RESUMEN
The U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP) is tasked with assessing chemicals for their potential to perturb endocrine pathways, including those controlled by androgen receptor (AR). To address challenges associated with traditional testing strategies, EDSP is considering in vitro high-throughput screening assays to screen and prioritize chemicals more efficiently. The ability of these assays to accurately reflect chemical interactions in nonmammalian species remains uncertain. Therefore, a goal of the EDSP is to evaluate how broadly results can be extrapolated across taxa. To assess the cross-species conservation of AR-modulated pathways, computational analyses and systematic literature review approaches were used to conduct a comprehensive analysis of existing in silico, in vitro, and in vivo data. First, molecular target conservation was assessed across 585 diverse species based on the structural similarity of ARs. These results indicate that ARs are conserved across vertebrates and are predicted to share similarly susceptibility to chemicals that interact with the human AR. Systematic analysis of over 5000 published manuscripts was used to compile in vitro and in vivo cross-species toxicity data. Assessment of in vitro data indicates conservation of responses occurs across vertebrate ARs, with potential differences in sensitivity. Similarly, in vivo data indicate strong conservation of the AR signaling pathways across vertebrate species, although sensitivity may vary. Overall, this study demonstrates a framework for utilizing bioinformatics and existing data to build weight of evidence for cross-species extrapolation and provides a technical basis for extrapolating hAR-based data to prioritize hazard in nonmammalian vertebrate species.
Asunto(s)
Disruptores Endocrinos , Receptores Androgénicos , Animales , Estados Unidos , Humanos , Receptores Androgénicos/metabolismo , United States Environmental Protection Agency , Sistema Endocrino/química , Sistema Endocrino/metabolismo , Disruptores Endocrinos/toxicidad , Disruptores Endocrinos/química , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
U.S. regulatory and research agencies use ecotoxicity test data to assess the hazards associated with substances that may be released into the environment, including but not limited to industrial chemicals, pharmaceuticals, pesticides, food additives, and color additives. These data are used to conduct hazard assessments and evaluate potential risks to aquatic life (e.g., invertebrates, fish), birds, wildlife species, or the environment. To identify opportunities for regulatory uses of non-animal replacements for ecotoxicity tests, the needs and uses for data from tests utilizing animals must first be clarified. Accordingly, the objective of this review was to identify the ecotoxicity test data relied upon by U.S. federal agencies. The standards, test guidelines, guidance documents, and/or endpoints that are used to address each of the agencies' regulatory and research needs regarding ecotoxicity testing are described in the context of their application to decision-making. Testing and information use, needs, and/or requirements relevant to the regulatory or programmatic mandates of the agencies taking part in the Interagency Coordinating Committee on the Validation of Alternative Methods Ecotoxicology Workgroup are captured. This information will be useful for coordinating efforts to develop and implement alternative test methods to reduce, refine, or replace animal use in chemical safety evaluations.
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Agencias Gubernamentales , Plaguicidas , Animales , EcotoxicologíaRESUMEN
BACKGROUND: Novel in vitro methods are being developed to identify chemicals that may interfere with estrogen receptor (ER) signaling, but the results are difficult to put into biological context because of reliance on reference chemicals established using results from other in vitro assays and because of the lack of high-quality in vivo reference data. The Organisation for Economic Co-operation and Development (OECD)-validated rodent uterotrophic bioassay is considered the "gold standard" for identifying potential ER agonists. OBJECTIVES: We performed a comprehensive literature review to identify and evaluate data from uterotrophic studies and to analyze study variability. METHODS: We reviewed 670 articles with results from 2,615 uterotrophic bioassays using 235 unique chemicals. Study descriptors, such as species/strain, route of administration, dosing regimen, lowest effect level, and test outcome, were captured in a database of uterotrophic results. Studies were assessed for adherence to six criteria that were based on uterotrophic regulatory test guidelines. Studies meeting all six criteria (458 bioassays on 118 unique chemicals) were considered guideline-like (GL) and were subsequently analyzed. RESULTS: The immature rat model was used for 76% of the GL studies. Active outcomes were more prevalent across rat models (74% active) than across mouse models (36% active). Of the 70 chemicals with at least two GL studies, 18 (26%) had discordant outcomes and were classified as both active and inactive. Many discordant results were attributable to differences in study design (e.g., injection vs. oral dosing). CONCLUSIONS: This uterotrophic database provides a valuable resource for understanding in vivo outcome variability and for evaluating the performance of in vitro assays that measure estrogenic activity. CITATION: Kleinstreuer NC, Ceger PC, Allen DG, Strickland J, Chang X, Hamm JT, Casey WM. 2016. A curated database of rodent uterotrophic bioactivity. Environ Health Perspect 124:556-562; http://dx.doi.org/10.1289/ehp.1510183.
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Receptores de Estrógenos/metabolismo , Pruebas de Toxicidad , Animales , Bioensayo , Relación Dosis-Respuesta a Droga , Estrógenos/toxicidad , Femenino , Técnicas In Vitro , Ratas , Útero/efectos de los fármacosRESUMEN
Human bronchial cells are one of the first cell types exposed to environmental toxins. Toxins often activate nuclear factor-kappaB (NF-kappaB) and protein kinase C (PKC). We evaluated the hypothesis that cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke, activates PKC-alpha and NF-kappaB, and concomitantly disrupts the F-actin cytoskeleton, induces apoptosis and alters cell function in BEAS-2B human bronchial epithelial cells. Compared to controls, exposure of BEAS-2B cells to doses of 30mug/ml CSC significantly activated PKC-alpha, while CSC doses above 20mug/ml CSC significantly activated NF-kappaB. As NF-kappaB was activated, cell number decreased. CSC treatment of BEAS-2B cells induced a decrease in cell size and an increase in cell surface extensions including filopodia and lamellipodia. CSC treatment of BEAS-2B cells induced F-actin rearrangement such that stress fibers were no longer prominent at the cell periphery and throughout the cells, but relocalized to perinuclear regions. Concurrently, CSC induced an increase in the focal adhesion protein vinculin at the cell periphery. CSC doses above 30mug/ml induced a significant increase in apoptosis in BEAS-2B cells evidenced by an increase in activated caspase 3, an increase in mitochondrial mass and a decrease in mitochondrial membrane potential. As caspase 3 increased, cell number decreased. CSC doses above 30mug/ml also induced significant concurrent changes in cell function including decreased cell spreading and motility. CSC initiates a signaling cascade in human bronchial epithelial cells involving PKC-alpha, NF-kappaB and caspase 3, and consequently decreases cell spreading and motility. These CSC-induced alterations in cell structure likely prevent cells from performing their normal function thereby contributing to smoke-induced diseases.
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Bronquios/efectos de los fármacos , Nicotiana , Transducción de Señal/efectos de los fármacos , Humo/efectos adversos , Fumar/efectos adversos , Actinas/metabolismo , Apoptosis/efectos de los fármacos , Bronquios/metabolismo , Bronquios/patología , Caspasa 3/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Concentración 50 Inhibidora , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Proteína Quinasa C-alfa/metabolismo , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/metabolismo , Vinculina/metabolismoRESUMEN
Metabolomics is a technology for identifying and quantifying numerous biochemicals across metabolic pathways. Using this approach, we explored changes in biochemical profiles of human alveolar epithelial carcinoma (A549) cells following in vitro exposure to mainstream whole smoke (WS) aerosol as well as to wet total particulate matter (WTPM) or gas/vapor phase (GVP), the two constituent phases of WS from 2R4F Kentucky reference cigarettes. A549 cells were exposed to WTPM or GVP (expressed as WTPM mass equivalent GVP volumes) at 0, 5, 25, or 50 microg/mL or to WS from zero, two, four, and six cigarettes for 1 or 24 h. Cell pellets were analyzed for perturbations in biochemical profiles, with named biochemicals measured, analyzed, and reported in a heat map format, along with biochemical and physiological interpretations (mSelect, Metabolon Inc.). Both WTPM and GVP exposures likely decreased glycolysis (based on decreased glycolytic intermediaries) and increased oxidative stress and cell damage. Alterations in the Krebs cycle and the urea cycle were unique to WTPM exposure, while induction of hexosamines and alterations in lipid metabolism were unique to GVP exposure. WS altered glutathione (GSH) levels, enhanced polyamine and pantothenate levels, likely increased beta-oxidation of fatty acids, and increased phospholipid degradation marked by an increase in phosphoethanolamine. GSH, glutamine, and pantothenate showed the most significant changes with cigarette smoke exposure in A549 cells based on principal component analysis. Many of the changed biochemicals were previously reported to be altered by cigarette exposure, but the global metabolomic approach offers the advantage of observing changes to hundreds of biochemicals in a single experiment and the possibility for new discoveries. The metabolomic approach may thus be used as a screening tool to evaluate conventional and novel tobacco products offering the potential to reduce risks of smoking.
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Células Epiteliales/metabolismo , Pulmón/citología , Metaboloma/efectos de los fármacos , Nicotiana , Humo/efectos adversos , Línea Celular Tumoral , Células Epiteliales/efectos de los fármacos , HumanosRESUMEN
Adipose triglyceride lipase (ATGL) is a recently described adipose-enriched protein with triglyceride-specific lipase activity. ATGL shares the greatest sequence homology with adiponutrin, a nutritionally regulated protein of unclear biological function. Here we present a functional analysis of ATGL and adiponutrin and describe their regulation by insulin. Retroviral-mediated overexpression of ATGL in 3T3-L1 adipocytes increased basal and isoproterenol-stimulated glycerol and nonesterified fatty acid (NEFA) release, whereas siRNA-mediated knockdown of ATGL had the opposite effect. In contrast, siRNA-mediated knockdown of adiponutrin in 3T3-L1 adipocytes had no effect on glycerol or NEFA release. In mice, both ATGL and adiponutrin are nutritionally regulated in adipose tissue, with ATGL being upregulated and adiponutrin being downregulated by fasting. In 3T3-L1 adipocytes, insulin decreased ATGL and increased adiponutrin expression in a dose- and time-dependent manner, suggesting that insulin directly mediates this nutritional regulation. In addition, adipose expression of ATGL was increased by insulin deficiency and decreased by insulin replacement in streptozotocin-induced diabetic mice and was increased in fat-specific insulin receptor knockout mice, whereas adiponutrin showed the opposite pattern. These data suggest that murine ATGL but not adiponutrin contributes to net adipocyte lipolysis and that ATGL and adiponutrin are oppositely regulated by insulin both in vitro and in vivo.
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Tejido Adiposo/enzimología , Insulina/farmacología , Lipasa/metabolismo , Proteínas de la Membrana/metabolismo , Células 3T3-L1 , Animales , Diabetes Mellitus Experimental , Ácidos Grasos/metabolismo , Regulación Enzimológica de la Expresión Génica , Insulina/metabolismo , Lipasa/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Interferencia de ARN , Receptor de Insulina/genéticaRESUMEN
Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear hormone receptor that is critical for adipogenesis and insulin sensitivity. Ligands for PPARgamma include some polyunsaturated fatty acids and prostanoids and the synthetic high affinity antidiabetic agents thiazolidinediones. However, the identity of a biologically relevant endogenous PPARgamma ligand is unknown, and limited insight exists into the factors that may regulate production of endogenous PPARgamma ligands during adipocyte development. To address this question, we created a line of 3T3-L1 preadipocytes that carry a beta-galactosidase-based PPARgamma ligand-sensing vector system. In this system, induction of adipogenesis resulted in elevated beta-galactosidase activity that signifies activation of PPARgamma via its ligand-binding domain (LBD) and suggests generation and/or accumulation of a ligand moiety. The putative endogenous ligand appeared early in adipogenesis in response to increases in cAMP, accumulated in the medium, and dissipated later in adipogenesis. Organically extracted and high pressure liquid chromatography-fractionated conditioned media from differentiating cells, but not from mature adipocytes, were enriched in this activity. One or more components within the organic extract activated PPARgamma through interaction with its LBD, induced lipid accumulation in 3T3-L1 cells as efficiently as the differentiation mixture, and competed for binding of rosiglitazone to the LBD of PPARgamma. The active species appears to be different from other PPARgamma ligands identified previously. Our findings suggest that a novel biologically relevant PPARgamma ligand is transiently produced in 3T3-L1 cells during adipogenesis.
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Adipocitos/fisiología , Diferenciación Celular/fisiología , PPAR gamma/fisiología , Células 3T3-L1 , Adipocitos/citología , Animales , Sitios de Unión , Ligandos , Ratones , Transducción de Señal , beta-Galactosidasa/fisiologíaRESUMEN
Pregnant Long Evans rats received 1.0 microg/kg of dioxin toxic equivalents (TEQ) by oral gavage on the 15th gestational day (GD 15), using a dosing mixture that contained two polychlorinated dioxins, four polychlorinated furans and three non-ortho polychlorinated biphenyls (PCBs). Rats were sacrificed on GD 16, GD 21 and postnatal day 4 (PND 4). The lipid content of fetus, pup, placenta and maternal liver, serum and adipose tissue were determined. Treated GD 16 and GD 21 fetuses had identical lipid content to the control group, yet the lipid content of treated pups on PND 4 was 32% higher than that of the control group. On the other hand, the lipid content of placenta, liver, and serum from the treated dams was 44-50%, 24%, and 38% lower than that of the control group, respectively. Thus, a low-dose mixture of dioxin-like compounds can cause changes in lipid content. The lipid content of offspring was not affected until they were exposed via lactation.
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Benzofuranos/efectos adversos , Contaminantes Ambientales/efectos adversos , Lípidos/análisis , Intercambio Materno-Fetal , Bifenilos Policlorados/efectos adversos , Dibenzodioxinas Policloradas/análogos & derivados , Dibenzodioxinas Policloradas/efectos adversos , Contaminantes del Suelo/efectos adversos , Tejido Adiposo/química , Administración Oral , Animales , Dibenzofuranos Policlorados , Femenino , Lactancia , Hígado/química , Embarazo , Ratas , Ratas Long-EvansRESUMEN
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure during gestation has revealed reproductive anomalies in rat offspring, including inconclusive reports of stunted mammary development in females (Brown et al., 1998, Carcinogenesis 19, 1623-1629; Lewis et al., 2001, TOXICOL: Sci. 62, 46-53). The current studies were designed to examine mammary-gland development in female offspring exposed in utero and lactationally to TCDD, and to determine a critical exposure period and cellular source of these effects. Long-Evans rats were exposed to 1 microg TCDD/kg body weight (bw) or vehicle on gestation day (GD) 15. TCDD-exposed females sacrificed on postnatal days (PND) 4, 25, 33, 37, 45, and 68 weighed significantly less than control litter mates, and peripubertal animals exhibited delayed vaginal opening and persistent vaginal threads, yet did not display altered estrous cyclicity. Mammary glands taken from TCDD-exposed animals on PND 4 demonstrated reduced primary branches, decreased epithelial elongation, and significantly fewer alveolar buds and lateral branches. This phenomenon persisted through PND 68 when, unlike fully developed glands of controls, TCDD-exposed rats retained undifferentiated terminal structures. Glands of offspring exposed to TCDD or oil on gestation days 15 and 20 or lactation days 1, 3, 5, and 10 were examined on PND 4 or 25 to discern that GD 15 was a critical period for consistent inhibition of epithelial development. Experiments using mammary epithelial transplantation between control and TCDD-exposed females suggested that the stroma plays a major role in the retarded development of the mammary gland following TCDD exposure. Our data suggest that exposure to TCDD prior to migration of the mammary bud into the fat pad permanently alters mammary epithelial development in female rat offspring.