RESUMO
Environmental chemical (EC) exposures and our interactions with them has significantly increased in the recent decades. Toxicity associated biological characterization of these chemicals is challenging and inefficient, even with available high-throughput technologies. In this report, we describe a novel computational method for characterizing toxicity, associated biological perturbations and disease outcome, called the Chemo-Phenotypic Based Toxicity Measurement (CPTM). CPTM is used to quantify the EC "toxicity score" (Zts), which serves as a holistic metric of potential toxicity and disease outcome. CPTM quantitative toxicity is the measure of chemical features, biological phenotypic effects, and toxicokinetic properties of the ECs. For proof-of-concept, we subject ECs obtained from the Environmental Protection Agency's (EPA) database to the CPTM. We validated the CPTM toxicity predictions by correlating 'Zts' scores with known toxicity effects. We also confirmed the CPTM predictions with in-vitro, and in-vivo experiments. In in-vitro and zebrafish models, we showed that, mixtures of the motor oil and food additive 'Salpn' with endogenous nuclear receptor ligands such as Vitamin D3, dysregulated the nuclear receptors and key transcription pathways involved in Colorectal Cancer. Further, in a human patient derived cell organoid model, we found that a mixture of the widely used pesticides 'Tetramethrin' and 'Fenpropathrin' significantly impacts the population of patient derived pancreatic cancer cells and 3D organoid models to support rapid PDAC disease progression. The CPTM method is, to our knowledge, the first comprehensive toxico-physicochemical, and phenotypic bionetwork-based platform for efficient high-throughput screening of environmental chemical toxicity, mechanisms of action, and connection to disease outcomes.
Assuntos
Neoplasias Colorretais , Neoplasias Pancreáticas , Praguicidas , Animais , Colecalciferol , Humanos , Praguicidas/toxicidade , Peixe-ZebraRESUMO
An approach has been developed that allows the synthesis of submicron spherical silica particles with a controlled micro-mesoporous structure possessing a large specific surface area (up to 1300 m2g-1). Particle synthesis is carried out by the hydrolysis of a mixture of various organosilanes mostly associated either with CTAB or with each other. A change in the concentration of CTAB in the reaction mixture apparently leads to a change in the formation mechanism of nuclei for the silica particle growth, which allows for varying the diameter of the synthesized particles in the range from 40-450 nm. The effect of the composition of the silica precursor ([3-(methacryloyloxy)propyl]trimethoxysilane, (3-aminopropyl)triethoxysilane and tetraethoxysilane) on the formation process and porosity of the resulting particles is studied. It was shown that by simply varying the ratio of organosilanes in the composition of the precursor, one can control the pore diameter of the particles in a wide range from 0.6-15 nm. The large-pore (up to 15 nm) silica particles are used as a matrix for the spatial distribution of luminescent carbon dots. The incorporation of carbon dots into SiO2particles prevents their aggregation leading to emission quenching after drying, thus allowing us to obtain highly luminescent composite particles. LEDs based on the obtained composite material show bright visible luminescence with spectral characteristics similar to that of a commercial cold white LED.
RESUMO
Environmental pollution is a threat to humans and wildlife species. Of particular concern are endocrine disrupting chemicals (EDCs). An important target of EDCs is nuclear receptors (NRs) that control endocrine and metabolic responses through transcriptional regulation. Owing in part to structural differences of NRs, adverse effects of EDCs vary significantly among species. Here, we describe a multiplexed reporter assay (the Ecotox FACTORIAL) enabling parallel assessment of compounds' effects on estrogen, androgen, thyroid, and PPARγ receptors of representative mammals, birds, reptiles, amphibians, and fish. The Ecotox FACTORIAL is a single-well assay comprising a set of species-specific, one-hybrid GAL4-NR reporter constructs transiently transfected into test cells. To harmonize cross-species assessments, we used a combination of two approaches. First, we used the same type of test cells for all reporters; second, we implemented a parallel detection of reporter RNAs. The assay demonstrated excellent quality, reproducibility, and insignificant intra-assay variability. Importantly, the EC50 values for NR ligands were consistent with those reported for conventional assays. Using the assay allowed ranking the hazard potential of environmental pollutants (e.g., bisphenols, polycyclic aromatic hydrocarbons, and synthetic progestins) across species. Furthermore, the assay permitted detecting taxa-specific effects of surface water samples. Therefore, the Ecotox FACTORIAL enables harmonized assessment of the endocrine and metabolic disrupting activity of chemicals and surface water in humans as well as in wildlife species.
Assuntos
Disruptores Endócrinos , Poluentes Ambientais , Animais , Bioensaio , Disruptores Endócrinos/toxicidade , Sistema Endócrino , Poluentes Ambientais/farmacologia , Humanos , Reprodutibilidade dos TestesRESUMO
While chemical analysis of contaminant mixtures remains an essential component of environmental monitoring, bioactivity-based assessments using in vitro systems increasingly are used in the detection of biological effects. Historically, in vitro assessments focused on a few biological pathways, for example, aryl hydrocarbon receptor (AhR) or estrogen receptor (ER) activities. High-throughput screening (HTS) technologies have greatly increased the number of biological targets and processes that can be rapidly assessed. Here we screened extracts of surface waters from a nationwide survey of United States streams for bioactivities associated with 69 different end points using two multiplexed HTS assays. Bioactivity of extracts from 38 streams was evaluated and compared with concentrations of over 700 analytes to identify chemicals contributing to observed effects. Eleven primary biological end points were detected. Pregnane X receptor (PXR) and AhR-mediated activities were the most commonly detected. Measured chemicals did not completely account for AhR and PXR responses. Surface waters with AhR and PXR effects were associated with low intensity, developed land cover. Likewise, elevated bioactivities frequently associated with wastewater discharges included endocrine-related end points ER and glucocorticoid receptor. These results underscore the value of bioassay-based monitoring of environmental mixtures for detecting biological effects that could not be ascertained solely through chemical analyses.
Assuntos
Rios , Poluentes Químicos da Água , Misturas Complexas , Monitoramento Ambiental , Inquéritos e Questionários , Estados UnidosRESUMO
Popularized on social media, hand-moldable plastics are formed by consumers into tools, trinkets, and dental prosthetics. Despite the anticipated dermal and oral contact, manufacturers share little information with consumers about these materials, which are typically sold as microplastic-sized resin pellets. Inherent to their function, moldable plastics pose a risk of dermal and oral exposure to unknown leachable substances. We analyzed 12 moldable plastics advertised for modeling and dental applications and determined them to be polycaprolactone (PCL) or thermoplastic polyurethane (TPU). The bioactivities of the most popular brands advertised for modeling applications of each type of polymer were evaluated using a zebrafish embryo bioassay. While water-borne exposure to the TPU pellets did not affect the targeted developmental end points at any concentration tested, the PCL pellets were acutely toxic above 1 pellet/mL. The aqueous leachates of the PCL pellets demonstrated similar toxicity. Methanolic extracts from the PCL pellets were assayed for their bioactivity using the Attagene FACTORIAL platform. Of the 69 measured end points, the extracts activated nuclear receptors and transcription factors for xenobiotic metabolism (pregnane X receptor, PXR), lipid metabolism (peroxisome proliferator-activated receptor γ, PPARγ), and oxidative stress (nuclear factor erythroid 2-related factor 2, NRF2). By nontargeted high-resolution comprehensive two-dimensional gas chromatography (GC × GC-HRT), we tentatively identified several compounds in the methanolic extracts, including PCL oligomers, a phenolic antioxidant, and residues of suspected antihydrolysis and cross-linking additives. In a follow-up zebrafish embryo bioassay, because of its stated high purity, biomedical grade PCL was tested to mitigate any confounding effects due to chemical additives in the PCL pellets; it elicited comparable acute toxicity. From these orthogonal and complementary experiments, we suggest that the toxicity was due to oligomers and nanoplastics released from the PCL rather than chemical additives. These results challenge the perceived and assumed inertness of plastics and highlight their multiple sources of toxicity.
Assuntos
Poliésteres , Peixe-Zebra , Peixe-Zebra/embriologia , Animais , Poliésteres/química , Poliésteres/toxicidade , Poliuretanos/química , Poliuretanos/toxicidade , Plásticos/química , Plásticos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , HumanosRESUMO
It is well established that catecholamine-stimulated thermogenesis in brown fat requires beta-adrenergic elevations in cyclic AMP (cAMP) to increase expression of the uncoupling protein 1 (UCP1) gene. However, little is known about the downstream components of the signaling cascade or the relevant transcription factor targets thereof. Here we demonstrate that cAMP- and protein kinase A-dependent activation of p38 mitogen-activated protein kinase (MAPK) in brown adipocytes is an indispensable step in the transcription of the UCP1 gene in mice. By phosphorylating activating transcription factor 2 (ATF-2) and peroxisome proliferator-activated receptor gamma (PPARgamma) coativator 1alpha (PGC-1alpha), members of two distinct nuclear factor families, p38 MAPK controls the expression of the UCP1 gene through their respective interactions with a cAMP response element and a PPAR response element that both reside within a critical enhancer motif of the UCP1 gene. Activation of ATF-2 by p38 MAPK additionally serves as the cAMP sensor that increases expression of the PGC-1alpha gene itself in brown adipose tissue. In conclusion, our findings illustrate that by orchestrating the activity of multiple transcription factors, p38 MAPK is a central mediator of the cAMP signaling mechanism of brown fat that promotes thermogenesis.
Assuntos
Tecido Adiposo Marrom/fisiologia , Proteínas de Transporte/genética , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transcrição Gênica , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Células Cultivadas , Temperatura Baixa , Elementos Facilitadores Genéticos , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Humanos , Canais Iônicos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais , Distribuição Aleatória , Transdução de Sinais/fisiologia , Termogênese/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Desacopladores , Proteína Desacopladora 1 , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The beta(3)-adrenergic receptor (beta(3)AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of beta(3)AR activation is the substantial elevation in interleukin-6 (IL-6) levels. Although the beta(3)AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific beta(3)AR agonist CL316243 in adipocytes, cells that exhibit the highest expression of beta(3)ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated via the G(s)-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-kappaB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block beta(3)AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the beta(3)AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the beta(3)AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways.
Assuntos
Adipócitos Brancos/metabolismo , Interleucina-6/biossíntese , Receptores Adrenérgicos beta 3/fisiologia , Fator 1 Ativador da Transcrição/metabolismo , Adipócitos Brancos/citologia , Agonistas de Receptores Adrenérgicos beta 3 , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dioxóis/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Regulação da Expressão Gênica , Humanos , Camundongos , NF-kappa B/fisiologia , Proteína Quinase C/fisiologia , Elementos de Resposta , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The adipocyte integrates crucial information about metabolic needs in order to balance energy intake, storage, and expenditure. Whereas white adipose tissue stores energy, brown adipose tissue is a major site of energy dissipation through adaptive thermogenesis mediated by uncoupling protein 1 (UCP1) in mammals. In both white and brown adipose tissue, nuclear receptors and their coregulators, such as peroxisome proliferator-activated receptor gamma (PPARgamma) and PPARgamma coactivator 1alpha (PGC-1alpha), play key roles in regulating their development and metabolic functions. Here we show the unexpected role of liver X receptor alpha (LXRalpha) as a direct transcriptional inhibitor of beta-adrenergic receptor-mediated, cyclic AMP-dependent Ucp1 gene expression through its binding to the critical enhancer region of the Ucp1 promoter. The mechanism of inhibition involves the differential recruitment of the corepressor RIP140 to an LXRalpha binding site that overlaps with the PPARgamma/PGC-1alpha response element, resulting in the dismissal of PPARgamma. The ability of LXRalpha to dampen energy expenditure in this way provides another mechanism for maintaining a balance between energy storage and utilization.
Assuntos
Adipócitos Marrons/metabolismo , Proteínas de Ligação a DNA/fisiologia , Canais Iônicos/fisiologia , Proteínas Mitocondriais/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Temperatura Corporal/genética , Temperatura Corporal/fisiologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Colforsina/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Canais Iônicos/biossíntese , Canais Iônicos/genética , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína 1 de Interação com Receptor Nuclear , Receptores Nucleares Órfãos , Consumo de Oxigênio , PPAR gama/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Proteína Desacopladora 1RESUMO
Catecholamine-stimulated lipolysis is primarily a beta-adrenergic and cAMP-dependent event. In previous studies we established that the beta(3)-adrenergic receptor (beta(3)AR) in adipocytes utilizes a unique mechanism to stimulate extracellular signal-regulated kinases 1 and 2 (ERK) by direct recruitment and activation of Src kinase. Therefore, we investigated the role of the ERK pathway in adipocyte metabolism and found that the beta(3)AR agonist CL316,243 regulates lipolysis through both cAMP-dependent protein kinase (PKA) and ERK. Inhibition of PKA activity completely eliminated lipolysis at low (subnanomolar) CL316,243 concentrations and by 75-80% at higher nanomolar concentrations. The remaining 20-25% of PKA-independent lipolysis, as well as ERK activation, was abolished by inhibiting the activity of either Src (PP2 or small interfering RNA), epidermal growth factor receptor (EGFR with AG1478 or small interfering RNA), or mitogen-activated protein kinase kinase 1 or 2 (MKK1/2 with PD098059). PD098059 inhibited lipolysis by 53% in mice as well. Finally, the effect of estradiol, a reported acute activator of ERK and lipolysis, was also totally prevented by PP2, AG1478, and PD098059. These results suggest that ERK activation by beta(3)AR depends upon Src and epidermal growth factor receptor kinase activities and is responsible for the PKA-independent portion of the lipolytic response. Together these results illustrate the distinct and complementary roles for PKA and ERK in catecholamine-stimulated lipolysis.
Assuntos
Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lipólise/fisiologia , Receptores Adrenérgicos beta 3/metabolismo , Quinases da Família src/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Agonistas de Receptores Adrenérgicos beta 3 , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dioxóis/farmacologia , Ativação Enzimática , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Estradiol/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Isoquinolinas/farmacologia , Lipólise/efeitos dos fármacos , Camundongos , Quinazolinas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Sulfonamidas/farmacologia , Tirfostinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
One of the phenotypes of mice with targeted disruption of the uncoupling protein-2 gene (Ucp2-/-) is greater macrophage phagocytic activity and free radical production, resulting in a striking resistance to infectious microorganisms. In this study, the molecular mechanisms of this enhanced immune response were investigated. We found that levels of nitric oxide measured in either plasma or isolated macrophages from Ucp2-/- mice are significantly elevated in response to bacterial lipopolysaccharide challenge compared with similarly treated Ucp2+/+ mice. Likewise, expression of inducible nitric-oxide synthase and inflammatory cytokines is higher in Ucp2-/- mice in vivo and in vitro. Key steps in the activation cascade of nuclear factor (NF)-kappa B, including I kappa B kinase and nuclear translocation of NF-kappa B subunits, are all remarkably enhanced in Ucp2-/- mice, most notably even under basal conditions. The elevated basal activity of I kappa B kinase in macrophages from Ucp2-/- mice can be blocked by cell-permeable inhibitors of superoxide and hydrogen peroxide generation, but not by a specific inhibitor for inducible nitric-oxide synthase. Isolated mitochondria from Ucp2-/- cells produced more superoxide/hydrogen peroxide. We conclude that mitochrondrially derived reactive oxygen from Ucp2-/- cells constitutively activates NF-kappa B, resulting in a "primed" state to both potentiate and amplify the inflammatory response upon subsequent stimulation.
Assuntos
Citocinas/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Algoritmos , Animais , Northern Blotting , Western Blotting , Ciclo-Oxigenase 2 , Citosol/metabolismo , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Radicais Livres , Genótipo , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Quinase I-kappa B , Inflamação , Canais Iônicos , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Modelos Biológicos , NADPH Oxidases/química , Nitratos/química , Nitratos/metabolismo , Óxido Nítrico/química , Óxido Nítrico Sintase/metabolismo , Nitritos/química , Nitritos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/metabolismo , Fenótipo , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteínas Serina-Treonina Quinases/química , Baço/metabolismo , Frações Subcelulares , Superóxidos/metabolismo , Fatores de Tempo , Proteína Desacopladora 2RESUMO
Current evidence suggests that uncoupling protein-2 (UCP2) is a regulator of insulin secretion. It is also known that chronic exposure of pancreatic islets to free fatty acids (FFAs) blunts glucose-stimulated insulin secretion and is accompanied by elevated levels of UCP2. However, the mechanisms regulating expression of UCP2 in beta-cells are unknown. Here, we show that UCP2 mRNA and protein levels were increased after a 48-h exposure of INS-1(832/13) beta-cells to oleic acid (0.5 mm) by activation of the UCP2 promoter. Furthermore, progressive deletions of the mouse UCP2 promoter (from -7.3 kb to +12 bp) indicated that an enhancer region (-86/-44) was responsible for both basal and FFA-stimulated UCP2 gene transcription. This enhancer contains tightly clustered Sp1, sterol regulatory element (SRE), and double E-Box elements. While all three sequence motifs were required for basal activity of the UCP2 promoter, the mutations in either the SRE or the E-Box elements eliminated the response to FFAs. The SRE and sterol regulatory element binding protein-1 (SREBP1) appear to be crucial for the response of the UCP2 gene to FFAs, since overexpression of the nuclear forms of the SREBPs increased UCP2 promoter activity by 7-10-fold and restored the ability of E-Box mutants to respond to oleic acid. These data support a model in which SREBP is the major modulator of UCP2 gene transcription by FFA, while E-Box binding factors play a supportive role.