RESUMEN
The aryl hydrocarbon receptor (AhR) is a nuclear receptor that facilitates a wide transcriptional response and causes a variety of adaptive and maladaptive physiological functions. Such functions are entirely dependent on the type of ligand activating it, and therefore, the nuances in the activation of this receptor at the single-cell level have become a research interest for different pharmacological and toxicological applications. Here, we investigate the activation of the AhR by diverse classes of compounds in a Hepa1c1c7-based murine hepatoma cell line. The exogenous compounds analyzed produced different levels of ultrasensitivity in AhR activation as measured by XRE-coupled EGFP production and analyzed by both flow cytometric and computational simulation techniques. Interestingly, simulation experiments reported herein were able to reproduce and quantitate the natural single-cell stochasticity inherent to mammalian cell lines as well as the ligand-specific differences in ultrasensitivity. Classical AhR modulators 2,3,7,8-tetrachlorodibenzodioxin (10- 1-105 pM), PCB-126 (10- 1-107 pM), and benzo[a]pyrene (10- 1-107 pM) produced the greatest levels of single-cell ultrasensitivity and most maximal responses, while consumption-based ligands indole-3-carbinol (103-109 pM), 3,3'-diindolylmethane (103-108 pM), and cannabidiol (103-108 pM) caused low-level AhR activation in more purely graded single-cell fashions. All compounds were tested and analyzed over a 24 h period for consistency. The comparative quantitative results for each compound are presented within. This study aids in defining the disparity between different types of AhR modulators that produce distinctly different physiological outcomes. In addition, the simulation tool developed for this study can be used in future studies to predict the quantitative effects of diverse types of AhR ligands in the context of pharmacological therapies or toxicological concerns.
Asunto(s)
Sustancias Peligrosas/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Benzo(a)pireno/toxicidad , Carcinoma Hepatocelular , Línea Celular Tumoral , Indoles/toxicidad , Neoplasias Hepáticas , Ratones , Bifenilos Policlorados/toxicidad , Dibenzodioxinas Policloradas/toxicidadRESUMEN
Pharmaceutical companies develop specialized therapies to treat late stage cancer. In order to accelerate life-saving treatments and reduce animal testing, compounds to treat life-threatening malignancies are allowed modified requirements for preclinical toxicology testing. Limited data packages in early drug development can present product quality challenges at multi-product manufacturing facilities. The present analysis established an endpoint-specific threshold of toxicological concern (TTC) for developmental and reproductive toxicity (DART) for anticancer compounds. A comprehensive database was created consisting of over 300 no-observed adverse effect levels (NOAELs) for DART of 108 anticancer compounds. The 5th percentile NOAEL for developmental and reproductive toxicity was 0.005 mg/kg/day (300 µg/day), resulting in a human exposure threshold of 3 µg/day assuming standard uncertainty factors and a 60 kg human bodyweight. The analysis shows this threshold is protective for developmental and reproductive toxicity of highly potent groups of anticancer compounds. There were similar TTC values calculated for direct-acting and indirect-acting anticancer compounds.
Asunto(s)
Antineoplásicos/toxicidad , Medición de Riesgo/métodos , Animales , Relación Dosis-Respuesta a Droga , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Fetal/efectos de los fármacos , Humanos , Reproducción/efectos de los fármacos , Pruebas de ToxicidadRESUMEN
Alzheimer's disease (AD) is the leading cause of dementia and one of the most common causes of death worldwide. As an age-dependent multifactorial disease, the causative triggers of AD are rooted in spontaneous declines in cellular function and metabolic capacity with increases in protein stressors such as the tau protein. This multitude of age-related processes that cause neurons to change from healthy states to ones vulnerable to the damage seen in AD are difficult to simultaneously investigate and even more difficult to quantify. Here we aimed to diminish these gaps in our understanding of neuronal vulnerability in AD development by using simulation methods to theoretically quantify an array of cellular stress responses and signaling molecules. This temporally-descriptive molecular signature was produced using a novel multimethod simulation approach pioneered by our laboratory for biological research; this methodology combines hierarchical agent-based processes and continuous equation-based modeling in the same interface, all while maintaining intrinsic distributions that emulate natural biological stochasticity. The molecular signature was validated for a normal organismal aging trajectory using experimental longitudinal data from Caenorhabditis elegans and rodent studies. In addition, we have further predicted this aging molecular signature for cells impacted by the pathogenic tau protein, giving rise to distinct stress response conditions needed for cytoprotective aging. Interestingly, our simulation experiments showed that oxidative stress signaling (via daf-16 and skn-1 activities) does not substantially protect cells from all the early stressors of aging, but that it is essential in preventing a late-life degenerative cellular phenotype. Together, our simulation experiments aid in elucidating neurodegenerative triggers in the onset of AD for different genetic conditions. The long-term goal of this work is to provide more detailed diagnostic and prognostic tools for AD development and progression, and to provide more comprehensive preventative measures for this disease.
RESUMEN
The complex molecular events that occur within the host during the establishment of a Mycobacterium tuberculosis infection are poorly defined, thus preventing identification of predictive markers of disease progression and state. To identify such molecular markers during M. tuberculosis infection, global changes in transcriptional response in the host were assessed using mouse whole genome arrays. Bacterial load in the lungs, the lesions associated with infection, and gene expression profiling was performed by comparing normal lung tissue to lungs from mice collected at 20, 40, and 100 days after aerosol infection with the H37Rv strain of M. tuberculosis. Quantitative, whole lung gene expression identified signature profiles defining different signaling pathways and immunological responses characteristic of disease progression. This includes genes representing members of the interferon-associated gene families, chemokines and cytokines, MHC, and NOS2, as well as an array of cell surface markers associated with the activation of T cells, macrophages, and dendritic cells that participate in immunity to M. tuberculosis infection. More importantly, several gene transcripts encoding proteins that were not previously associated with the host response to M. tuberculosis infection, and unique molecular markers associated with disease progression and state, were identified.
Asunto(s)
Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Animales , Biomarcadores/metabolismo , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Interferón gamma/genética , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética , Tuberculosis/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
High doses of the commonly used herbicide atrazine have been shown to suppress luteinizing hormone (LH) release. To determine whether atrazine alters the function of gonadotropin-releasing hormone (GnRH) neurons, we examined the effects of atrazine on GnRH neuronal activation and the subsequent release of LH normally associated with ovulation. Ovariectomized adult Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle for 4 days. Animals were primed with estrogen and progesterone to induce an evening LH surge. Blood samples were obtained over the afternoon and evening using an indwelling right atrial cannula, and plasma was assayed for LH and FSH. Another cohort of animals was transcardially perfused in the afternoon to examine GnRH activation using FOS immunoreactivity. Results of these studies show that 4-day treatment with atrazine resulted in a significant reduction in the magnitude of the LH and FSH surges, and this corresponds to a decrease in GnRH neurons expressing FOS immunoreactivity. To determine if the effects of atrazine were long lasting, additional studies were performed examining LH levels and GnRH activation 2 days and 4 days after atrazine withdrawal. Within 4 days (but not 2 days) after cessation of atrazine treatment, measures of hypothalamic-pituitary-gonadal (HPG) activation returned to normal. These data indicate that atrazine affects neuroendocrine function in the female rat by actions at the level of the GnRH neuron and that the acute effects of high doses of atrazine can be reversed within 4 days after withdrawal of treatment.
Asunto(s)
Atrazina/administración & dosificación , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo/efectos de los fármacos , Neuronas/efectos de los fármacos , Análisis de Varianza , Animales , Recuento de Células , Relación Dosis-Respuesta a Droga , Estradiol/farmacología , Femenino , Hormona Folículo Estimulante/sangre , Herbicidas/administración & dosificación , Sistema Hipotálamo-Hipofisario/metabolismo , Hipotálamo/metabolismo , Inmunohistoquímica , Hormona Luteinizante/sangre , Neuronas/metabolismo , Ovariectomía , Progesterona/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Radioinmunoensayo , Ratas , Ratas Wistar , Recuperación de la FunciónRESUMEN
DJ-1 mutation induces early-onset Parkinson's disease, and conversely over-expression of DJ-1 is associated with cancer in numerous tissues. A gene-trap screening library conducted in embryonic stem cells was utilized for generation of a DJ-1 mutant mouse. Real-time PCR and immunoblotting were utilized to confirm functional mutation of the DJ-1 gene. Normal DJ-1 protein expression in adult mouse tissue was characterized and demonstrates high expression in brain tissue with wide systemic distribution. Primary astrocytes isolated from DJ-1(-/-) mice reveal a decreased nuclear localization of DJ-1 protein in response to rotenone or LPS, with a concomitant increase in mitochondrial localization of DJ-1 found only in the rotenone exposure. Resting mitochondrial membrane potential was significantly lower in DJ-1(-/-) astrocytes, as compared to controls. Our DJ-1 knockout mouse provides an exciting tool for exploring the molecular and physiological roles of DJ-1 to further explicate its functions in neurodegeneration.
Asunto(s)
Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Mutación , Proteínas Oncogénicas/genética , Animales , Astrocitos/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Genotipo , Lipopolisacáridos/farmacología , Potencial de la Membrana Mitocondrial , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Oncogénicas/metabolismo , Peroxirredoxinas , Fenotipo , Proteína Desglicasa DJ-1 , Transporte de Proteínas , Rotenona/farmacologíaRESUMEN
Inflammatory activation of glial cells is associated with neuronal injury in several degenerative movement disorders of the basal ganglia, including manganese neurotoxicity. Manganese (Mn) potentiates the effects of inflammatory cytokines on nuclear factor-kappaB (NF-kappaB)-dependent expression of nitric oxide synthase 2 (NOS2) in astrocytes, but the signaling mechanisms underlying this effect have remained elusive. It was postulated in the present studies that direct stimulation of cGMP synthesis and activation of mitogen-activated protein (MAP) kinase signaling pathways underlies the capacity of Mn to augment NF-kappaB-dependent gene expression in astrocytes. Exposure of primary cortical astrocytes to a low concentration of Mn (10 microM) potentiated expression of NOS2 mRNA and protein along with production of NO in response to interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), which was prevented by overexpression of dominant negative IkappaB alpha. Mn also potentiated IFNgamma- and TNFalpha-induced phosphorylation of extracellular response kinase (ERK), p38, and JNK, as well as cytokine-induced activation of a fluorescent NF-kappaB reporter construct in transgenic astrocytes. Activation of ERK preceded that of NF-kappaB and was required for maximal activation of NO synthesis. Independently of IFNgamma/TNFalpha, Mn-stimulated synthesis of cGMP in astrocytes and inhibition of soluble guanylate cyclase (sGC) abolished the potentiating effect of Mn on MAP kinase phosphorylation, NF-kappaB activation, and production of NO. These data indicate that near-physiological concentrations of Mn potentiate cytokine-induced expression of NOS2 and production of NO in astrocytes via activation of sGC, which promotes ERK-dependent enhancement of NF-kappaB signaling.
Asunto(s)
Astrocitos/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanilato Ciclasa/metabolismo , Manganeso/farmacología , FN-kappa B/fisiología , Óxido Nítrico Sintasa de Tipo II/genética , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Medios de Cultivo , GMP Cíclico/metabolismo , Activación Enzimática , Genes Reporteros , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico/metabolismo , ARN Mensajero/genéticaRESUMEN
Atrazine (ATRA) is metabolized by cytochrome P450s to the chlorinated metabolites, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (ETHYL), 2-chloro-4-amino-6-isopropylamino-1, 3, 5-triazine (ISO), and diaminochlorotriazine (DACT). Here, we develop a set of physiologically based pharmacokinetic (PBPK) models that describe the influence of oral absorption and oxidative metabolism on the blood time course curves of individual chlorotriazines (Cl-TRIs) in rat after oral dosing of ATRA. These models first incorporated in vitro metabolic parameters to describe time course plasma concentrations of DACT, ETHYL, and ISO after dosing with each compound. Parameters from each individual model were linked together into a final composite model in order to describe the time course of all 4 Cl-TRIs after ATRA dosing. Oral administration of ISO, ETHYL and ATRA produced double peaks of the compounds in plasma time courses that were described by multiple absorption phases from gut. An adequate description of the uptake and bioavailability of absorbed ATRA also required inclusion of additional oxidative metabolic clearance of ATRA to the mono-dealkylated metabolites occurring in GI a tract compartment. These complex processes regulating tissue dosimetry of atrazine and its chlorinated metabolites likely reflect limited compound solubility in the gut from dosing with an emulsion, and sequential absorption and metabolism along the GI tract at these high oral doses.
Asunto(s)
Atrazina/farmacocinética , Herbicidas/farmacocinética , Modelos Biológicos , Mucosa Bucal , Absorción , Administración Oral , Animales , Área Bajo la Curva , Atrazina/análogos & derivados , Atrazina/sangre , Atrazina/química , Atrazina/metabolismo , Circulación Sanguínea/fisiología , Femenino , Herbicidas/sangre , Herbicidas/química , Herbicidas/metabolismo , Fase I de la Desintoxicación Metabólica , Estructura Molecular , Mucosa Bucal/metabolismo , Mucosa Bucal/fisiología , Ratas , Ratas Sprague-Dawley , Distribución Tisular/fisiología , Triazinas/sangre , Triazinas/químicaRESUMEN
Genome-wide oligonucleotide DNA microarrays and real time RT-PCR were used to assess differential gene expression in rat glioma and hepatoma cell lines after exposure to the aryl hydrocarbon receptor (AhR) agonist 3,3',4,4',5-pentachlorobiphenyl (penta-CB). Under maximal inducing concentrations for cytochrome P450 1A1 (CYP1A1) in H4IIE rat hepatoma cells, both H4IIE and C6 rat glioma cells were exposed to sub-micromolar concentrations of penta-CB for 24h. Differential gene expression for approximately 28,000 gene probes were computationally analyzed and compared. As expected, penta-CB potently activated CYP1A1/2 transcription in liver-derived H4IIE hepatoma cells yet did not do so in brain-derived C6 glioma cells. Additionally, we show that penta-CB causes: (1) distinct patterns of gene expression between tumor cells derived from liver or brain; (2) robust transcriptional activation of select C6 glioma gene ontologies; (3) over-expression of H4IIE hepatoma genes associated with tumor progression in liver; (4) greater than 100-fold over-expression of C6 glioma genes associated with protein processing and programmed cell death and/or metastasis; (5) tissue-selective histone deacetylase inhibition in C6 glioma, but not H4IIE hepatoma cells as signaled by galectin-1 over-expression.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Cromatina/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas/metabolismo , Bifenilos Policlorados/toxicidad , Receptores de Hidrocarburo de Aril/agonistas , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Cromatina/ultraestructura , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , ADN Complementario/biosíntesis , ADN Complementario/genética , Inhibidores Enzimáticos/farmacología , Galectina 1/metabolismo , Glioma/genética , Inhibidores de Histona Desacetilasas , Ligandos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas Experimentales/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación Transcripcional/efectos de los fármacosRESUMEN
This study estimated the kinetic constants for oxidative metabolism of atrazine (ATRA) and its chlorotriazine (Cl-TRI) metabolites, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (ETHYL), 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine (ISO), and diaminochlorotriazine (DACT), using freshly isolated rat hepatocytes. Hepatocytes were incubated with 1.74, 44, 98, and 266 microM ATRA. Disappearance of ATRA and formation of the Cl-TRI metabolites were quantified over 90 min. At all incubation concentrations, ATRA was preferentially metabolized to ETHYL, producing ETHYL concentrations approximately 6 times higher than those of ISO. DACT concentrations peaked at 44 microM ATRA and decreased with increasing incubation concentrations, indicating non-linear metabolic behavior of ATRA with respect to DACT formation. A series of kinetic models were developed from these data to describe the dose and time-dependent oxidative metabolism of ATRA and the Cl-TRI metabolites. An integrated model for all the chloro-triazines included multi-substrate competitive inhibition of metabolism to describe the non-linear behavior of DACT production in relation to ATRA while simultaneously simulating the time-course behavior of the Cl-TRIs at all four ATRA concentrations. The maximal metabolic rate (V(max)) of ATRA metabolism and the Michaelis-Menten constant (K(M)) for the reaction were 1.6 microM/min and 30 microM, respectively. V(max) and K(M) values for ETHYL and ISO metabolism to DACT were also estimated using this modeling approach.
Asunto(s)
Atrazina/farmacología , Hepatocitos/efectos de los fármacos , Herbicidas/farmacología , Fase I de la Desintoxicación Metabólica/fisiología , Animales , Atrazina/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Hepatocitos/metabolismo , Herbicidas/metabolismo , Técnicas In Vitro , Cinética , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Triazinas/metabolismo , Triazinas/farmacologíaRESUMEN
Research in biogerontology has largely focused on the complex relationship between mitochondrial dysfunction and biological aging. In particular, the mitochondrial free radical theory of aging (MFRTA) has been well accepted. However, this theory has been challenged by recent studies showing minimal increases in reactive oxygen species (ROS) as not entirely deleterious in nature, and even beneficial under the appropriate cellular circumstances. To assess these significant and nonintuitive observations in the context of a functional system, we have taken an in silico approach to expand the focus of the MFRTA by including other key mitochondrial stress response pathways, as they have been observed in the nematode Caenorhabditis elegans. These include the mitochondrial unfolded protein response (UPRmt ), mitochondrial biogenesis and autophagy dynamics, the relevant DAF-16 and SKN-1 axes, and NAD+ -dependent deacetylase activities. To integrate these pathways, we have developed a multilevel hybrid-modeling paradigm, containing agent-based elements among stochastic system-dynamics environments of logically derived ordinary differential equations, to simulate aging mitochondrial phenotypes within a population of energetically demanding cells. The simulation experiments resulted in accurate predictions of physiological parameters over time that accompany normal aging, such as the declines in both NAD+ and ATP and an increase in ROS. Additionally, the in silico system was virtually perturbed using a variety of pharmacological (e.g., rapamycin, pterostilbene, paraquat) and genetic (e.g., skn-1, daf-16, sod-2) schemes to quantitate the temporal alterations of specific mechanistic targets, supporting insights into molecular determinants of aging as well as cytoprotective agents that may improve neurological or muscular healthspan.
Asunto(s)
Dinámicas Mitocondriales/genética , Envejecimiento , Humanos , Estrés OxidativoRESUMEN
Acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can suppress adaptive immunity. In this study, pre-exposure of Leishmania major-infected mice to TCDD caused a dose-dependent and unexpected decrease in parasite burdens on day 20 after infection. In contrast, TCDD-mediated lymphoid atrophy, suppressed antibody levels, and enhanced interleukin-2 production were observed as expected. These results suggest that TCDD may enhance resistance to L. major in the face of immune suppression.
Asunto(s)
Contaminantes Ambientales/farmacología , Leishmania major/efectos de los fármacos , Leishmaniasis Cutánea/parasitología , Dibenzodioxinas Policloradas/farmacología , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/efectos de los fármacos , Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inmunidad Celular/efectos de los fármacos , Leishmania major/crecimiento & desarrollo , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
Melioidosis is caused by the facultative intracellular bacterium Burkholderia pseudomallei and is potentially fatal. Despite a growing global burden and high fatality rate, little is known about the disease. Recent studies demonstrate that cyclooxygenase-2 (COX-2) inhibition is an effective post-exposure therapeutic for pulmonary melioidosis, which works by inhibiting the production of prostaglandin E2 (PGE2). This treatment, while effective, was conducted using an experimental COX-2 inhibitor that is not approved for human or animal use. Therefore, an alternative COX-2 inhibitor needs to be identified for further studies. Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) COX-2 inhibitor marketed outside of the United States for the treatment of migraines. While this drug was developed for COX-2 inhibition, it has been found to modulate other aspects of inflammation as well. In this study, we used RAW 264.7 cells infected with B pseudomallei to analyze the effect of TA on cell survival, PGE2 production and regulation of COX-2 and nuclear factor- kappaB (NF-ĸB) protein expression. To evaluate the effectiveness of post-exposure treatment with TA, results were compared to Ceftazidime (CZ) treatments alone and the co-treatment of TA with a sub-therapeutic treatment of CZ determined in a study of BALB/c mice. Results revealed an increase in cell viability in vitro with TA and were able to reduce both COX-2 expression and PGE2 production while also decreasing NF-ĸB activation during infection. Co-treatment of orally administered TA and a sub-therapeutic treatment of CZ significantly increased survival outcome and cleared the bacterial load within organ tissue. Additionally, we demonstrated that post-exposure TA treatment with sub-therapeutic CZ is effective to treat melioidosis in BALB/c mice.
Asunto(s)
Burkholderia pseudomallei/fisiología , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Melioidosis/tratamiento farmacológico , Melioidosis/inmunología , ortoaminobenzoatos/administración & dosificación , Animales , Burkholderia pseudomallei/inmunología , Ceftazidima/administración & dosificación , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Profilaxis PosexposiciónRESUMEN
Previous research from our laboratory has shown a switch-like response to PCB 126 mediated CYP1A1 induction in primary rat hepatocytes and in H4IIE rat hepatoma cells. On a single cell level, cells appear to be either "on" or "off" for CYP1A1 induction at a given dose; some cells never respond to PCB 126. These cells represent a non-responding population. Cells that are switched "on" by PCB 126 display varying levels of induction, much like the dimmer on a light switch. The goal of the present research is to begin to uncover the mechanism for this switch-like response to CYP1A1 induction in H4IIE rat hepatoma cells. The AhR pathway is modulated by multiple co-activators and by phosphorylation. This research focuses on the phosphorylation cascades initiated by PCB 126 and the role they play in CYP1A1 induction. Our research reveals a likely role for protein kinase C (PKC) in this switch response. Inhibition of PKC by H-7 dramatically reduced the percent of cells that express CYP1A1 in response to PCB 126 treatment, as determined by flow cytometry. The effect of H-7 was concentration dependent, decreasing the number of cells expressing CYP1A1 rather than decreasing the level of CYP1A1 in all cells. This finding provides further evidence for the switch-like behavior of CYP1A1 induction and implicates PKC in this response to PCB126. The protein kinase inhibitor, HA-1004, had only a minor effect on CYP1A1 induction. A high-throughput immunoblot screen for 40 proteins revealed the regulation of several proteins/phosphoproteins by PCB 126. Most importantly, two proteins containing phosphoserine/phoshothreonine residues were increased by PCB126 treatment. However, PKC translocation studies and activity studies failed to verify that PCB126 activates PKC. It is possible that constitutive PKC activity is sufficient to maintain phosphorylation of critical components of the AhR pathway. Immunoblotting studies showed that MAP kinases ERK and JNK are not activated by PCB 126 in H4IIE cells and the ERK inhibitor U0126 did not impair CYP1A1 induction. Additional studies are planned to further investigate the role of PKC in the switch-like response to PCB 126.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Citocromo P-450 CYP1A1/biosíntesis , Genes de Cambio/fisiología , Hepatocitos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Animales , Western Blotting , Recuento de Células , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Antagonistas de Estrógenos/toxicidad , Citometría de Flujo , Genes de Cambio/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Fosforilación , Bifenilos Policlorados/toxicidad , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/toxicidad , Ratas , Transducción de SeñalRESUMEN
The identification of molecular markers related to critical biological processes during carcinogenesis may aid in the evaluation of carcinogenic potentials of chemicals and chemical mixtures. Work from our laboratory demonstrated that a single treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhanced spontaneous malignant transformation of the human keratinocyte cell line RHEK-1. In contrast, chronic low-level exposure of cells to arsenic alone or in a mixture containing arsenic, cadmium, chromium, and lead inhibited malignant conversion. To identify changes in gene expression that influence these different outcomes, cDNA microarray technology was used. Analysis of multiple human arrays in MNNG-transformed RHEK-1 cells, designated OM3, and those treated with arsenic or the arsenic-containing metal mixture showed unique patterns of gene expression. Genes that were overexpressed in OM3 included oncogenes, cell cycle regulators, and those involved in signal transduction, whereas genes for DNA repair enzymes and inhibitors of transformation and metastasis were suppressed. In arsenic-treated cells, multiple DNA repair proteins were overexpressed. Mixture-treated cells showed increased expression of a variety of genes including metallothioneins and integrin 4. These cells showed decreased expression of oncogenes, DNA repair proteins, and genes involved in the mitogen-activated protein kinase pathway. For comparison we are currently analyzing gene expression changes in RHEK-1 cells transformed by other means. The goal of these studies is to identify common batteries of genes affected by chemical modulators of the carcinogenic process. Mechanistic studies may allow us to correlate alterations in their expression with sequential stages in the carcinogenic process and may aid in the risk assessment of other xenobiotics.
Asunto(s)
Arsénico/efectos adversos , Transformación Celular Neoplásica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Metilnitronitrosoguanidina/efectos adversos , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Cultivo de Célula , Reparación del ADN , Marcadores Genéticos , Humanos , Queratinocitos/efectos de los fármacos , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/farmacología , Medición de Riesgo , Xenobióticos/efectos adversosRESUMEN
In vivo induction of CYP1A1 in hepatocytes by aryl hydrocarbon receptor agonists is heterogeneous. Using immunohistochemistry, cells appear to be either induced or not induced as if the response of an individual cell is better represented as a switch. We have examined induction of CYP1A1 in vitro in primary rat hepatocytes to distinguish the responses of populations of cells and responses of individual cells. Cells were treated with various concentrations of the aryl hydrocarbon receptor agonist, 3,3',4,4',5-pentachlorobiphenyl. Concentration-response and time-course responses were determined for the population of cells by Western blotting for CYP1A1 protein and by real-time RT-PCR for CYP1A1 mRNA. Individual cell responses were visualized by immunocytochemistry (ICC) for protein and by in situ hybridization (ISH) for mRNA. CYP1A1 mRNA was quantified by frequency distribution analysis of grains observed on the ISH slides. Population responses showed time- and concentration-related increases in induction. Single cell responses appeared as all-or-none in the field, with cells appearing to be induced and others appearing to be not induced. Even at the highest concentrations (2.5 x 10(-7) M), some hepatocytes remained unresponsive. Distribution frequencies of single cell induction were more consistent with a switch with variable levels of induction in cells depending on treatment concentration. Combined with the reports from in vivo studies, our results support a switch with rheostat behavior for individual hepatocytes. Mechanistic studies in liver cell lines that are confirmed to exhibit switch-like induction of single cells will be necessary to assess the molecular pathways of this circuit element.
Asunto(s)
Citocromo P-450 CYP1A1/biosíntesis , Hepatocitos/enzimología , Bifenilos Policlorados/toxicidad , Animales , Bioensayo/métodos , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Hepatocitos/efectos de los fármacos , Masculino , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Hidrocarburo de Aril/agonistasRESUMEN
The shape of the dose-response curve may vary depending on whether one examines response at a population or a single cell level. Populations of cells may exhibit a graded response whereas single cell responses may have threshold or switch-like behavior. Studies in vivo and in vitro using primary hepatocyte cultures have shown that induction of CYP1A1 in the liver exhibits switch-like behavior in response to PCB 126 (3,3',4,4',5-pentachlorobiphenyl). The goal of the present study was to determine if two liver cell lines (H4IIE rat hepatoma and Hepa 1c1c7 mouse hepatoma) also show switch-like behavior and develop experimental models for studying mechanisms of these switch-like responses. Both cell lines were analyzed via concentration-response and time-course studies using quantitative real-time PCR, revealing a sigmoidal concentration-response curve for CYP1A1 mRNA induction at the population level. To study CYP1A1 protein induction on a single cell level, flow cytometry was employed. In both cell lines the distribution of fluorescence increased with increasing concentrations of PCB 126. The switch behavior was more pronounced in the H4IIE cells than in the Hepa 1c1c7 cells, exhibiting a well-defined shift of induction from the "off" to the "on" state. The concentration-response curve at the single cell level appeared more switch-like with two populations of cells-basal levels and maximally induced. Immunocytochemistry studies of individual cells also support these conclusions. Our data support the hypothesis that PCB 126 induces CYP1A1 in a switch-like fashion in H4IIE rat hepatoma cells. These cells can now be used to study the mechanism of the biological switch.
Asunto(s)
Citocromo P-450 CYP1A1/biosíntesis , Hígado/enzimología , Animales , Línea Celular Tumoral , Citocromo P-450 CYP1A1/genética , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Citometría de Flujo , Ratones , Bifenilos Policlorados/toxicidad , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Receptores de Hidrocarburo de Aril/agonistas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
High oral doses of atrazine (ATRA) disrupt normal neuroendocrine function, resulting in suppression of the luteinizing hormone (LH) surge in adult, ovariectomized (OVX) estrogen-primed female rats. While the mechanism by which ATRA inhibits LH secretion is not known, current data indicate that ATRA does have anti-estrogenic properties in vitro and in vivo. In the body, ATRA is rapidly converted to diaminochlorotriazine (DACT). The present study was conducted to investigate the effects of ATRA and DACT on the estradiol benzoate (EB)/progesterone (P) induced LH surge and to determine if such changes correlate with impaired estrogen receptor (ER) function. ATRA, administered by gavage for five consecutive days to adult OVX, female Sprague-Dawley rats, caused a dose-dependent suppression of the EB/P induced LH surge. Although to a lesser degree than ATRA, DACT significantly suppressed total plasma LH and peak LH surge levels in EB/P primed animals by 60 and 58%, respectively. DACT treatment also decreased release of LH from the pituitary in response to exogenous gonadotropin releasing hormone (GnRH) by 47% compared to control. Total plasma LH secretion was reduced by 37% compared to control, suggesting that in addition to potential hypothalamic dysfunction, pituitary function is altered. To further investigate the mechanism by which hypothalamic function might be altered, potential anti-estrogenicity of ATRA and DACT were assessed by evaluating ER function treated rats. Using an in vitro receptor binding assay, ATRA, but not DACT, inhibited binding of [(3)H]-estradiol to ER. In contrast, ATRA, administered to female rats under dosing conditions which suppressed the LH surge, neither changed the levels of unoccupied ER nor altered the estrogen induced up-regulation of progesterone receptor mRNA. Collectively, these results indicate that although ATRA is capable of binding ER in vitro, the suppression of LH after treatment with high doses of ATRA is not due to alterations of hypothalamic ER function.
Asunto(s)
Atrazina/farmacología , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Hormona Luteinizante/biosíntesis , Hipófisis/efectos de los fármacos , Receptores de Progesterona/efectos de los fármacos , Animales , Núcleo Hipotalámico Anterior/efectos de los fármacos , Núcleo Hipotalámico Anterior/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Hormona Liberadora de Gonadotropina/farmacología , Hormona Luteinizante/antagonistas & inhibidores , Hormona Luteinizante/sangre , Ovariectomía , Ovario/efectos de los fármacos , Ovario/metabolismo , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/metabolismo , Hipófisis/metabolismo , Área Preóptica , Progesterona , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Triazinas/farmacologíaRESUMEN
We have identified a new mutant mouse that we have named new mouse neurological mutant 3 (NM3); it may be a useful model to understand the underlying molecular and genetic basis of Parkinson's disease (PD). A mouse carrying the NM3 mutation arose spontaneously in an RIIIS/J breeding colony and was identified as having a movement disorder. Upon neurological examination of these mice, their movement was found to be slow and abnormal, with characteristic choreaform and bradykinetic-type movements, typical of PD. The importance of the gene mutation in NM3 in the molecular pathway involved in this pathology is underscored by the fact that these mice do not survive past weaning age if they are homozygous for the genetic mutation. We localized the gene mutation by positional cloning and genetic mapping to mouse chromosome 2 in an area that corresponds to human chromosome 2q24-31, which does not contain any known genes associated with PD. However, there was a significant decrease of 15-20% in the levels of dopamine, and its principal metabolite, 3,4-dihydroxyphenylacetic acid, in the midbrain of affected mice. Low concentrations of these substances are associated with PD in human patients, making these mutant mice candidates for studies of this disease.
Asunto(s)
Química Encefálica/genética , Modelos Animales de Enfermedad , Ratones Mutantes Neurológicos/genética , Enfermedad de Parkinson/genética , Ácido 3,4-Dihidroxifenilacético/análisis , Animales , Mapeo Cromosómico , Dopamina/análisis , RatonesRESUMEN
Expression of an estrogen-regulated reporter gene, growth of MCF-7 cells in the presence of 17beta-estradiol (E2) or E2 plus TCDD, and DNA microarray plus real time quantitative PCR analyses of gene expression in MCF-7 cells were used to evaluate the effects of TCDD, a known E2 antagonist, on E2-regulated gene expression in human cells. TCDD added simultaneously with E2 exhibited significantly decreased E2-associated upregulation of reporter gene expression compared with cells treated with E2 alone, and decreased E2 enhancement of mitosis in MCF-7 cells. MCF-7 cells treated with E2 or E2 plus TCDD and DNA microarray-evaluated to determine patterns of gene expression, showed substantial differences in gene expression in TCDD-treated cells compared with E2-treated cells. Of the 2400 genes on the Perkin Elmer global array microchip utilized for this analysis, a minimum of 317 were significantly upregulated and 488 were significantly downregulated. Of these, the gene encoding insulin receptor substrate-1 (IRS-1), the protein product of which has been previously reported to be decreased, missing, altered, or defective in persons with type 2 diabetes mellitus, was evaluated by real time quantitative PCR to corroborate the array data. An evaluation of the potential consequences of TCDD-altered IRS-1 downregulation is presented.