RESUMO
11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been identified as the primary enzyme responsible for the activation of hepatic cortisone to cortisol in specific peripheral tissues resulting in the concomitant antagonism of insulin action within these tissues. Dysregulation of 11ß-HSD1, particularly in adipose tissues, has been associated with metabolic syndrome and type 2 diabetes mellitus. Therefore, inhibition of 11ß-HSD1 with a small nonsteroidal molecule is therapeutically desirable. Implementation of a scaffold-hopping approach revealed a three-point pharmacophore for 11ß-HSD1 that was utilized to design a steroid mimetic scaffold. Reiterative optimization provided valuable insight into the bioactive conformation of our novel scaffold and led to the discovery of INCB13739. Clinical evaluation of INCB13739 confirmed for the first time that tissue-specific inhibition of 11ß-HSD1 in patients with type 2 diabetes mellitus was efficacious in controlling glucose levels and reducing cardiovascular risk factors.
Assuntos
Diabetes Mellitus Tipo 2 , Síndrome Metabólica , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Hidrocortisona/metabolismo , Síndrome Metabólica/metabolismoRESUMO
The randomized, double-blind, double-dummy, phase 3b RELIEF trial evaluated polycythaemia vera (PV)-related symptoms in patients who were well controlled with a stable dose of hydroxycarbamide (also termed hydroxyurea) but reported PV-related symptoms. Patients were randomized 1:1 to ruxolitinib 10 mg BID (n = 54) or hydroxycarbamide (prerandomization dose/schedule; n = 56); crossover to ruxolitinib was permitted after Week 16. The primary endpoint, ≥50% improvement from baseline in myeloproliferative neoplasm -symptom assessment form total symptom score cytokine symptom cluster (TSS-C; sum of tiredness, itching, muscle aches, night sweats, and sweats while awake) at Week 16, was achieved by 43·4% vs. 29·6% of ruxolitinib- and hydroxycarbamide-treated patients, respectively (odds ratio, 1·82; 95% confidence interval, 0·82-4·04; P = 0·139). The primary endpoint was achieved by 34% of a subgroup who maintained their hydroxycarbamide dose from baseline to Weeks 13-16. In a post hoc analysis, the primary endpoint was achieved by more patients with stable screening-to-baseline TSS-C scores (ratio ≤ 2) receiving ruxolitinib than hydroxycarbamide (47·4% vs. 25·0%; P = 0·0346). Ruxolitinib treatment after unblinding was associated with continued symptom score improvements. Adverse events were primarily grades 1/2 with no unexpected safety signals. Ruxolitinib was associated with a nonsignificant trend towards improved PV-related symptoms versus hydroxycarbamide, although an unexpectedly large proportion of patients who maintained their hydroxycarbamide dose reported symptom improvement.
Assuntos
Substituição de Medicamentos , Hidroxiureia/uso terapêutico , Policitemia Vera/tratamento farmacológico , Pirazóis/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Cross-Over , Método Duplo-Cego , Fadiga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nitrilas , Pirimidinas , Qualidade de Vida , Resultado do Tratamento , Adulto JovemRESUMO
Combined Janus kinase 1 (JAK1) and JAK2 inhibition therapy effectively reduces splenomegaly and symptom burden related to myelofibrosis but is associated with dose-dependent anemia and thrombocytopenia. In this open-label phase II study, we evaluated the efficacy and safety of three dose levels of INCB039110, a potent and selective oral JAK1 inhibitor, in patients with intermediate- or high-risk myelofibrosis and a platelet count ≥50×109/L. Of 10, 45, and 32 patients enrolled in the 100 mg twice-daily, 200 mg twice-daily, and 600 mg once-daily cohorts, respectively, 50.0%, 64.4%, and 68.8% completed week 24. A ≥50% reduction in total symptom score was achieved by 35.7% and 28.6% of patients in the 200 mg twice-daily cohort and 32.3% and 35.5% in the 600 mg once-daily cohort at week 12 (primary end point) and 24, respectively. By contrast, two patients (20%) in the 100 mg twice-daily cohort had ≥50% total symptom score reduction at weeks 12 and 24. For the 200 mg twice-daily and 600 mg once-daily cohorts, the median spleen volume reductions at week 12 were 14.2% and 17.4%, respectively. Furthermore, 21/39 (53.8%) patients who required red blood cell transfusions during the 12 weeks preceding treatment initiation achieved a ≥50% reduction in the number of red blood cell units transfused during study weeks 1-24. Only one patient discontinued for grade 3 thrombocytopenia. Non-hematologic adverse events were largely grade 1 or 2; the most common was fatigue. Treatment with INCB039110 resulted in clinically meaningful symptom relief, modest spleen volume reduction, and limited myelosuppression.
Assuntos
Azetidinas/uso terapêutico , Ácidos Isonicotínicos/uso terapêutico , Janus Quinase 1/antagonistas & inibidores , Mielofibrose Primária/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Azetidinas/administração & dosagem , Azetidinas/efeitos adversos , Citocinas/metabolismo , Feminino , Frequência do Gene , Humanos , Ácidos Isonicotínicos/administração & dosagem , Ácidos Isonicotínicos/efeitos adversos , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
BACKGROUND: Treatment options for myelofibrosis are limited. We evaluated the efficacy and safety of ruxolitinib, a potent and selective Janus kinase (JAK) 1 and 2 inhibitor, as compared with the best available therapy, in patients with myelofibrosis. METHODS: We assigned 219 patients with intermediate-2 or high-risk primary myelofibrosis, post-polycythemia vera myelofibrosis, or post-essential thrombocythemia myelofibrosis to receive oral ruxolitinib or the best available therapy. The primary end point and key secondary end point of the study were the percentage of patients with at least a 35% reduction in spleen volume at week 48 and at week 24, respectively, as assessed with the use of magnetic resonance imaging or computed tomography. RESULTS: A total of 28% of the patients in the ruxolitinib group had at least a 35% reduction in spleen volume at week 48, as compared with 0% in the group receiving the best available therapy (P<0.001); the corresponding percentages at week 24 were 32% and 0% (P<0.001). At 48 weeks, the mean palpable spleen length had decreased by 56% with ruxolitinib but had increased by 4% with the best available therapy. The median duration of response with ruxolitinib was not reached, with 80% of patients still having a response at a median follow-up of 12 months. Patients in the ruxolitinib group had an improvement in overall quality-of-life measures and a reduction in symptoms associated with myelofibrosis. The most common hematologic abnormalities of grade 3 or higher in either group were thrombocytopenia and anemia, which were managed with a dose reduction, interruption of treatment, or transfusion. One patient in each group discontinued treatment owing to thrombocytopenia, and none discontinued owing to anemia. Nonhematologic adverse events were rare and mostly grade 1 or 2. Two cases of acute myeloid leukemia were reported with the best available therapy. CONCLUSIONS: Continuous ruxolitinib therapy, as compared with the best available therapy, was associated with marked and durable reductions in splenomegaly and disease-related symptoms, improvements in role functioning and quality of life, and modest toxic effects. An influence on overall survival has not yet been shown. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT00934544.).
Assuntos
Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Mielofibrose Primária/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Esplenomegalia/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Causas de Morte , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Nitrilas , Tamanho do Órgão , Mielofibrose Primária/mortalidade , Mielofibrose Primária/patologia , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/efeitos adversos , Pirimidinas , Qualidade de Vida , Baço/efeitos dos fármacos , Baço/patologia , Análise de SobrevidaRESUMO
Ruxolitinib is a potent Janus kinase (JAK)1/JAK2 inhibitor that has demonstrated rapid reductions in splenomegaly and marked improvement in disease-related symptoms and quality of life in patients with myelofibrosis (MF). The present analysis reports the 3-year follow-up (median, 151 weeks) of the efficacy and safety of Controlled Myelofibrosis Study With Oral Janus-associated Kinase (JAK) Inhibitor Treatment-II (the COMFORT-II Trial), comparing ruxolitinib with the best available therapy (BAT) in 219 patients with intermediate-2 and high-risk MF. In the ruxolitinib arm, with continued therapy, spleen volume reductions of ≥35% by magnetic resonance imaging (equivalent to approximately 50% reduction by palpation) were sustained for at least 144 weeks, with the probability of 50% (95% confidence interval [CI], 36-63) among patients achieving such degree of response. At the time of this analysis, 45% of the patients randomized to ruxolitinib remained on treatment. Ruxolitinib continues to be well tolerated. Anemia and thrombocytopenia were the main toxicities, but they were generally manageable, improved over time, and rarely led to treatment discontinuation (1% and 3.6% of patients, respectively). No single nonhematologic adverse event led to definitive ruxolitinib discontinuation in more than 1 patient. Additionally, patients randomized to ruxolitinib showed longer overall survival than those randomized to BAT (hazard ratio, 0.48; 95% CI, 0.28-0.85; log-rank test, P = .009).
Assuntos
Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/mortalidade , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Anemia/induzido quimicamente , Anemia/tratamento farmacológico , Anemia/enzimologia , Anemia/mortalidade , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Masculino , Nitrilas , Mielofibrose Primária/enzimologia , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/efeitos adversos , Pirimidinas , Taxa de Sobrevida , Trombocitopenia/induzido quimicamente , Trombocitopenia/tratamento farmacológico , Trombocitopenia/enzimologia , Trombocitopenia/mortalidade , Fatores de TempoRESUMO
Prior to Janus kinase inhibitors, available therapies for myelofibrosis were generally supportive and did not improve survival. This analysis compares efficacy outcomes of patients with myelofibrosis in the control arms (placebo [n=154] and best available therapy [n=73]) from the two phase 3 COntrolled MyeloFibrosis study with ORal JAK inhibitor Treatment (COMFORT) studies. Spleen volume was assessed by magnetic resonance imaging/computed tomography at baseline and every 12 weeks through week 72; spleen length was assessed by palpation at each study visit. Health-related quality of life and symptoms were assessed using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 Items at baseline and in weeks 4, 8, 12, 16 and 24 in COMFORT-I and in weeks 8, 16, 24 and 48 in COMFORT-II. The demographic and baseline characteristics were similar between the control arms of the two studies. One patient who received placebo and no patients who received best available therapy had a ≥35% reduction in spleen volume from baseline at week 24. At 24 weeks, neither placebo nor best available therapy had produced clinically meaningful changes in global quality of life or symptom scales. Non-hematologic adverse events were mostly grade 1/2; the most frequently reported adverse events in each group were abdominal pain, fatigue, peripheral edema and diarrhea. These data suggest that non-Janus kinase inhibitor therapies provide little improvement in splenomegaly, symptoms or quality of life as compared with placebo. Both COMFORT-I (NCT00952289) and COMFORT-II (NCT00934544) studies have been appropriately registered with clinicaltrials.gov.
Assuntos
Inibidores de Proteínas Quinases/administração & dosagem , Qualidade de Vida , Baço , Adulto , Idoso , Idoso de 80 Anos ou mais , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/patologia , Mielofibrose Primária/fisiopatologia , Baço/patologia , Baço/fisiopatologia , Esplenomegalia/tratamento farmacológico , Esplenomegalia/patologia , Esplenomegalia/fisiopatologia , Fatores de TempoRESUMO
Combustion of mixed materials during open air burning of refuse or structural fires in the wildland urban interface produces emissions that worsen air quality, contaminate rivers and streams, and cause poor health outcomes including developmental effects. The zebrafish, a freshwater fish, is a useful model for quickly screening the toxicological and developmental effects of agents in such species and elicits biological responses that are often analogous and predictive of responses in mammals. The purpose of this study was to compare the developmental toxicity of smoke derived from the burning of 5 different burn pit-related material types (plywood, cardboard, plastic, a mixture of the three, and the mixture plus diesel fuel as an accelerant) in zebrafish larvae. Larvae were exposed to organic extracts of increasing concentrations of each smoke 6-to-8-hr post fertilization and assessed for morphological and behavioral toxicity at 5 days post fertilization. To examine chemical and biological determinants of toxicity, responses were related to emissions concentrations of polycyclic hydrocarbons (PAH). Emissions from plastic and the mixture containing plastic caused the most pronounced developmental effects, including mortality, impaired swim bladder inflation, pericardial edema, spinal curvature, tail kinks, and/or craniofacial deformities, although all extracts caused concentration-dependent effects. Plywood, by contrast, altered locomotor responsiveness to light changes to the greatest extent. Some morphological and behavioral responses correlated strongly with smoke extract levels of PAHs including 9-fluorenone. Overall, the findings suggest that material type and emissions chemistry impact the severity of zebrafish developmental toxicity responses to burn pit-related smoke.
Assuntos
Antineoplásicos/uso terapêutico , Policitemia Vera/tratamento farmacológico , Pirazóis/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Resistencia a Medicamentos Antineoplásicos , Humanos , Hidroxiureia/uso terapêutico , Pessoa de Meia-Idade , Nitrilas , Pirimidinas , Resultado do TratamentoRESUMO
Patients with myelofibrosis (MF) have significant debilitating symptoms, physical disabilities, and poor health-related quality of life (HRQoL). Here, we report post-hoc analyses of the impact of ruxolitinib, a potent and selective JAK1 and JAK2 inhibitor, on disease-related symptoms and HRQoL in MF patients from the large phase 3 COMFORT-II study (N = 219). During the follow-up period of 48 weeks, HRQoL and MF-associated symptoms improved from baseline for ruxolitinib-treated patients but remained the same or worsened for best available therapy (BAT)-treated patients. Based on the European Organization for Research and Treatment of Cancer QoL Questionnaire core 30 items (EORTC QLQ-C30), treatment-induced differences in physical and role functioning, fatigue, and appetite loss significantly favoured ruxolitinib versus BAT from week 8 (P < 0·05) up to week 48 (P < 0·05). Ruxolitinib resulted in significantly higher response rates in global health status/QoL and Functional Assessment of Cancer Therapy-Lymphoma (FACT-Lym) summary scores versus BAT at most time points (P < 0·05). Significant improvements in the Lymphoma subscale (including symptoms of pain, fever, itching, fatigue, weight loss, loss of appetite, and other patient concerns), FACT-General, FACT-Lym trial outcome index, and FACT-Lym total were also observed with ruxolitinib versus BAT starting at week 8 and continuing thereafter. Overall, these data demonstrated that ruxolitinib improved HRQoL in MF patients and further support the use of ruxolitinib for the treatment of symptomatic MF.
Assuntos
Mielofibrose Primária/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Idoso , Feminino , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Masculino , Nitrilas , Mielofibrose Primária/enzimologia , Mielofibrose Primária/patologia , Pirimidinas , Qualidade de Vida , Inquéritos e Questionários , Resultado do TratamentoRESUMO
New approaches in developmental neurotoxicity (DNT) screening are needed due to the tens of thousands of chemicals requiring hazard assessments. Zebrafish (Danio rerio) are an alternative vertebrate model for DNT testing, but without a standardized protocol for larval behavioral assays, comparison of results among laboratories is challenging. To evaluate the congruence of protocols across laboratories, we conducted a literature review of DNT studies focusing on larval zebrafish behavior assays and cataloged experimental design consistencies. Our review focused on 51 unique method variables in publications where chemical exposure occurred in early development and subsequent larval locomotor evaluation focused on assays that included a light/dark photoperiod transition. We initially identified 94 publications, but only 31 exclusively met our inclusion criteria which focused on parameters that are important to an assay employed by our laboratory. No publication reported 100% of the targeted variables; only 51 to 86% of those variables were reported in the reviewed publications, with some aspects of the experimental design consistent among laboratories. However, no protocol was exactly the same for any two publications. Many of these variables had more than one parameter/design reported, highlighting the inconsistencies among methods. Overall, there is not only a strong need for the development of a standardized testing protocol for larval zebrafish locomotor assays, but there is also a need for a standardized protocol for reporting experimental variables in the literature. Here we include an extensive guideline checklist for conducting larval zebrafish developmental behavior assays.
Assuntos
Atividade Motora , Peixe-Zebra , Animais , Larva , Comportamento Animal , Projetos de PesquisaRESUMO
The use of larval zebrafish developmental testing and assessment, specifically larval zebrafish locomotor activity, has been recognized as a higher throughput testing strategy to identify developmentally toxic and neurotoxic chemicals. There are, however, no standardized protocols for this type of assay, which could result in confounding variables being overlooked. Two chemicals commonly employed during early-life stage zebrafish assays, methylene blue (antifungal agent) and dimethyl sulfoxide (DMSO, a commonly used vehicle) have been reported to affect the morphology and behavior of freshwater fish. In this study, we conducted developmental toxicity (morphology) and neurotoxicity (behavior) assessments of commonly employed concentrations for both chemicals (0.6-10.0 µM methylene blue; 0.3%-1.0% v/v DMSO). A light-dark transition behavioral testing paradigm was applied to morphologically normal, 6 days postfertilization (dpf) zebrafish larvae kept at 26°C. Additionally, an acute DMSO challenge was administered based on early-life stage zebrafish assays typically used in this research area. Results from developmental toxicity screens were similar between both chemicals with no morphological abnormalities detected at any of the concentrations tested. However, neurodevelopmental results were mixed between the two chemicals of interest. Methylene blue resulted in no behavioral changes up to the highest concentration tested, 10.0 µM. By contrast, DMSO altered larval behavior following developmental exposure at concentrations as low as 0.5% (v/v) and exhibited differential concentration-response patterns in the light and dark photoperiods. These results indicate that developmental DMSO exposure can affect larval zebrafish locomotor activity at routinely used concentrations in developmental neurotoxicity assessments, whereas methylene blue does not appear to be developmentally or neurodevelopmentally toxic to larval zebrafish at routinely used concentrations. These results also highlight the importance of understanding the influence of experimental conditions on larval zebrafish locomotor activity that may ultimately confound the interpretation of results.
Assuntos
Dimetil Sulfóxido , Peixe-Zebra , Animais , Peixe-Zebra/fisiologia , Dimetil Sulfóxido/toxicidade , Azul de Metileno/toxicidade , Comportamento Animal , Locomoção , LarvaRESUMO
With the abundance of chemicals in the environment that could potentially cause neurodevelopmental deficits, there is a need for rapid testing and chemical screening assays. This study evaluated the developmental toxicity and behavioral effects of 61 chemicals in zebrafish (Danio rerio) larvae using a behavioral Light/Dark assay. Larvae (n = 16-24 per concentration) were exposed to each chemical (0.0001-120 µM) during development and locomotor activity was assessed. Approximately half of the chemicals (n = 30) did not show any gross developmental toxicity (i.e., mortality, dysmorphology or non-hatching) at the highest concentration tested. Twelve of the 31 chemicals that did elicit developmental toxicity were toxic at the highest concentration only, and thirteen chemicals were developmentally toxic at concentrations of 10 µM or lower. Eleven chemicals caused behavioral effects; four chemicals (6-aminonicotinamide, cyclophosphamide, paraquat, phenobarbital) altered behavior in the absence of developmental toxicity. In addition to screening a library of chemicals for developmental neurotoxicity, we also compared our findings with previously published results for those chemicals. Our comparison revealed a general lack of standardized reporting of experimental details, and it also helped identify some chemicals that appear to be consistent positives and negatives across multiple laboratories.
RESUMO
The C-C motif chemokine receptor-2 (CCR2) regulates monocyte and macrophage recruitment and is necessary for macrophage-dependent inflammatory responses and the development of atherosclerosis. Although adipose tissue expression and circulating concentrations of CCL2 (also known as MCP1), a high-affinity ligand for CCR2, are elevated in obesity, the role of CCR2 in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity, has not been studied. To determine what role CCR2 plays in the development of metabolic phenotypes, we studied the effects of Ccr2 genotype on the development of obesity and its associated phenotypes. Genetic deficiency in Ccr2 reduced food intake and attenuated the development of obesity in mice fed a high-fat diet. In obese mice matched for adiposity, Ccr2 deficiency reduced macrophage content and the inflammatory profile of adipose tissue, increased adiponectin expression, ameliorated hepatic steatosis, and improved systemic glucose homeostasis and insulin sensitivity. In mice with established obesity, short-term treatment with a pharmacological antagonist of CCR2 lowered macrophage content of adipose tissue and improved insulin sensitivity without significantly altering body mass or improving hepatic steatosis. These data suggest that CCR2 influences the development of obesity and associated adipose tissue inflammation and systemic insulin resistance and plays a role in the maintenance of adipose tissue macrophages and insulin resistance once obesity and its metabolic consequences are established.
Assuntos
Gorduras na Dieta , Resistência à Insulina , Obesidade/fisiopatologia , Receptores de Quimiocinas/fisiologia , Tecido Adiposo/fisiopatologia , Animais , Quimiocina CCL2/fisiologia , Cruzamentos Genéticos , Feminino , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR2 , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genéticaRESUMO
Exposure to fine particulate matter (PM) air pollution causes adverse cardiopulmonary outcomes. Yet, the limited capacity to readily identify contributing PM sources and associated PM constituents in any given ambient air shed impedes risk assessment efforts. The health effects of PM have been attributed in part to its capacity to elicit irritant responses. A variety of chemicals trigger irritant behavior responses in zebrafish that can be easily measured. The purposes of this study were to examine the utility of zebrafish locomotor responses in the toxicity assessment of fine PM and its chemical fractions and uncover mechanisms of action. Locomotor responses were recorded in 6-day-old zebrafish exposed for 60 min in the dark at 26 °C to the extractable organic matter of a compressor-generated diesel exhaust PM (C-DEP) and 4 of its fractions (F1-F4) containing varying chemical classes of increasing polarity. The role of the transient receptor potential (TRP) cation channel TRPA1, a chemical sensor in mammals and zebrafish, in locomotor responses to C-DEP, was also examined. Acrolein, an environmental irritant and known activator of TRPA1, and all extracts induced concentration-dependent locomotor responses whose potencies ranked as follows: polar F3 > weakly polar F2 > C-DEP > highly polar F4 > nonpolar F1, indicating that polar and weakly polar fractions that included nitro- and oxy-polyaromatic hydrocarbons (PAHs), drove C-DEP responses. Irritant potencies in fish positively correlated with mutagenic potencies of the same extracts in strains of Salmonella sensitive to nitro- and oxy-PAHs, further implicating these chemical classes in the zebrafish responses to C-DEP. Pharmacologic inhibition of TRPA1 blocked locomotor responses to acrolein and the extracts. Taken together, these data indicate that the zebrafish locomotor assay may help expedite toxicity screening of fine PM sources, identify causal chemical classes, and uncover plausible biological mechanisms.
Assuntos
Poluentes Atmosféricos/toxicidade , Comportamento Animal/efeitos dos fármacos , Humor Irritável/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Material Particulado/toxicidade , Canal de Cátion TRPA1/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Relação Dose-Resposta a Droga , Testes de Mutagenicidade , Salmonella/efeitos dos fármacos , Salmonella/genética , Canal de Cátion TRPA1/antagonistas & inibidores , Emissões de Veículos/toxicidade , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
Unregulated proliferation of mesenchymal cells in leiomyomas, lipomas, hamartomas,and other diseases has been linked to the high mobility group (HMGA) family of DNA architectural proteins. HMGA genes are primarily expressed during embryonal development and silenced in adult tissues but can become reactivated in neoplasia as a result of chromosomal rearrangements. Although the genetic data suggesting a role for HMGA proteins in tumorigenesis are compelling, the biological role of these proteins in mesenchymal proliferation and differentiation is incompletely defined. Uterine myometria and spontaneous leiomyomas from the Eker rat, which carries a germ-line mutation in the tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene, were analyzed for genetic defects in and expression of the Tsc2 and HMGA proteins. Eker leiomyomas exhibited a 50% incidence of loss of the wild-type Tsc2 allele and an almost uniform loss of protein expression, implicating loss of function of the Tsc2 gene in these tumors. Concomitantly, HMGA2 protein, which was completely absent in normal myometria, was expressed in 16 of 19 Eker leiomyomas. HMGA1 was expressed in both leiomyoma and normal myometria. No structural alterations were observed at the HMGA2 locus in either primary rat leiomyomas or leiomyoma-derived cell lines that expressed HMGA2. These data support a role for HMGA2 in the development of smooth muscle neoplasms and suggest HMGA2 expression is a point of convergence between the human disease and the Eker rat model. Furthermore, these data indicate that aberrant HMGA2 expression can result from dysfunction of the Tsc2 tumor suppressor gene, in the absence of structural alterations involving the HMGA2 locus.
Assuntos
Genes Supressores de Tumor/fisiologia , Proteína HMGA2/biossíntese , Leiomioma/genética , Proteínas Repressoras/genética , Neoplasias Uterinas/genética , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Proteína HMGA2/genética , Leiomioma/metabolismo , Perda de Heterozigosidade , Ratos , Proteínas Repressoras/biossíntese , Translocação Genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor , Regulação para Cima , Neoplasias Uterinas/metabolismoRESUMO
Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome characterized by seizures, mental retardation, autism, and tumors of the brain, kidney, heart, retina, and skin. TSC is caused by mutations in either TSC1 or TSC2, both of which are tumor suppressor genes. Hamartin, the protein product of TSC1, was found to interact with the ezrin-radixin-moesin family of cytoskeletal proteins and to activate the small GTPase Rho. To determine whether tuberin, the TSC2 product, can also activate Rho, we stably expressed full-length human tuberin in two cell types: MDCK cells and ELT3 cells. ELT3 cells lack endogenous tuberin expression. We found that expression of human tuberin in both MDCK and ELT3 cells was associated with an increase in the amount of Rho-GTP, but not in Rac1-GTP or cdc42-GTP. Tuberin expression increased cell adhesion in both cell types, and decreased chemotactic cell migration in ELT3 cells. In MDCK cells, there was a decrease in the amount of total Focal Adhesion Kinase (FAK) and an increase in the fraction of phosphorylated FAK. These findings demonstrate for the first time that tuberin activates Rho and regulates cell adhesion and migration. Pathways involving Rho activation may have relevance to the clinical manifestations of TSC, including pulmonary lymphangioleiomyomatosis.
Assuntos
Adesão Celular/genética , Genes Supressores de Tumor , Proteínas Repressoras/genética , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Movimento Celular/genética , Cães , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas/genética , Ratos , Transfecção , Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de TumorRESUMO
Cognitive and motor impairment often follow acute poisoning with an organophosphorous (OP) pesticide. However, the persistence of these effects and the conditions necessary for their appearance are not clear: two specific concerns are whether symptomatic poisoning is necessary for persistent effects, and whether inhibition of cholinesterase (ChE) activity is a protective metric of OP exposure. This study examined the effects of chronic dietary and repeated high-level acute exposure to the pesticide chlorpyrifos (diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate, CPF) on learning and attention. Beginning at 3 months of age, male Long-Evans rats received dietary CPF at a daily dose of 0, 1, or 5 mg/kg for 1 year. Half of each dietary group also received an acute oral dose of CPF (initial dose at 60 mg/kg, 5 doses at 45 mg/kg) every 2 months. Beginning 2 weeks before the fourth acute dose, behavioral assessments were conducted on the eight rats in each of the six exposure groups (0-Oil, 0-CPF, 1-Oil, 1-CPF, 5-Oil, and 5-CPF). Using an auto-shaping procedure, the groups learned to press a lever for food in the following order: 5-Oil, 5-CPF, 1-Oil, and 0-Oil. The 0-CPF and 1-CPF groups did not learn the response in three 50-trial sessions. Chronic CPF did not affect acquisition of other behaviors required by a signal detection task (SDT) designed to assess sustained attention. The sixth acute CPF dose significantly disrupted the SDT in all dosed groups. Two months after the end of dosing, performance of the SDT was impaired in the 5-CPF group. These data suggest that learning the contingency between an action and reward may be accelerated by chronic exposure to CPF and inhibited by previous symptomatic exposure to CPF, and that persistent cognitive impairment may follow if CPF exposure inhibits brain ChE activity and is accompanied by acute doses sufficient to induce signs of toxicity.
Assuntos
Atenção/efeitos dos fármacos , Clorpirifos/toxicidade , Inibidores da Colinesterase/toxicidade , Inseticidas/toxicidade , Aprendizagem/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Clorpirifos/administração & dosagem , Inibidores da Colinesterase/administração & dosagem , Condicionamento Operante/efeitos dos fármacos , Dieta , Discriminação Psicológica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inseticidas/administração & dosagem , Masculino , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Ratos Long-Evans , Detecção de Sinal Psicológico/efeitos dos fármacos , Percepção Visual/efeitos dos fármacosRESUMO
Very little is known about the effects of chronic exposure to relatively low levels of anticholinesterase insecticides or how the effects of chronic exposure compare to those of higher, intermittent exposure. To that end, adult male rats were fed an anticholinesterase insecticide, chlorpyrifos (CPF), for 1 year at three levels of dietary exposure: 0, 1, or 5 mg/kg/day (0+oil, 1+oil, and 5+oil). In addition, half of each of these groups also received a bolus dosage of CPF in corn oil ("spiked" animals; 60 mg/kg initially and 45 mg/kg thereafter) every 2 months (0+CPF, 1+CPF, 5+CPF). Animals were analyzed after 6 or 12 months of dosing, and again 3 months after cessation of dosing (i.e., "recovery" animals-six experimental groups with n = 4-6/group/time point). Cholinesterase (ChE) activity was measured in retina, whole blood, plasma, red blood cells, diaphragm, and brain [pons, striatum, and the rest of the brain (referred to simply as "brain")]. Muscarinic receptor density was assessed in retina, pons, and brain, whereas dopamine transporter density and the levels of dopamine and its metabolites were assessed in striatum. Cholinesterase activity at 6 and 12 months was not different in any of the tissues, indicating that a steady state had been reached prior to 6 months. The 1+oil group animals showed ChE inhibition only in the blood, whereas the 5+oil group exhibited > or = 50% ChE inhibition in all tissues tested. One day after the bolus dose, all three groups (0+CPF, 1+CPF, 5+CPF) showed > or = 70% ChE inhibition in all tissues. Muscarinic receptor density decreased only in the brain of the 5+oil and 5+CPF groups, whereas dopamine transporter density increased only at 6 months in all three spiked groups. Striatal dopamine or dopamine metabolite levels did not change at any time. Three months after CPF dosing ended, all end points had returned to control levels. These data indicate that, although chronic feeding with or without intermittent spiked dosages with CPF produces substantial biochemical changes in a dose- and tissue-related manner, there are no persistent biochemical changes.
Assuntos
Clorpirifos/toxicidade , Inibidores da Colinesterase/toxicidade , Colinesterases/metabolismo , Inseticidas/toxicidade , Doenças do Sistema Nervoso/induzido quimicamente , Doença Aguda , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Clorpirifos/administração & dosagem , Inibidores da Colinesterase/administração & dosagem , Colinesterases/sangue , Doença Crônica , Dieta , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Inibidores da Captação de Dopamina/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Inseticidas/administração & dosagem , Masculino , Mazindol/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso/metabolismo , Ratos , Ratos Long-Evans , Receptores Muscarínicos/metabolismoRESUMO
Paclitaxel (Taxol) activates a number of signal transduction pathways that lead to apoptosis. In contrast, paclitaxel also activates cell survival pathways, such as the Raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway. Previously, we have shown that inhibition of MEK combined with paclitaxel treatment causes an impressive enhancement of apoptosis in various tumor cell lines. Here, we find that the combination of paclitaxel with a MEK inhibitor leads to a dramatic inactivation of the antiapoptotic Akt (protein kinase B) kinase. The decrease in Akt is not reflected at the protein or mRNA level but rather attributed to kinase inactivation. To confirm that inactivation of Akt is significant, a constitutively active Akt mutant was introduced and shown to reverse tumor cell apoptosis. Further analysis upstream of Akt shows that treatment with the combination of paclitaxel and MEK inhibitor down-regulates PI3K activity more than either agent alone. The direct pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) similarly enhances paclitaxel-induced tumor apoptosis in a dose-dependent manner. Our results suggest the combination of paclitaxel and MEK inhibitor leads to down-regulation of the PI3K-Akt signaling in addition to the proapoptotic effects of paclitaxel and MEK inhibitor alone. Overall, these findings render the combined use of paclitaxel with MEK inhibitors, or paclitaxel with PI3K inhibitors, as a promising new strategy for cancer chemotherapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Paclitaxel/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Células Tumorais Cultivadas/patologia , Northern Blotting , Butadienos/farmacologia , Cromonas/farmacologia , Quimioterapia Combinada , Fator de Crescimento Epidérmico/farmacologia , Humanos , Immunoblotting , Morfolinas/farmacologia , Neuregulina-1/farmacologia , Nitrilas/farmacologia , Fosforilação , Plasmídeos , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
As polybrominated diphenyl ethers are phased out, numerous compounds are emerging as potential replacement flame retardants for use in consumer and electronic products. Little is known, however, about the neurobehavioral toxicity of these replacements. This study evaluated the neurobehavioral effects of acute or developmental exposure to t-butylphenyl diphenyl phosphate (BPDP), 2-ethylhexyl diphenyl phosphate (EHDP), isodecyl diphenyl phosphate (IDDP), isopropylated phenyl phosphate (IPP), tricresyl phosphate (TMPP; also abbreviated TCP), triphenyl phosphate (TPHP; also abbreviated TPP), tetrabromobisphenol A (TBBPA), tris (2-chloroethyl) phosphate (TCEP), tris (1,3-dichloroisopropyl) phosphate (TDCIPP; also abbreviated TDCPP), tri-o-cresyl phosphate (TOCP), and 2,2-,4,4'-tetrabromodiphenyl ether (BDE-47) in zebrafish (Danio rerio) larvae. Larvae (n≈24 per dose per compound) were exposed to test compounds (0.4-120 µM) at sub-teratogenic concentrations either developmentally or acutely, and locomotor activity was assessed at 6 days post fertilization. When given developmentally, all chemicals except BPDP, IDDP and TBBPA produced behavioral effects. When given acutely, all chemicals produced behavioral effects, with TPHP, TBBPA, EHDP, IPP, and BPDP eliciting the most effects at the most concentrations. The results indicate that these replacement flame retardants may have developmental or pharmacological effects on the vertebrate nervous system.