purkinje cells are shown to involve an intimate interaction between brain-derived nerve growth factor (BDNF) and neurotrophin-3 (NT3) acting primarily through their specific tyrosine-kinase (Trk) receptors. We believe that the specific ethanol vulnerability, and the timing of this vulnerability result from alterations in the BDNF-NT3 interplay. We hypothesize that disruption of TrkB and/or TrkC mediated neurotrophin communication is, in part, responsible for the ethanol-induced loss of Purkinje cells during development. The current study was undertaken to define the impact of ethanol exposure at the onset of ethanol vulnerability on the relative concentrations of mRNA encoding the neurotrophic factor receptors TrkB and TrkC. The reverse transcriptase (RT) polymerase chain reaction (PCR) amplification technique was used to identify the relative expression levels of mRNA specific to these receptors as well as the truncated TrkB receptor isoforms. We identify a specific decrease in overall TrkB receptor mRNA expression that is primarily a function of the TrkB-T2 receptor isoform. Concurrent decreases in mRNA specific to BDNF were also identified. No significant alterations to the expression of TrkC mRNA were found indicating that ethanol-exposure appears to act selectively on the BDNF communication system.}, number={1}, journal={Molecular Brain Research}, author={Light, K.E. and Ge, Y. and Belcher, S.M.}, year={2001}, pages={46–55} }
 @article{jakab_wong_belcher_2001, title={Estrogen receptor β immunoreactivity in differentiating cells of the developing rat cerebellum}, volume={430}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035847559&partnerID=MN8TOARS}, DOI={10.1002/1096-9861(20010212)430:3<396::AID-CNE1039>3.0.CO;2-0}, abstractNote={Estrogen receptors (ER) play a significant role in the development of some regions of the mammalian brain. Recently, ER‐beta (ERβ) mRNA and protein were shown to be expressed in the rat cerebellum. In the present study, the ontogeny of ERβ protein expression was examined in the rat cerebellum during postnatal development. Western blot analysis indicated that a single ERβ‐like immunoreactive species of ∼55 kDa was present in protein lysates prepared from the cerebella of female and male Sprague‐Dawley rat pups. Immunocytochemical analysis of cerebellar sections from the midline vermis revealed that during development, the expression of ERβ varied with age and cell‐type, but not sex. In the developing cerebellum, highest levels of ERβ‐immunoreactivity (IR) were detected in neurons during neurite growth, and in some glia during migration. Throughout the first postnatal week, ERβ‐IR was localized to differentiating granule cells in the external germinal layer and to migrating glia. Differentiating granule cells expressed detectable levels of ERβ throughout development. In Purkinje cells, ERβ‐IR was first detected on postnatal day 6 (P6), with peak intensities of immunostaining coinciding with the initiation of axonal and dendritic growth that occurs between P7 and P8. Expression of ERβ‐IR remained high during maturation of Purkinje cell dendrites, and then decreased to a lower level maintained in the adult. From the third postnatal week, ERβ‐IR was also detected in the later developing Golgi, stellate, and basket neurons. These results suggest that ERβ may play a role in growth‐related mechanisms during differentiation of cerebellar neurons and glia. J. Comp. Neurol. 430:396–409, 2001. © 2001 Wiley‐Liss, Inc.}, number={3}, journal={Journal of Comparative Neurology}, author={Jakab, R.L. and Wong, J.K. and Belcher, S.M.}, year={2001}, pages={396–409} }
 @article{belcher_zsarnovszky_2001, title={Estrogenic actions in the brain: Estrogen, phytoestrogens, and rapid intracellular signaling mechanisms}, volume={299}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0034768705&partnerID=MN8TOARS}, number={2}, journal={Journal of Pharmacology and Experimental Therapeutics}, author={Belcher, S.M. and Zsarnovszky, A.}, year={2001}, pages={408–414} }
 @article{zsarnovszky_belcher_2001, title={Identification of a developmental gradient of estrogen receptor expression and cellular localization in the developing and adult female rat primary somatosensory cortex}, volume={129}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035939097&partnerID=MN8TOARS}, DOI={10.1016/S0165-3806(01)00180-8}, abstractNote={Immunohistochemistry was used to investigate the spatiotemporal distribution of estrogen receptor alpha and beta (ER alpha, ER beta) in the posteromedial barrel subfield (PMBS) of the cerebral cortex in developing and adult female rats. Counting of immunopositive cells in predefined areas from each layer of the PMBS showed that at PN3, ER alpha immunoreactivity (IR) was present in every cell, whereas ER beta-IR was not detected. At PN6, about 59% of the cells were ER alpha immunopositive and low levels of ER beta-IR were observed in scattered cells. At PN18 the proportion of ER alpha-IR cells decreased to 49%; however, ER beta-IR became widespread and was detected in 39% of cells. By PN25 only faint ER alpha-IR was observed and in the adults ER alpha-IR was not detected. In contrast, at PN25 and in adults, ER beta-IR was detected in about half the cells of the PMBS. Regarding the cellular localization of ER-IR, at PN3 an outside-in gradient of cytoplasmic to nuclear localization of ER alpha-IR was observed. At PN18 and in adults ER beta-IR was preferentially localized to the nucleus of principal neurons, and to the cytoplasm of small, stellate-shaped interneurons. Together, these observations reveal a developmental transition of ER expression in the PMBS; ER alpha is expressed during early development, ER alpha and ER beta are co-expressed at later developmental times, and only ER beta is expressed in adults. These changes in ER expression and localization suggest that ER alpha and ER beta may play important, but different roles in the formation and function of the PMBS region of the primary somatosensory cortex.}, number={1}, journal={Developmental Brain Research}, author={Zsarnovszky, A. and Belcher, S.M.}, year={2001}, pages={39–46} }
 @article{wong_kennedy_belcher_2001, title={Simplified serum- and steroid-free culture conditions for high-throughput viability analysis of primary cultures of cerebellar granule neurons}, volume={110}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035975671&partnerID=MN8TOARS}, DOI={10.1016/S0165-0270(01)00419-8}, abstractNote={A serum- and steroid-free primary culture system was developed for the maintenance and automated analysis of cerebellar granule cell viability. Conventional poly-lysine coated 96-well tissue culture plates serve as a platform for growth, experimental manipulation and subsequent automated analysis of these primary cultured neurons. Cerebellar granule neurons were seeded at densities ranging from 2 x 10(4) to 1.25 x 10(6) cells/cm(2) and maintained in serum- and steroid-free culture conditions for 7 days. Viability was subsequently determined by the reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and the degree of cell death occurring over that period was determined by the release of lactate dehydrogenase (LDH). At appropriate cell densities, the results of the MTS reduction and LDH release assays were directly proportional to the initial number of cerebellar granule cells plated. Those results indicate that an initial cell density of 0.5 - 1.0 x 10(5) cells per well (0.32 cm(2)) was appropriate for simultaneous analysis with the MTS reduction and LDH release assays. Both assays were then used to demonstrate the utility of this model system for analysis of tert-butyl-hydroperoxide and hydrogen peroxide induced oxidative stress. Additionally, the MTS reduction assay was used to demonstrate that the NMDA-receptor selective antagonist MK-801 was neuroprotective against glutamate-mediated excitotoxicity. This study defines a powerful and flexible primary culture system for cerebellar neurons that is useful for high-throughput analysis of factors that influence neuronal viability.}, number={1-2}, journal={Journal of Neuroscience Methods}, author={Wong, J.K. and Kennedy, P.R. and Belcher, S.M.}, year={2001}, pages={45–55} }
 @article{belcher_1999, title={Regulated expression of estrogen receptor α and β mRNA in granule cells during development of the rat cerebellum}, volume={115}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033536057&partnerID=MN8TOARS}, DOI={10.1016/S0165-3806(99)00050-4}, abstractNote={A semi-quantitative RT-PCR approach was used to characterize expression of the mRNA encoding estrogen receptors in developing cerebellar granule cells of the rat. Evidence is presented for expression of both ERalpha and ERbeta transcripts in granule cells throughout the first 15 postnatal days. While transcripts encoding both ERalpha and ERbeta were expressed in granule cells, the relative levels of expression varied significantly during the first two postnatal weeks of cerebellar development. The ERalpha mRNA was expressed at the lowest level on the first day following birth; whereas expression of ERbeta was highest on that day. On the fourth postnatal day the expression of ERalpha increased, while there was a significant decrease in ERbeta expression. Between postnatal day 4 and 15, the expression of the mRNA of each receptor varied in a similar fashion; expression decreased slightly between days 4 and 10 and then increased significantly on day 15. Alternative splicing of the ERbeta transcripts was also investigated and was likewise found to vary during granule cell development. Initially, the mRNA encoding the beta1 isoform was predominant, but by day 4, the beta2 isoform was the major isoform expressed. On postnatal days 7 and 10, there was not a significant difference between the level of beta1 and beta2 expressed. By day 15, beta1 was again the predominant ERbeta isoform accounting for nearly 90% of all ERbeta transcripts expressed.}, number={1}, journal={Developmental Brain Research}, author={Belcher, S.M.}, year={1999}, pages={57–69} }
 @article{pemberton_belcher_ripellino_howe_1998, title={High-affinity kainate-type ion channels in rat cerebellar granule cells}, volume={510}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0032527976&partnerID=MN8TOARS}, DOI={10.1111/j.1469-7793.1998.401bk.x}, abstractNote={1 Patch‐clamp recordings were made from rat cerebellar granule cells in primary culture. In cells pre‐exposed to concanavalin A (ConA) to remove kainate receptor desensitization, concentration‐response data for kainate showed two components. The EC50 value for the high‐affinity component (4 μM) was consistent with activation of kainate‐type channels. ConA enhanced the apparent potency of the kainate receptor ligand SYM 2081 by 100‐fold. 2 In ConA‐treated granule cells, currents evoked by 10 μM kainate were not significantly reduced by the AMPA receptor antagonist GYKI 53655, nor were these currents significantly reduced by the co‐application of 100 μM AMPA. Currents activated by low concentrations of kainate in the presence of AMPA were completely inhibited by 10 μM La3+. 3 Single‐cell reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis indicated that granule cells express both unedited (Q) and edited (R) versions of GluR5, with the majority of the GluR5 transcripts being unedited. In contrast, GluR6(R) was detected in seven cells and GluR6(Q) was detected in one granule cell. 4 Whole‐cell current‐voltage curves for kainate‐type currents in granule cells were measured and the ratio of the slope conductances at +40 mV and −40 mV was used as an index of rectification. The mean +40 mV/‐40 mV ratio determined from thirty‐six granule cells was 1.3 ± 0.1. Spectral density analysis of kainate‐evoked whole‐cell current noise gave values for the apparent single‐channel conductance, γnoise, that were on average about 1 pS. 5 To compare further the properties of recombinant kainate channels with the native kainate‐type channels in granule cells, we determined EC50 and γnoise values for SYM 2081 in stable cell lines expressing either GluR6(R) or GluR6(R) and KA2. Co‐expression of KA2 with GluR6(R) shifts the EC50 and γnoise values determined for SYM 2081 closer to the values typically found for native kainate‐type channels in granule cells. 6 The results demonstrate that cerebellar granule cells in culture express functional kainate‐type channels and that in most cells these channels show properties that are similar to those determined for heteromeric channels formed from GluR6(R) and KA2. However, the results also suggest that different granule cells express different repertoires of kainate‐type channels with different, and perhaps variable, subunit composition.}, number={2}, journal={Journal of Physiology}, author={Pemberton, K.E. and Belcher, S.M. and Ripellino, J.A. and Howe, J.R.}, year={1998}, pages={401–420} }
 @article{mrzljak_levey_belcher_goldman-rakic_1998, title={Localization of the m2 muscarinic acetylcholine receptor protein and mRNA in cortical neurons of the normal and cholinergically deafferented rhesus monkey}, volume={390}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0031881557&partnerID=MN8TOARS}, DOI={10.1002/(SICI)1096-9861(19980105)390:1<112::AID-CNE10>3.0.CO;2-Z}, abstractNote={The m2 muscarinic acetylcholine receptor in the cerebral cortex has traditionally been thought of as an autoreceptor located on cholinergic fibers that originate from neurons in the nucleus basalis of Meynert. We now provide evidence for widespread localization of the m2 receptor in noncholinergic neurons and fibers of the cerebral cortex. The cellular and subcellular distribution of the m2 receptor protein and mRNA were examined in normal monkeys and in monkeys in which the cortical cholinergic afferents were selectively lesioned by injection of the specific immunotoxin, anti‐p75NTR‐saporin into the nucleus basalis. Both in normal and immunolesioned monkeys, the m2 mRNA and protein were localized in pyramidal and nonpyramidal neurons. In pyramidal neurons, membrane‐associated receptor immunoreactivity was found exclusively in dendritic spines receiving asymmetric synapses, indicating that the m2 receptor may modulate excitatory neurotransmission at these sites. In nonpyramidal neurons, the m2 immunoreactivity was present along the cytoplasmic surface of membranes in cell bodies, dendrites and axons. Both in pyramidal and nonpyramidal neurons of normal and lesioned monkeys, the m2 receptor was located peri‐ and extra‐synaptically, suggesting that it may be contacted by acetylcholine via volume transmission. The localization of the m2 receptor in cortical neurons and the sparing of m2 immunoreactivity in lesioned monkeys indicates that the m2 receptor is synthesized largely within the cortex and/or is localized to noncholinergic terminals of either intrinsic or extrinsic origin. These findings open the possibility that the loss of the m2 receptor in Alzheimer's disease may in part be due to degenerative changes in m2 positive neurons of the cortex rather than entirely due to the loss of autoreceptors. J. Comp. Neurol. 390:112–132, 1998. © 1998 Wiley‐Liss, Inc.}, number={1}, journal={Journal of Comparative Neurology}, author={Mrzljak, L. and Levey, A.I. and Belcher, S. and Goldman-Rakic, P.S.}, year={1998}, pages={112–132} }
 @article{schmidt_maue_lehmann_belcher_stahl_perlman_1998, title={Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the catalytic core of a mitochondrial group II intron}, volume={282}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0032566594&partnerID=MN8TOARS}, DOI={10.1006/jmbi.1998.2021}, abstractNote={Previous studies show that some yeast strains carrying point mutations of domain 5 that block splicing of a mitochondrial group II intron yield spontaneous revertants in which splicing is partially restored by dominant mutations of nuclear genes. Here we cloned and sequenced the suppressor allele of one such gene, and found it to be a missense mutation of the MRS2 gene (MRS2-L232F). The MRS2 gene was first implicated in group II intron splicing by the finding that overexpression of the wild-type gene weakly suppresses the splicing defect of a mutation of another intron. Tetrad analysis showed that independently isolated suppressors of two other domain 5 mutations are also allelles of the MRS2 gene and DNA sequencing identified a new missense mutation in each strain (MRS2-T230I and MRS2-L213M). All three suppressor mutations cause a temperature-sensitive respiration defect that is dominant negative in heterozygous diploids, but those strains splice the mutant intron at the elevated temperature. The three mutations are in a domain of the protein that is likely to be a helix-turn-helix region, so that effects of the mutations on protein-protein interactions may contribute to these phenotypes. These mutations suppress the splicing defect of many, but not all, of the available splicing defective mutations of aI5gamma, including mutations of several intron domains. Protein and RNA blot experiments show that the level of the protein encoded by the MRS2 gene, but not the mRNA, is elevated by these mutations. Interestingly, overexpression of the wild-type protein restores much lower levels of splicing than were obtained with similar elevated levels of the mutated Mrs2 proteins. The splicing phenotypes of these strains suggest a direct role for Mrs2 protein on group II intron splicing, but an indirect effect is not yet ruled out.}, number={3}, journal={Journal of Molecular Biology}, author={Schmidt, U. and Maue, I. and Lehmann, K. and Belcher, S.M. and Stahl, U. and Perlman, P.S.}, year={1998}, pages={525–541} }
 @article{belcher_howe_1997, title={Characterization of RNA editing of the glutamate-receptor subunits GluR5 and GluR6 in granule cells during cerebellar development}, volume={52}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0030780391&partnerID=MN8TOARS}, DOI={10.1016/S0169-328X(97)00252-0}, abstractNote={The non-NMDA class of ionotropic glutamate receptors are subject to RNA editing resulting in single amino acid changes within individual subunits that make up these oligomeric receptors. These amino acid changes result in significant alterations of important channel properties. Both edited and unedited versions of the kainate-receptor subunits GluR5 and GluR6 are present in brain, but whether this reflects the expression of both versions in individual types of neurons or differences in editing between different cell types is unclear. To characterize editing in a single identified type of central neuron, we have determined the extent to which GluR5 and GluR6 mRNAs are edited in acutely isolated cerebellar granule cells. RT-PCR analysis revealed that editing at each site in GluR5 and GluR6 increased during early postnatal development. The Q/R site was predominantly unedited in GluR5, whereas GluR6 was mostly edited. The Q/R and Y/C sites of GluR6 were edited to similar extents, whereas a smaller percentage of transcripts were edited at the I/V site. The expression of two double-stranded RNA adenosine deaminases implicated in GluR editing (DRADA and RED1) increased in granule cells between postnatal days 1 and 15. Finally, cerebellar granule cells express a previously unreported variant of RED1 which appears to arise from developmentally regulated alternative splicing.}, number={1}, journal={Molecular Brain Research}, author={Belcher, S.M. and Howe, J.R.}, year={1997}, pages={130–138} }
 @article{beckers_ernst_belcher_howe_levenson_gros_1996, title={A new sodium channel α-subunit gene (Scn9a) from Schwann cells maps to the Scn1a, Scn2a, Scn3a cluster of mouse chromosome 2}, volume={36}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0030586911&partnerID=MN8TOARS}, DOI={10.1006/geno.1996.0447}, abstractNote={We have used a total of 27 AXB/BXA recombinant inbred mouse strains to determine the chromosomal location of a newly identified gene encoding an alpha-subunit isoform of the sodium channel from Schwann cells, Scn9a. Linkage analysis established that Scn9a mapped to the proximal segment of mouse chromosome 2. The segregation of restriction fragment length polymorphisms in 145 progeny from a Mus spretus x C57BL/6J backcross indicates that Scn9a is very tightly linked to Scn1a (gene encoding the type I sodium channel alpha-subunit of the brain) and forms part of a cluster of four Scna genes located on mouse chromosome 2.}, number={1}, journal={Genomics}, author={Beckers, M.-C. and Ernst, E. and Belcher, S. and Howe, J. and Levenson, R. and Gros, P.}, year={1996}, pages={202–205} }
 @article{belcher_howe_1996, title={Cloning of the cDNA encoding the sodium channel β1 subunit from rabbit}, volume={170}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0029972385&partnerID=MN8TOARS}, DOI={10.1016/0378-1119(95)00871-3}, abstractNote={Here, we report the nucleotide (nt) sequence of the cDNA encoding the sodium channel β1 subunit from rabbit (oβ1). Cloning of the oβ1 cDNA was accomplished by reverse transcription-polymerase chain reaction using rabbit brain RNA as a template for cDNA synthesis. The nt sequence of the oβ1 cDNA predicts a 218-amino-acid polypeptide which is 96.3 and 97.3% identical to the β1 from human (hβ1) and rat (rβ1), respectively.}, number={2}, journal={Gene}, author={Belcher, S.M. and Howe, J.R.}, year={1996}, pages={285–286} }
 @book{butow_henke_moran_belcher_perlman_1996, title={[24] Transformation of Saccharomyces cerevisiae mitochondria using the biolistic gun}, volume={264}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0029873586&partnerID=MN8TOARS}, journal={Methods in Enzymology}, author={Butow, R.A. and Henke, R.M. and Moran, J.V. and Belcher, S.M. and Perlman, P.S.}, year={1996}, pages={265–278} }
 @article{belcher_zerillo_levenson_ritchie_howe_1995, title={Cloning of a sodium channel α subunit from rabbit Schwann cells}, volume={92}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0028834635&partnerID=MN8TOARS}, DOI={10.1073/pnas.92.24.11034}, abstractNote={Overlapping cDNA clones spanning the entire coding region of a Na-channel alpha subunit were isolated from cultured Schwann cells from rabbits. The coding region predicts a polypeptide (Nas) of 1984 amino acids exhibiting several features characteristic of Na-channel alpha subunits isolated from other tissues. Sequence comparisons showed that the Nas alpha subunit resembles most the family of Na channels isolated from brain (approximately 80% amino acid identity) and is least similar (approximately 55% amino acid identity) to the atypical Na channel expressed in human heart and the partial rat cDNA, NaG. As for the brain II and III isoforms, two variants of Nas exist that appear to arise by alternative splicing. The results of reverse transcriptase-polymerase chain reaction experiments suggest that expression of Nas transcripts is restricted to cells in the peripheral and central nervous systems. Expression was detected in cultured Schwann cells, sciatic nerve, brain, and spinal cord but not in skeletal or cardiac muscle, liver, kidney, or lung.}, number={24}, journal={Proceedings of the National Academy of Sciences of the United States of America}, author={Belcher, S.M. and Zerillo, C.A. and Levenson, R. and Ritchie, J.M. and Howe, J.R.}, year={1995}, pages={11034–11038} }
 @article{boulanger_belcher_schmidt_dib-hajj_schmidt_perlman_1995, title={Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron}, volume={15}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0029055177&partnerID=MN8TOARS}, DOI={10.1128/MCB.15.8.4479}, abstractNote={Domain 5 (D5) is a highly conserved, largely helical substructure of group II introns that is essential for self-splicing. Only three of the 14 base pairs present in most D5 structures (A2.U33, G3.U32, and C4.G31) are nearly invariant. We have studied effects of point mutations of those six nucleotides on self-splicing and in vivo splicing of aI5 gamma, an intron of the COXI gene of Saccharomyces cerevisiae mitochondria. Though none of the point mutations blocked self-splicing under one commonly used in vitro reaction condition, the most debilitating mutations were at G3 and G4. Following mitochondrial Biolistic transformation, it was found that mutations at A2, G3, and C4 blocked respiratory growth and splicing while mutations at the other sites had little effect on either phenotype. Intra-D5 second-site suppressors showed that pairing between nucleotides at positions 2 and 33 and 4 and 31 is especially important for D5 function. At the G3.U32 wobble pair, the mutant A.U pair blocks splicing, but a revertant of that mutant that can form an A+.C base pair regains some splicing. A dominant nuclear suppressor restores some splicing to the G3A mutant but not the G3U mutant, suggesting that a purine is required at position 3. These findings are discussed in terms of the hypothesis of Madhani and Guthrie (H. D. Madhani and C. Guthrie, Cell 71:803-817, 1992) that helix 1 formed between yeast U2 and U6 small nuclear RNAs may be the spliceosomal cognate of D5.}, number={8}, journal={Molecular and Cellular Biology}, author={Boulanger, S.C. and Belcher, S.M. and Schmidt, U. and Dib-Hajj, S.D. and Schmidt, T. and Perlman, P.S.}, year={1995}, pages={4479–4488} }
 @article{howe_skryabin_belcher_zerillo_schmauss_1995, title={The responsiveness of a tetracycline-sensitive expression system differs in different cell lines}, volume={270}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0029009922&partnerID=MN8TOARS}, DOI={10.1074/jbc.270.23.14168}, abstractNote={A tetracycline-sensitive inducible expression system was used to regulate the expression of neurotransmitter receptor genes in two mammalian cell lines. The dopamine D3-receptor was stably expressed in GH3 cells, and GluR6 (a glutamate receptor subunit) was stably expressed in human embryonic kidney (HEK 293) cells. Three striking differences were found. 1) In the inactive state, virtually no D3-receptor expression was found in GH3 cells, whereas substantial levels of GluR6 expression were found in HEK 293 cells. 2) The induction of expression obtained upon removal of tetracycline was robust in GH3 cells but only modest in HEK 293 cells. 3) Whereas in each clonal cell line, the expression of a co-transfected hybrid transactivator is clearly regulated in a tetracycline-responsive manner, in the induced state, its mRNA levels were found to be very low in GH3 cells and very high in HEK 293 cells. The results indicate that, in contrast to GH3 cells, HEK 293 cells do not provide a cellular environment in which the expression of a heterologous gene can be tightly controlled in a tetracycline-responsive manner.}, number={23}, journal={Journal of Biological Chemistry}, author={Howe, J.R. and Skryabin, B.V. and Belcher, S.M. and Zerillo, C.A. and Schmauss, C.}, year={1995}, pages={14168–14174} }
 @article{moran_mecklenburg_sass_belcher_mahnke_lewin_perlman_1994, title={Splicing defective mutants of the COXI gene of yeast mitochondrial DNA: Initial definition of the maturase domain of the group II intron AI2}, volume={22}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0028225133&partnerID=MN8TOARS}, DOI={10.1093/nar/22.11.2057}, abstractNote={Six mutations blocking the function of a seven intron form of the mitochondrial gene encoding subunit I of cytochrome c oxidase (COXI) and mapping upstream of exon 3 were isolated and characterized. A cis-dominant mutant of the group IIA intron 1 defines a helical portion of the C1 substructure of domain 1 as essential for splicing. A trans-recessive mutant confirms that the intron 1 reading frame encodes a maturase function. A cis-dominant mutant in exon 2 was found to have no effect on the splicing of intron 1 or 2. A trans-recessive mutant, located in the group IIA intron 2, demonstrates for the first time that intron 2 encodes a maturase. A genetic dissection of the five missense mutations present in the intron 2 reading frame of that strain demonstrates that the maturase defect results from one or both of the missense mutations in a newly-recognized conserved sequence called domain X.}, number={11}, journal={Nucleic Acids Research}, author={Moran, J.V. and Mecklenburg, K.L. and Sass, P. and Belcher, S.M. and Mahnke, D. and Lewin, A. and Perlman, P.}, year={1994}, pages={2057–2064} }
 @article{peebles_belcher_zhang_dietrich_perlman_1993, title={Mutation of the conserved first nucleotide of a group II intron from yeast mitochondrial DNA reduces the rate but allows accurate splicing}, volume={268}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0027156992&partnerID=MN8TOARS}, number={16}, journal={Journal of Biological Chemistry}, author={Peebles, C.L. and Belcher, S.M. and Zhang, M. and Dietrich, R.C. and Perlman, P.S.}, year={1993}, pages={11929–11938} }
 @article{hebbar_belcher_perlman_1992, title={A maturase-encoding group MA intron of yeast mitochondria self-splices in vitro}, volume={20}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0026555469&partnerID=MN8TOARS}, DOI={10.1093/nar/20.7.1747}, abstractNote={Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.}, number={7}, journal={Nucleic Acids Research}, author={Hebbar, S.K. and Belcher, S.M. and Perlman, P.S.}, year={1992}, pages={1747–1754} }