RESUMEN
The human testis is sensitive to toxicant-induced injury but current methods for detecting adverse effects are limited, insensitive and unreliable. Animal studies use sensitive histopathological endpoints to assess toxicity, but require testicular tissue that is not available during human clinical trials. More sensitive and reliable molecular biomarkers of testicular injury are needed to better monitor testicular toxicity in both clinical and preclinical. Adult male Wistar Han rats were exposed for 4weeks to compounds previously associated with testicular injury, including cisplatin (0, 0.2, 0.3, or 0.4mg/kg/day), BI665915 (0, 20, 70, 100mg/kg/d), BI665636 (0, 20, 100mg/kg/d) or BI163538 (0, 70, 150, 300mg/kg/d) to evaluate reproductive toxicity and assess changes in sperm mRNA levels. None of the compounds resulted in any significant changes in body, testis or epididymis weights, nor were there decreases in testicular homogenization resistant spermatid head counts. Histopathological evaluation found that only BI665915 treatment caused any testicular effects, including minor germ cell loss and disorganization of the seminiferous tubule epithelium, and an increase in the number of retained spermatid heads. A custom PCR-array panel was used to assess induced changes in sperm mRNA. BI665915 treatment resulted in a significant increase in clusterin (Clu) levels and decreases in GTPase, IMAP family member 4 (Gimap4), prostaglandin D2 synthase (Ptgds) and transmembrane protein with EGF like and two follistatin like domains 1 (Tmeff1) levels. Correlation analysis between transcript levels and quantitative histopathological endpoints found a modest association between Clu with retained spermatid heads. These results demonstrate that sperm mRNA levels are sensitive molecular indicators of testicular injury that can potentially be translated into a clinical setting.
Asunto(s)
Acetamidas/toxicidad , Cisplatino/toxicidad , Oxadiazoles/toxicidad , ARN Mensajero/biosíntesis , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Testículo/efectos de los fármacos , Testículo/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Masculino , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/fisiología , Ratas , Ratas Wistar , Espermatogénesis/efectos de los fármacos , Espermatogénesis/fisiología , Espermatozoides/patología , Testículo/patologíaRESUMEN
Empagliflozin, a selective inhibitor of the renal tubular sodium-glucose cotransporter 2, was developed for treatment of type 2 diabetes mellitus. Nonclinical safety of empagliflozin was studied in a battery of tests to support global market authorization. Safety pharmacology studies indicated no effect of empagliflozin on measures of respiratory or central nervous system function in rats or cardiovascular safety in telemeterized dogs. In CD-1 mouse, Wistar Han rat, or beagle dogs up to 13, 26, or 52 weeks of treatment, respectively, empagliflozin exhibited a toxicity profile consistent with secondary supratherapeutic pharmacology related to glucose loss and included decreased body weight and body fat, increased food consumption, diarrhea, dehydration, decreased serum glucose and increases in other serum parameters reflective of increased protein catabolism, gluconeogenesis, and electrolyte imbalances, and urinary changes such as polyuria and glucosuria. Microscopic changes were consistently observed in kidney and included tubular nephropathy and interstitial nephritis (dog), renal mineralization (rat) and tubular epithelial cell karyomegaly, single cell necrosis, cystic hyperplasia, and hypertrophy (mouse). Empagliflozin was not genotoxic. Empagliflozin was not carcinogenic in female mice or female rats. Renal adenoma and carcinoma were induced in male mice only at exposures 45 times the maximum clinical dose. These tumors were associated with a spectrum of nonneoplastic changes suggestive of a nongenotoxic, cytotoxic, and cellular proliferation-driven mechanism. In male rats, testicular interstitial cell tumors and hemangiomas of the mesenteric lymph node were observed; both tumors are common in rats and are unlikely to be relevant to humans. These studies demonstrate the nonclinical safety of empagliflozin.
Asunto(s)
Compuestos de Bencidrilo/toxicidad , Glucósidos/toxicidad , Hipoglucemiantes/toxicidad , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Perros , Evaluación Preclínica de Medicamentos , Femenino , Riñón/efectos de los fármacos , Riñón/patología , Neoplasias Renales/inducido químicamente , Neoplasias Renales/patología , Masculino , Ratones , Pruebas de Mutagenicidad , Ratas Wistar , Pruebas de Toxicidad Crónica , Pruebas de Toxicidad SubcrónicaRESUMEN
Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.
Asunto(s)
Proteínas Sanguíneas/química , Transformación Celular Neoplásica/genética , Mutación/genética , Neoplasias/genética , Fosfoproteínas/química , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/genética , Homología de Secuencia de Aminoácido , Animales , Neoplasias de la Mama/genética , Neoplasias Colorrectales/genética , Análisis Mutacional de ADN , Activación Enzimática/genética , Femenino , Humanos , Leucemia/genética , Ratones , Modelos Moleculares , Neoplasias/patología , Neoplasias Ováricas/genética , Estructura Terciaria de Proteína/genética , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
BACKGROUND: Health care associated Clostridium difficile infections are a major cause of morbidity and mortality in hospitals. In the United States, from 2000 through 2009, discharge diagnoses from hospitals in the United States that included C. difficile increased from 139,000 to 336,600, and the yearly national excess hospital cost associated with hospital-onset C. difficile is estimated to be upwards of $1.3 billion. METHODS: A hospitalwide, multidisciplinary approach was undertaken at Rhode Island Hospital (Providence), a tertiary care hospital. The hospital plan entailed six interventions: (1) develop a C. difficile hospital infection control plan based on a risk assessment; (2) monitor hospitalwide morbidity and mortality associated with C. difficile infection; (3) improve sensitivity of C. difficile toxin detection in stool specimens using a polymerase chain reaction-based nucleic acid amplification assay; (4) enhance environmental cleaning of patient rooms and equipment; (5) develop a C. difficile infection treatment plan; and (6) conduct other interventions. The incidence of health care-associated C. difficile infection was assessed from January 2006 through the third quarter of 2012; the number of colectomies and mortality associated with C. difficile infection were determined from January 2005 through the third quarter of 2012. RESULTS: The incidence of health care-associated C. difficile infection decreased from a peak of 12.2/1,000 discharges during the second quarter of 2006 to 3.6/1,000 discharges during the third quarter of 2012. The yearly mortality in patients with health care-associated C. difficile infection was reduced from a peak of 52 in 2006 to 19 in 2011, with 13 such cases in the first three quarters of 2012. CONCLUSIONS: A hospitalwide multidisciplinary approach can reduce health care-associated C. difficile infection morbidity and mortality.
Asunto(s)
Clostridioides difficile , Infecciones por Clostridium/prevención & control , Colectomía/estadística & datos numéricos , Infección Hospitalaria/prevención & control , Mortalidad Hospitalaria , Control de Infecciones/organización & administración , Antibacterianos/uso terapéutico , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/mortalidad , Infecciones por Clostridium/transmisión , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/mortalidad , Infección Hospitalaria/transmisión , Heces/microbiología , Servicio de Limpieza en Hospital/organización & administración , Humanos , Incidencia , Reacción en Cadena de la Polimerasa , Calidad de la Atención de Salud , Rhode Island , Trastornos Relacionados con Sustancias , Centros de Atención TerciariaRESUMEN
In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G(2)/M transition. We find that activation of Aurora A first occurs at centrosomes at late G(2) and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Aurora Quinasa A , Aurora Quinasa B , Aurora Quinasas , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , División Celular , Fase G2 , Células HeLa , Histonas/química , Humanos , Ratones , Mitosis , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Treonina/química , Venas Umbilicales/citología , Quinasa Tipo Polo 1RESUMEN
Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Neoplasias/terapia , Biosíntesis de Proteínas/genética , Animales , Apoptosis , Secuencia de Bases , Células Cultivadas , Células Endoteliales/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Humanos , Ratones , Neoplasias/irrigación sanguínea , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Fusion of the MOZ and TIF2 genes by an inv (8) (p11q13) translocation has been identified in patients with acute mixed-lineage leukemia. Characterization of the molecular structure of the MOZ-TIF2 fusion protein suggested that the fusion protein would effect on nuclear receptor signaling. RESULTS: A series of deletions from the N-terminus of the MOZ-TIF2 fusion protein demonstrated that the MOZ portion is essential for nuclear localization of the fusion protein. Transient expression of MOZ-TIF2 dramatically decreased both basal and estradiol inducible reporter gene activity in an estrogen receptor element (ERE) driven luciferase reporter system and decreased androgen-inducible reporter gene activity in an androgen receptor element (ARE) luciferase reporter system. Deletions in the MOZ portion of the MOZ-TIF2 fusion protein reduced the suppression in the ER reporter system. Stable expression of MOZ-TIF2 inhibited retinoic acid (RA) inducible endogenous CD11b and C/EBPbeta gene response. The suppression of the reporter systems was released with either a CID domain deletion or with mutations of leucine-rich repeats in the TIF2 portion of MOZ-TIF2. The co-expression of TIF2, but not CBP, with MOZ-TIF2 partially restored the inhibition of the reporter systems. In addition, analysis of protein interactions demonstrated MOZ-TIF2 interaction with the C-terminus of CBP through both the MOZ and TIF2 portions of the fusion protein. CONCLUSION: MOZ-TIF2 inhibited nuclear receptor-mediated gene response by aberrant recruitment of CBP and both the MOZ and TIF2 portions are required for this inhibition.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Coactivador 2 del Receptor Nuclear/metabolismo , Proteínas de Fusión Oncogénica/genética , Receptores Citoplasmáticos y Nucleares/genética , Transcripción Genética , Enfermedad Aguda , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Corticosterona , Genes Reporteros , Histona Acetiltransferasas/genética , Humanos , Leucemia Mieloide/genética , Coactivador 2 del Receptor Nuclear/genética , Proteínas de Fusión Oncogénica/química , Receptores Androgénicos/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/genéticaRESUMEN
High-content imaging (HCI) provides researchers with a powerful tool for understanding cellular processes. Although phenotypic analysis generated through HCI is a potent technique to determine the overall cellular effects of a given treatment, it frequently produces complex data sets requiring extensive interpretation. The authors developed statistical analyses to decrease the time spent to determine the outcome of each HCI assay and to better understand complex phenotypic changes. To test these tools, the authors performed a comparison experiment between 2 types of oligonucleotide-mediated gene silencing (OMGS), antisense oligonucleotides (ASOs), and short, double-stranded RNAs (siRNAs). Although similar in chemical structure, these 2 methods differ in cellular mechanism of action and off-target effects. Using a library of 50 validated ASOs and siRNAs to the same targets, the authors characterized the differential effects of these 2 technologies using a HeLa cell G2-M cell cycle assay. Although knockdown of a variety of targets by ASOs or siRNAs affected the cell cycle profile, few of those targets were affected by both ASOs and siRNAs. Distribution analysis of population changes induced through target knockdown led to the identification of targets that, when inhibited, could affect the G2-M transition in the cell cycle in a statistically significant manner. The distinctly different mechanisms of action of these 2 forms of gene silencing may help define the use of these treatments in both clinical and research environments.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Oligonucleótidos Antisentido/farmacología , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Perfilación de la Expresión Génica , Células HeLa , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Organismos Modificados GenéticamenteRESUMEN
BACKGROUND: Toxicogenomics experiments often reveal thousands of transcript alterations that are related to multiple processes, making it difficult to identify key gene changes that are related to the toxicity of interest. OBJECTIVES: The objective of this study was to compare gene expression changes in a nontarget tissue to the target tissue for toxicity to help identify toxicity-related genes. METHODS: Male rats were given the hepatotoxicant methapyrilene at two dose levels, with livers and kidneys removed 24 hr after one, three, and seven doses for gene expression analysis. To identify gene changes likely to be related to toxicity, we analyzed genes on the basis of their temporal pattern of change using a program developed at the National Institute of Environmental Health Sciences, termed "EPIG" (extracting gene expression patterns and identifying co-expressed genes). RESULTS: High-dose methapyrilene elicited hepatic damage that increased in severity with the number of doses, whereas no treatment-related lesions were observed in the kidney. High-dose methapyrilene elicited thousands of gene changes in the liver at each time point, whereas many fewer gene changes were observed in the kidney. EPIG analysis identified patterns of gene expression correlated to the observed toxicity, including genes associated with endoplasmic reticulum stress and the unfolded protein response. CONCLUSIONS: By factoring in dose level, number of doses, and tissue into the analysis of gene expression elicited by methapyrilene, we were able to identify genes likely to not be implicated in toxicity, thereby allowing us to focus on a subset of genes to identify toxicity-related processes.
Asunto(s)
Perfilación de la Expresión Génica , Antagonistas de los Receptores Histamínicos H1/toxicidad , Hígado/efectos de los fármacos , Metapirileno/toxicidad , Regulación hacia Arriba/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Riñón/efectos de los fármacos , Riñón/patología , Hígado/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Long-term administration of methotrexate (MTX) for management of chronic inflammatory diseases is associated with risk of liver damage. In this study, we examined the transcriptional profiles of livers from patients treated with MTX. The possibility that expression signatures correlate with grade of fibrosis or underlying rheumatic disease was evaluated. Twenty-seven patients taking MTX were accrued for this study. Ten non-MTX-exposed normal liver specimens were used as controls. Global mRNA expression was assayed using oligonucleotide arrays. A total of 205 genes were significantly altered in MTX-exposed livers. Six of these genes were validated by qPCR. Two genes, CLN8 and ANKH that map to chromosomal locations previously associated with rheumatoid arthritis, were found to be elevated in MTX-exposed samples. Subsequent pathway analysis indicates that MTX exposure is associated with the following key alterations: (1) upregulation of lipid biosynthetic genes, consistent with MTX-induced steatosis, (2) downregulation of proinflammatory chemokines, consistent with the anti-inflammatory effects of MTX, and (3) elevation of complement pathway gene expression. Complement 5, shown earlier to be correlated with liver fibrosis in mice, was found to be elevated (twofold) in MTX-exposed livers. In conclusion, we have found the expression of a number of genes associated with rheumatic disease and/or MTX exposure to be significantly different. Differences in complement expression provide the rationale for future correlative studies between MTX-induced liver fibrosis and C5 alleles in order to identify patients with increased risk for fibrosis.
Asunto(s)
Antagonistas del Ácido Fólico/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Metotrexato/efectos adversos , Adulto , Anciano , Biopsia , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Análisis por Conglomerados , Activación de Complemento/efectos de los fármacos , Femenino , Antagonistas del Ácido Fólico/uso terapéutico , Humanos , Hígado/patología , Cirrosis Hepática/patología , Masculino , Metotrexato/uso terapéutico , Persona de Mediana Edad , Datos de Secuencia Molecular , Psoriasis/complicaciones , Psoriasis/tratamiento farmacológico , ARN/biosíntesis , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedades Reumáticas/complicaciones , Enfermedades Reumáticas/tratamiento farmacológicoRESUMEN
The transforming growth factor-beta (TGF-beta) superfamily of ligands has a pivotal role in the regulation of a wide variety of physiological processes from development to pathogenesis. Since the discovery of the prototypic member, TGF-beta, almost 20 years ago, there have been tremendous advances in our understanding of the complex biology of this superfamily. Deregulation of TGF-beta has been implicated in the pathogenesis of a variety of diseases, including cancer and fibrosis. Here we present the rationale for evaluating TGF-beta signalling inhibitors as cancer therapeutics, the structures of small-molecule inhibitors that are in development and the targeted drug discovery model that is being applied to their development.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Diseño de Fármacos , Humanos , Neoplasias/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Intervention of cancer cell mitosis by antitubulin drugs is among the most effective cancer chemotherapies. However, antitubulin drugs have dose-limiting side effects due to important functions of microtubules in resting normal cells and are often rendered ineffective by rapid emergence of resistance. Antimitotic agents with different mechanisms of action and improved safety profiles are needed as new treatment options. Mitosis-specific kinesin Eg5 represents an attractive anticancer target for discovering such new antimitotic agents, because Eg5 is essential only in mitotic progression and has no roles in resting, nondividing cells. Here, we show that a novel selective Eg5 inhibitor, LY2523355, has broad target-mediated anticancer activity in vitro and in vivo. LY2523355 arrests cancer cells at mitosis and causes rapid cell death that requires sustained spindle-assembly checkpoint (SAC) activation with a required threshold concentration. In vivo efficacy of LY2523355 is highly dose/schedule-dependent, achieving complete remission in a number of xenograft tumor models, including patient-derived xenograft (PDX) tumor models. We further establish that histone-H3 phosphorylation of tumor and proliferating skin cells is a promising pharmacodynamic biomarker for in vivo anticancer activity of LY2523355.
Asunto(s)
Apoptosis/efectos de los fármacos , Cinesinas/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Sulfonamidas/farmacología , Tiadiazoles/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Células HCT116 , Células HT29 , Células HeLa , Humanos , Immunoblotting , Cinesinas/metabolismo , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/patología , Factores de Tiempo , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The application of gene expression profiling technology to examine multiple genes and signaling pathways simultaneously promises a significant advance in understanding toxic mechanisms to ultimately aid in protection of public health. Public and private efforts in the new field of toxicogenomics are focused on populating databases with gene expression profiles of compounds where toxicological and pathological endpoints are well characterized. The validity and utility of a toxicogenomics is dependent on whether gene expression profiles that correspond to different chemicals can be distinguished. The principal hypothesis underlying a toxicogenomic or pharmacogenomic strategy is that chemical-specific patterns of altered gene expression will be revealed using high-density microarray analysis of tissues from exposed organisms. Analyses of these patterns should allow classification of toxicants and provide important mechanistic insights. This report provides a verification of this hypothesis. Patterns of gene expression corresponding to liver tissue derived from chemically exposed rats revealed similarity in gene expression profiles between animals treated with different agents from a common class of compounds, peroxisome proliferators [clofibrate (ethyl-p-chlorophenoxyisobutyrate), Wyeth 14,643 ([4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid), and gemfibrozil (5-2[2,5-dimethylphenoxy]2-2-dimethylpentanoic acid)], but a very distinct gene expression profile was produced using a compound from another class, enzyme inducers (phenobarbital).
Asunto(s)
Perfilación de la Expresión Génica/métodos , Expresión Génica , Genómica , Proliferadores de Peroxisomas/toxicidad , Fenobarbital/toxicidad , Animales , Clofibrato/química , Clofibrato/toxicidad , Biología Computacional , ADN Complementario/análisis , Gemfibrozilo/química , Gemfibrozilo/toxicidad , Perfilación de la Expresión Génica/clasificación , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Reconocimiento de Normas Patrones Automatizadas , Proliferadores de Peroxisomas/química , Fenobarbital/química , Pirimidinas/química , Pirimidinas/toxicidad , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-ActividadRESUMEN
DNA microarrays, used to measure the gene expression of thousands of genes simultaneously, hold promise for future application in efficient screening of therapeutic drugs. This will be aided by the development and population of a database with gene expression profiles corresponding to biological responses to exposures to known compounds whose toxicological and pathological endpoints are well characterized. Such databases could then be interrogated, using profiles corresponding to biological responses to drugs after developmental or environmental exposures. A positive correlation with an archived profile could lead to some knowledge regarding the potential effects of the tested compound or exposure. We have previously shown that cDNA microarrays can be used to generate chemical-specific gene expression profiles that can be distinguished across and within compound classes, using clustering, simple correlation, or principal component analyses. In this report, we test the hypothesis that knowledge can be gained regarding the nature of blinded samples, using an initial training set comprised of gene expression profiles derived from rat liver exposed to clofibrate, Wyeth 14,643, gemfibrozil, or phenobarbital for 24 h or 2 weeks of exposure. Highly discriminant genes were derived from our database training set using approaches including linear discriminant analysis (LDA) and genetic algorithm/K-nearest neighbors (GA/KNN). Using these genes in the analysis of coded liver RNA samples derived from 24-h, 3-day, or 2-week exposures to phenytoin, diethylhexylpthalate, or hexobarbital led to successful prediction of whether these samples were derived from livers of rats exposed to enzyme inducers or to peroxisome proliferators. This validates our initial hypothesis and lends credibility to the concept that the further development of a gene expression database for chemical effects will greatly enhance the hazard identification processes.
Asunto(s)
Bases de Datos Factuales , Perfilación de la Expresión Génica/métodos , Genómica , Biosíntesis de Proteínas , Xenobióticos/toxicidad , Algoritmos , Animales , Biología Computacional , ADN Complementario/análisis , Dietilhexil Ftalato/toxicidad , Análisis Discriminante , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica/clasificación , Hexobarbital/toxicidad , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Reconocimiento de Normas Patrones Automatizadas , Fenitoína/toxicidad , Valor Predictivo de las Pruebas , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Método Simple Ciego , Relación Estructura-Actividad , Xenobióticos/químicaRESUMEN
Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1mg/kg per day for 1, 7 and 14 days), methapyrilene (100mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and (3) develop hypotheses regarding mechanisms of toxicity.
Asunto(s)
Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Mutágenos/toxicidad , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Molecular techniques, such as cDNA microarrays, are being used to aid in the elucidation of the mechanisms of toxicity of a variety of compounds. In this study, we evaluate the molecular effects of furan in the rat liver. Sprague-Dawley rats were exposed to 4 or 40 mg/kg furan for up to 14 days. Furan induced an initial degenerative and necrotic phenotype that was followed by inflammation and fibrosis, consistent with previous observations for this compound. RNA was harvested from each lobe of the liver at several time points to observe whether lobe-specific gene expression effects occurred. Similar gene expression changes were observed in all lobes, however the magnitude of gene expression change was more pronounced in the right lobe. Finally, to help determine the correlation between gene expression changes and liver pathology, we applied traditional microarray visualization tools to the assessment of clinical chemistry and pathology parameters.
Asunto(s)
Furanos/toxicidad , Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Animales , ADN Complementario , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-DawleyRESUMEN
Calcineurin inhibitor (CI) therapy has been associated with chronic nephrotoxicity, which limits its long-term utility for suppression of allograft rejection. In order to understand the mechanisms of the toxicity, we analyzed gene expression changes that underlie the development of CI immunosuppressant-mediated nephrotoxicity in male Sprague-Dawley rats dosed daily with cyclosporine (CsA; 2.5 or 25 mg/kg/day), FK506 (0.6 or 6 mg/kg/day), or rapamycin (1 or 10 mg/kg/day) for 1, 7, 14, or 28 days. A significant increase in blood urea nitrogen was observed in animals treated with CsA (high) or FK506 (high) for 14 and 28 days. Histopathological examination revealed tubular basophilia and mineralization in animals given CsA (high) or FK506 (low and high). We identified a group of genes whose expression in rat kidney is correlated with CI-induced kidney injury. Among these genes are two genes, Slc12a3 and kidney-specific Wnk1 (KS-Wnk1), that are known to be involved in sodium transport in the distal nephrons and could potentially be involved in the mechanism of CI-induced nephrotoxicity. The downregulation of NCC (the Na-Cl cotransporter coded by Slc12a3) in rat kidney following CI treatment was confirmed by immunohistochemical staining, and the downregulation of KS-Wnk1 was confirmed by quantitative real-time-polymerase chain reaction (qRT-PCR). We hypothesize that decreased expression of Slc12a3 and KS-Wnk1 could alter the sodium chloride reabsorption in the distal tubules and contribute to the prolonged activation of the renin-angiotensin system, a demonstrated contributor to the development of CI-induced nephrotoxicity in both animal models and clinical settings. Therefore, if validated as biomarkers in humans, SLC12A3 and KS-WNK1 could potentially be useful in the early detection and reduction of CI-related nephrotoxicity in immunosuppressed transplant patients when monitoring the health of kidney xenographs in clinical practice.
Asunto(s)
Inhibidores de la Calcineurina , Inmunosupresores/toxicidad , Enfermedades Renales/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores de Droga/genética , Simportadores/genética , Animales , Enfermedad Crónica , Regulación hacia Abajo , Diagnóstico Precoz , Marcadores Genéticos , Rechazo de Injerto/prevención & control , Inmunohistoquímica , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/inducido químicamente , Enfermedades Renales/patología , Enfermedades Renales/fisiopatología , Trasplante de Riñón , Masculino , Antígenos de Histocompatibilidad Menor , Ratas , Ratas Sprague-Dawley , Sistema Renina-Angiotensina/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12 , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
The cardiovascular injury of the type III selective PDE inhibitor SK&F 95654 was investigated in SHR. Twenty-four hours after a single sc injection of 100 or 200 mg/kg of the drug, rats exhibited cardiomyocyte necrosis and apoptosis, interstitial inflammation, hemorrhage and edema, as well as mesenteric arterial hemorrhage and necrosis, periarteritis, EC and VSMC apoptosis, EC activation, and MC activation and degranulation. Elevated serum levels of cTnT and decreased cTnT immunoperoxidase staining on cardiomyocytes were detected in the drug-treated rats. Serum levels of alpha2-macroglobulin and IL-6 were significantly elevated following drug treatment. NMR spectral patterns of urine samples are significantly different between the drug-treated and control rats. These results indicate that measurement of serum cTnT, acute phase proteins, and cytokines as well as metabonomic urine profiles may serve as potential biomarkers for drug-induced cardiovascular injury in rats. Increased expression of CD63 on MC (tissue biomarker of MC), of nitrotyrosine on MC and EC (an indirect indicator of NO in vivo), and of iNOS on MC and EC (source of NO) suggest that NO produced by activated and degranulated MC as well as activated EC play an important role in SK&F 95654-induced mesenteric vascular injury.
Asunto(s)
Biomarcadores/análisis , Enfermedades Cardiovasculares/inducido químicamente , Sistema Cardiovascular/efectos de los fármacos , Inhibidores de Fosfodiesterasa/toxicidad , Piridazinas/toxicidad , Piridinas/toxicidad , Alanina Transaminasa/sangre , Animales , Apoptosis/efectos de los fármacos , Aspartato Aminotransferasas/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/orina , Sistema Cardiovascular/patología , Creatina/orina , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Hipuratos/orina , Inmunohistoquímica , Interleucina-6/sangre , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/patología , Miocitos Cardíacos/química , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Inhibidores de Fosfodiesterasa/farmacología , Piridazinas/farmacología , Piridinas/farmacología , Ratas , Ratas Endogámicas SHR , Taurina/orina , Troponina/análisis , Troponina/sangre , Factor de Necrosis Tumoral alfa/análisis , alfa-Macroglobulinas/análisisRESUMEN
Activation of p38alpha MAPK triggers G2/M checkpoint, thus inhibiting cell proliferation. In this study we found that depletion of p38alpha by RNAi also inhibited cell proliferation and caused mitotic arrest. However, treatment with selective small molecule p38 kinase inhibitors had no effect on cell cycle progression, even though the p38 kinase was completely inhibited, revealing p38alpha functions that are independent of its kinase activity. Indeed, ectopic expression of a kinase negative p38alpha rescued the lethality caused by RNAi-depletion of the endogenous p38alpha, thus providing further evidence for a kinase-independent function of p38alpha. In addition, we showed that overexpression of the wild type or kinase-negative p38alpha also strongly inhibited cell proliferation, similarly as RNAi depletion of p38alpha. Together the results demonstrate that, in addition to its kinase-dependent functions, such as in activation of G2/M checkpoint, p38alpha also has an essential, kinase-independent function.
Asunto(s)
Proteína Quinasa 14 Activada por Mitógenos/química , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Mitosis/fisiología , Animales , División Celular/fisiología , Proliferación Celular , Fase G2/fisiología , Inhibidores de Crecimiento/antagonistas & inhibidores , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/fisiología , Células HeLa , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/fisiología , Ratones , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Células 3T3 NIH , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARNRESUMEN
Methapyrilene (MP) exposure of animals can result in an array of adverse pathological responses including hepatotoxicity. This study investigates gene expression and histopathological alterations in response to MP treatment in order to 1) utilize computational approaches to classify samples derived from livers of MP treated rats based on severity of toxicity incurred in the corresponding tissue, 2) to phenotypically anchor gene expression pattems, and 3) to gain insight into mechanism(s) of methapyrilene hepatotoxicity. Large-scale differential gene expression levels associated with the exposure of male Sprague-Dawley rats to the rodent hepatic carcinogen MP for 1, 3, or 7 days after daily dosage with 10 or 100 mg/kg/day were monitored. Hierarchical clustering and principal component analysis were successful in classifying samples in agreement with microscopic observations and revealed low-dose effects that were not observed histopathologically. Data from cDNA microarray analysis corroborated observed histopathological alterations such as hepatocellular necrosis, bile duct hyperplasia, microvesicular vacuolization, and portal inflammation observed in the livers of MP exposed rats and provided insight into the role of specific genes in the studied toxicological processes.