RESUMO
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with metabolic syndrome (MetS) in humans and elicits pathologies in rodents that resemble non-alcoholic fatty liver disease (NAFLD) in humans through activation of the aryl hydrocarbon receptor (AHR) pathway. Dysregulation of cholesterol homeostasis, an aspect of MetS, is linked to NAFLD pathogenesis. TCDD exposure is also linked to the suppression of genes that encode key cholesterol biosynthesis steps and changes in serum cholesterol levels. In a previous experiment, treating mice with TCDD in the presence of simvastatin, a 3-Hydroxy-3-Methylglutaryl-CoA Reductase competitive inhibitor, altered lipid and glycogen levels, AHR-battery gene expression, and liver injury in male mice compared to TCDD alone. The aim of this study was to deduce a possible mechanism(s) for the metabolic changes and increased injury using single-nuclei RNA sequencing in mouse liver. We demonstrated that co-treated mice experienced wasting and increased AHR activation compared to TCDD alone. Furthermore, relative proportions of cell (sub)types were different between TCDD alone and co-treated mice including important mediators of NAFLD progression like hepatocytes and immune cell populations. Analysis of non-overlapping differentially expressed genes identified several pathways where simvastatin co-treatment significantly impacted TCDD-induced changes, which may explain the differences between treatments. Overall, these results demonstrate a connection between dysregulation of cholesterol homeostasis and toxicant-induced metabolic changes.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Dibenzodioxinas Policloradas , Humanos , Camundongos , Masculino , Animais , Dibenzodioxinas Policloradas/toxicidade , Sinvastatina/farmacologia , Sinvastatina/metabolismo , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fígado/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Expressão Gênica , Colesterol/metabolismoRESUMO
Cellular homeostasis requires the use of multiple environmental sensors that can respond to a variety of endogenous and exogenous compounds. The aryl hydrocarbon receptor (AHR) is classically known as a transcription factor that induces genes that encode drug metabolizing enzymes when bound to toxicants such as 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD). The receptor has a growing number of putative endogenous ligands, such as tryptophan, cholesterol, and heme metabolites. Many of these compounds are also linked to the translocator protein (TSPO), an outer mitochondrial membrane protein. Given a portion of the cellular pool of the AHR has also been localized to mitochondria and the overlap in putative ligands, we tested the hypothesis that crosstalk exists between the two proteins. CRISPR/Cas9 was used to create knockouts for AHR and TSPO in a mouse lung epithelial cell line (MLE-12). WT, AHR-/-, and TSPO-/- cells were then exposed to AHR ligand (TCDD), TSPO ligand (PK11195), or both and RNA-seq was performed. More mitochondrial-related genes were altered by loss of both AHR and TSPO than would have been expected just by chance. Some of the genes altered included those that encode for components of the electron transport system and the mitochondrial calcium uniporter. Both proteins altered the activity of the other as AHR loss caused the increase of TSPO at both the mRNA and protein level and loss of TSPO significantly increased the expression of classic AHR battery genes after TCDD treatment. This research provides evidence that AHR and TSPO participate in similar pathways that contribute to mitochondrial homeostasis.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Dibenzodioxinas Policloradas , Receptores de Hidrocarboneto Arílico , Animais , Camundongos , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Células Epiteliais/metabolismo , Ligantes , Pulmão/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor in the Per-Arnt-Sim superfamily of environmental sensors that is linked to several metabolic diseases, including nonalcoholic fatty liver disease. Much remains unknown regarding the impact of genetic variation in AHR-driven disease, as past studies have focused on a small number of inbred strains. Recently, the presence of a wide range of interindividual variability amongst humans was reported in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical ligand of the AHR. In this study, a panel of 14 diverse mouse strains was exposed to TCDD for 10 days to characterize the AHR-mediated response across genetic backgrounds. Responses to TCDD are heavily dependent on genetic background. Although mice carry 1 of 4 Ahr alleles known to impact the affinity to AHR-ligands, we observed significant intra-allelic variability suggesting the presence of novel genetic modifiers of AHR signaling. A regression-based approach was used to scan for genes regulated by the AHR and/or associated with TCDD-induced phenotypes. The approach identified 7 genes, 2 of which are novel, that are likely regulated by the AHR based on association with hepatic TCDD burden (p ≤ .05). Finally, we identified 1 gene, Dio1, which was associated with change in percent body fat across the diverse set of strains (p ≤ .05). Overall, the results in this study exemplify the power of genetics-based approaches in identifying novel genes that are putatively regulated by the AHR.
Assuntos
Dibenzodioxinas Policloradas , Receptores de Hidrocarboneto Arílico , Animais , Humanos , Fígado/metabolismo , Camundongos , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de SinaisRESUMO
INTRODUCTION: Animal-based studies are essential for assessing toxicity to environmental pollutants, especially when the potential targets are specific developmental time points, teratogenic, or multi-organ systems that cannot be modeled in vitro. Orogastric gavage is a widely used technique for exposure because of its increased accuracy of dose administration over free feeding. However, repeated use of this method has been reported to cause physiological stress on the exposed animals that could interfere with interpretation of results. Previous studies have shown that genetic background also contributes to the level of stress and can affect individual response. METHODS: To evaluate the impact of stress on repeated orogastric gavage, we exposed C67BL/6J and 129S1/SvImJ inbred mouse strains to 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), a potent xenobiotic that has been extensively studied in vivo. Pregnant females were dosed for ten days after mating using orogastric gavage with olive oil as vehicle or through diet using peanut butter as vehicle. Serum corticosterone levels, body weight, and reproduction endpoints were measured to evaluate levels of stress induced by the dosing technique. RESULTS: The levels of stress caused by orogastric gavage was strongly dependent on strain background and on the phenotypic endpoint. Orogastric gavage-induced stress was more detrimental in 129S1/SvlmJ pregnant female mice than in C57BL/6J. CONCLUSION: These results show that administration of xenobiotics via controlled diet can improve the reproducibility and rigor of exposure studies requiring orogastric delivery.
Assuntos
Arachis , Dibenzodioxinas Policloradas , Animais , Camundongos , Camundongos Endogâmicos C57BL , Dibenzodioxinas Policloradas/toxicidade , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
With recent advances in sequencing technologies, the scientific community has begun to probe the potential genetic bases behind complex phenotypes in humans and model organisms. In many cases, the genomes of genetically distinct strains of model organisms, such as the mouse (Mus musculus), have not been fully sequenced. Here, we report on a tool designed to use single-nucleotide polymorphism (SNP) and insertion-deletion (indel) data to predict gene, mRNA, and protein sequences for up to 36 genetically distinct mouse strains. By automated querying of freely accessible databases through a graphical interface, the software requires no data and little computational experience. As a proof of concept, we predicted the gene and amino acid sequence of the aryl hydrocarbon receptor (Ahr) for all inbred mouse strains of which variant data were currently available through Mouse Genome Project. Predicted sequences were compared with fully sequenced genomes to show that the tool is effective in predicting gene and protein sequences.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Análise de Sequência/métodos , Software , Sequência de Aminoácidos , Animais , Mutação INDEL , Camundongos Endogâmicos , Polimorfismo de Nucleotídeo Único , RNA Mensageiro , Alinhamento de SequênciaRESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor. The prototypical ligand of the AHR is an environmental contaminant called 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD exposure is associated with many adverse health outcomes in humans including non-alcoholic fatty liver disease (NAFLD). Previous studies suggest that AHR ligands alter cholesterol homeostasis in mice through repression of genes involved in cholesterol biosynthesis, such as Hmgcr, which encodes the rate-limiting enzyme of cholesterol biosynthesis called 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR). In this study, we sought to characterize the impact of HMGCR repression in TCDD-induced liver injury. C57BL/6 mice were exposed to TCDD in the presence or absence of simvastatin, a competitive inhibitor of HMGCR. Simvastatin exposure decreased TCDD-induced hepatic lipid accumulation in both sexes, but was most prominent in females. Simvastatin and TCDD (S + T) co-treatment increased hepatic AHR-battery gene expression and liver injury in male, but not female, mice. In addition, the S + T co-treatment led to an increase in hepatic glycogen content that coincides with heavier liver in female mice. Results from this study suggest that statins, which are amongst the most prescribed pharmaceuticals, may protect from AHR-mediated steatosis, but alter glycogen metabolism and increase the risk of TCDD-elicited liver damage in a sex-specific manner.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Feminino , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dibenzodioxinas Policloradas/toxicidadeRESUMO
2,3,7,8-Tetrachlordibenzo- p-dioxin (TCDD) is an environmental pollutant that can cause various toxic effects, including chloracne, metabolic syndrome, and immune suppression. Most of the toxicity associated with TCDD is mediated through activation of the aryl hydrocarbon receptor (AHR). Recent research has suggested the presence of a wide-range of interindividual variability in TCDD-mediated suppression of the Immunoglobulin-M (IgM) response across the human population. In an attempt to identify putative modifiers of AHR-mediated immunosuppression beyond the AHR, B cells were isolated from a panel of genetically diverse mouse strain to scan for modulators that drive interstrain differences in TCDD-mediated suppression of the IgM response. Results implicated a region of mouse Chromosome 1 near a gene encoding serine peptidase inhibitor, clade B, member 2 ( Serpinb2) whose human ortholog is plasminogen activator inhibitor 2 (PAI2). Further downstream analyses indicated that Serpinb2 is dysregulated by TCDD and, furthermore, that B cells from Serpinb2 -/- mice are significantly more sensitive to TCDD-mediated suppression as compared to littermate controls. This study suggests a protective role of Serpinb2 within TCDD-mediated immunosuppression and, furthermore, a novel function of Serpinb2-related activity in the IgM response.
Assuntos
Linfócitos B/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Serpinas/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Poluentes Ambientais/química , Poluentes Ambientais/toxicidade , ELISPOT , Imunoglobulina M/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Filogenia , Dibenzodioxinas Policloradas/química , Locos de Características Quantitativas , Receptores de Hidrocarboneto Arílico/classificação , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Serpinas/química , Serpinas/genéticaRESUMO
Humans respond to chemical exposures differently due to many factors, such as previous and concurrent stressors, age, sex, and genetic background. The vast majority of laboratory-based toxicology studies, however, have not considered the impact of population-level variability within dose-response relationships. The lack of data dealing with the influence of genetic diversity on the response to chemical exposure provides a difficult challenge for risk assessment as individuals within the population will display a wide-range of responses following toxicant challenge. Notably, the genetic background of individuals plays a major role in the variability seen in a population-level response to a drug or chemical and, thus, there is growing interest in including genetic diversity into laboratory-models. Here we outline several laboratory-based models that can be used to assay the influence of genetic variability on an individual's response to chemicals: 1) genetically-diverse cell lines, 2) human primary cells, 3) and genetically-diverse mouse panels. We also provide a succinct review for several seminal studies to highlight the capability, feasibility, and power of each of these models. This article is intended to highlight the need to include population-level genetic diversity into toxicological study designs via laboratory-based models with the goal to provide and supplement evidence in assessing the risk posed by chemicals to the human population. As such, incorporation of genetic variability will positively impact human-based risk assessment and provide empirical data to aid and influence decision-making processes in relation to chemical exposures.
Assuntos
Variação Genética/genética , População , Toxicologia/métodos , Animais , Humanos , Camundongos , Modelos Animais , Medição de RiscoRESUMO
The influence of interindividual variability is not typically assessed in traditional toxicological studies. Given that chemical exposures occur in heterogeneous populations, this knowledge gap has the potential to cause undue harm within the realms of public health and industrial and municipal finances. A recent report from the National Research Council (NRC) suggests that when accounting for interindividual variation in responses, traditionally assumed nonlinear dose-response relationships (DRRs) for noncancer-causing endpoints would better be explained with a linear relationship within the low-dose region. To address this knowledge gap and directly test the NRC's assumption, this study focused on assessing the DRR between 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) exposure and immune suppression in a cohort of unique human donors. Human B cells were isolated from 51 individual donors and treated with logarithmically increasing concentrations of TCDD (0-30 nM TCDD). Two endpoints sensitive to TCDD were assessed: (1) number of IgM-secreting B cells and (2) quantity of IgM secreted. The results show that TCDD significantly suppressed both the number of IgM-secreting B cells and the quantity of IgM secreted (P < .05). Statistical model comparisons indicate that the low-dose region of the two DRRs is best explained with a nonlinear relationship. Rather than assuming low-dose linearity for all noncancer-causing DRRs, our study suggests the need to consider the specific mode of action of toxicants and pharmaceuticals during risk-management decision making.
Assuntos
Linfócitos B/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Linfócitos B/metabolismo , Ligante de CD40/imunologia , Técnicas de Cocultura , Estudos de Coortes , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina M/metabolismo , Ativação LinfocitáriaRESUMO
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most widely studied ligand of the aryl hydrocarbon receptor (AHR). The AHR-dependent TCDD-induced mitochondrial hyperpolarization (Tappenden et al., 2011) [1] and reduced oxygen consumption rate of intact mouse hepatoma cells (Huang et al., in press) [2] in the previous studies suggest that these alterations can be related to enzymatic activities of the electron transport chain (ETC) and ATP synthase in oxidative phosphorylation (OXPHOS) system. Here, we evaluated the activity of each complex in the OXPHOS system using in vitro enzymatic assays. The calculated enzymatic activity of each complex was normalized against the activity of citrate synthase. To combine each value from an independent experiment, normalized enzyme activities from cells exposed to TCDD were converted to fold changes via comparison to the activity relative to time-matched vehicle control. The averaged fold change for each treatment suggests more replicates are needed in order to clearly evaluate a difference between treatments.
RESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is the principal regulator of a cell׳s response to many polyaromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To gain a better understanding of the impact of TCDD on the mitochondrial proteome, a stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic analysis was performed. We used two mouse hepatoma cell lines that differ in AHR expression levels, hepa1c1c7 (AHR-expressing) and hepac12 (AHR-deficient). The cell lines were exposed to TCDD (10 nM) for 72 h; each treatment was assayed in triplicate and were analyzed as separate runs on the mass-spectrometer. Mitochondria were then isolated and mitochondrial proteins were separated by SDS-PAGE and subject to mass spectrometry. The data presented were collected from four independent SILAC experiments. Within each experiment, three isotopes were employed to compare protein ratios via mass-spectrometry: (1) light l-arginine/l-lysine HCl (Arg0, Lys0), (2) medium (15)N4-l-arginin/(13)C6l-lysine HCl (Arg4, Lys6), and (3) heavy (13)C6 (15)N4l-arginine/(13)C6 (15)N2l-lysine HCl (Arg10, Lys8). The raw data includes approximately 2500 annotated proteins. The datasets provided by this study can be a reference to other toxicologists investigating TCDD-induced mitochondrial dysfunction. The data presented here are associated with the research article, "Mitochondrial-targeted Aryl Hydrocarbon Receptor and the Impact of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Cellular Respiration and the Mitochondrial Proteome" (Hwang et al. (2016) [1]).
RESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor within the Per-Arnt-Sim (PAS) domain superfamily. Exposure to the most potent AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is associated with various pathological effects including metabolic syndrome. While research over the last several years has demonstrated a role for oxidative stress and metabolic dysfunction in AHR-dependent TCDD-induced toxicity, the role of the mitochondria in this process has not been fully explored. Our previous research suggested that a portion of the cellular pool of AHR could be found in the mitochondria (mitoAHR). Using a protease protection assay with digitonin extraction, we have now shown that this mitoAHR is localized to the inter-membrane space (IMS) of the organelle. TCDD exposure induced a degradation of mitoAHR similar to that of cytosolic AHR. Furthermore, siRNA-mediated knockdown revealed that translocase of outer-mitochondrial membrane 20 (TOMM20) was involved in the import of AHR into the mitochondria. In addition, TCDD altered cellular respiration in an AHR-dependent manner to maintain respiratory efficiency as measured by oxygen consumption rate (OCR). Stable isotope labeling by amino acids in cell culture (SILAC) identified a battery of proteins within the mitochondrial proteome influenced by TCDD in an AHR-dependent manner. Among these, 17 proteins with fold changes≥2 are associated with various metabolic pathways, suggesting a role of mitochondrial retrograde signaling in TCDD-mediated pathologies. Collectively, these studies suggest that mitoAHR is localized to the IMS and AHR-dependent TCDD-induced toxicity, including metabolic dysfunction, wasting syndrome, and hepatic steatosis, involves mitochondrial dysfunction.
Assuntos
Mitocôndrias/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/biossíntese , Animais , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Consumo de Oxigênio/efeitos dos fármacos , Proteoma , RNA Interferente Pequeno , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Hypoxia is a state of decreased oxygen reaching the tissues of the body. During prenatal development, the fetus experiences localized occurrences of hypoxia that are essential for proper organogenesis and survival. The response to decreased oxygen availability is primarily regulated by hypoxia-inducible factors (HIFs), a family of transcription factors that modulate the expression of key genes involved in glycolysis, angiogenesis, and erythropoiesis. HIF-1α and HIF-2α, two key isoforms, are important in embryonic development, and likely are involved in lung morphogenesis. We have recently shown that the inducible loss of Hif-1α in lung epithelium starting at E4.5 leads to death within an hour of parturition, with symptoms similar to neonatal respiratory distress syndrome (RDS). In addition to Hif-1α, Hif-2α is also expressed in the developing lung, although the overlapping roles of Hif-1α and Hif-2α in this context are not fully understood. To further investigate the independent role of Hif-2α in lung epithelium and its ability to alter Hif-1α-mediated lung maturation, we generated two additional lung-specific inducible Hif-α knockout models (Hif-2α and Hif-1α+Hif-2α). The intrauterine loss of Hif-2α in the lungs does not lead to decreased viability or observable phenotypic changes in the lung. More interestingly, survivability observed after the loss of both Hif-1α and Hif-2α suggests that the loss of Hif-2α is capable of rescuing the neonatal RDS phenotype seen in Hif-1α-deficient pups. Microarray analyses of lung tissue from these three genotypes identified several factors, such as Scd1, Retlnγ, and Il-1r2, which are differentially regulated by the two HIF-α isoforms. Moreover, network analysis suggests that modulation of hormone-mediated, NF-κB, C/EBPα, and c-MYC signaling are central to HIF-mediated changes in lung development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Deleção de Genes , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Fenótipo , Síndrome do Desconforto Respiratório do Recém-Nascido/genética , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismo , Animais , Animais Recém-Nascidos , Redes Reguladoras de Genes , Genótipo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Síndrome do Desconforto Respiratório do Recém-Nascido/patologia , Transdução de Sinais , Análise de Sobrevida , Transcrição GênicaRESUMO
Hypoxia inducible factor-1 (HIF1) is a stress-responsive nuclear transcription factor that is activated with a decrease in oxygen availability. HIF1 regulates the expression of genes involved in a cell's adaptation to hypoxic stress, including those with mitochondrial specific function. To gain a more comprehensive understanding of the role of HIF1 in mitochondrial homeostasis, we studied the link between hypoxia, HIF1 transactivation, and electron transport chain (ETC) function. We established immortalized mouse embryonic fibroblasts (MEFs) for HIF1α wild-type (WT) and null cells and tested whether HIF1α regulates mitochondrial respiration by modulating gene expressions of nuclear-encoded ETC components. High-throughput quantitative real-time polymerase chain reaction was performed to screen nuclear-encoded mitochondrial genes related to the ETC to identify those whose regulation was HIF1α-dependent. Our data suggest that HIF1α regulates transcription of cytochrome c oxidase (CcO) heart/muscle isoform 7a1 (Cox7a1) under hypoxia, where it is induced 1.5-2.5-fold, whereas Cox4i2 hypoxic induction was HIF1α-independent. We propose that adaptation to hypoxic stress of CcO as the main cellular oxygen consumer is mediated by induction of hypoxia-sensitive tissue-specific isoforms. We suggest that HIF1 plays a central role in maintaining homeostasis in cellular respiration during hypoxic stress via regulation of CcO activity.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Indução Enzimática , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio , Animais , Hipóxia Celular , Células Cultivadas , Células Clonais , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Immunotoxicology assessments have historically focused on the effects that xenobiotics exhibit directly on immune cells. These studies are invaluable as they identify immune cell targets and help characterize mechanisms and/or adverse outcome pathways of xenobiotics within the immune system. However, leukocytes can receive environmental cues by cell-cell contact or via released mediators from cells of organs outside of the immune system. These organs include, but are not limited to, the mucosal areas such as the lung and the gut, the liver, and the central nervous system. Homeostatic perturbation in these organs induced directly by toxicants can initiate and alter the outcome of local and systemic immunity. This review will highlight some of the identified nonimmune influences on immune homeostasis and provide summaries of how immunotoxic mechanisms of selected xenobiotics involve nonimmune cells or mediators. Thus, this review will identify data gaps and provide possible alternative mechanisms by which xenobiotics alter immune function that could be considered during immunotoxicology safety assessment.
Assuntos
Comunicação Celular/efeitos dos fármacos , Sistema Imunitário/efeitos dos fármacos , Toxicologia/métodos , Xenobióticos/toxicidade , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/metabolismo , Medição de Risco , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/imunologia , Células Estromais/metabolismo , Timo/efeitos dos fármacos , Timo/imunologia , Timo/metabolismoRESUMO
Hard metal lung disease (HMLD) is an occupational lung disease specific to inhalation of cobalt-containing particles whose mechanism is largely unknown. Cobalt is a known hypoxia mimic and stabilizer of the alpha subunits of hypoxia-inducible factors (HIFs). Previous work revealed that though HIF1α contrib utes to cobalt toxicity in vitro, loss of HIF1α in the alveolar epithelial cells does not provide in vivo protection from cobalt-induced lung inflammation. HIF1α and HIF2α show unique tissue expression profiles, and HIF2α is known to be the predominant HIF mRNA isoform in the adult lung. Thus, if HIF2α activation by cobalt contributes to pathophysiology of HMLD, we hypothesized that loss of HIF2α in lung epithelium would provide protection from cobalt-induced inflammation. Mice with HIF2α-deficiency in Club and alveolar type II epithelial cells (ATIIs) (HIF2α(Δ/Δ)) were exposed to cobalt (60 µg/day) or saline using a subacute occupational exposure model. Bronchoalveolar lavage cellularity, cytokines, qRT-PCR, and histopathology were analyzed. Results show that loss of HIF2α leads to enhanced eosinophilic inflammation and increased goblet cell metaplasia. Additionally, control mice demonstrated a mild recovery from cobalt-induced lung injury compared with HIF2α(Δ/Δ) mice, suggesting a role for epithelial HIF2α in repair mechanisms. The expression of important cytokines, such as interleukin (IL)-5 and IL-10, displayed significant differences following cobalt exposure when HIF2α(Δ/Δ) and control mice were compared. In summary, our data suggest that although loss of HIF2α does not afford protection from cobalt-induced lung inflammation, epithelial HIF2α signaling does play an important role in modulating the inflammatory and repair response in the lung.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Cobalto/toxicidade , Lesão Pulmonar/induzido quimicamente , Alvéolos Pulmonares/efeitos dos fármacos , Eosinofilia Pulmonar/induzido quimicamente , Mucosa Respiratória/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Doxiciclina/farmacologia , Perfilação da Expressão Gênica , Imuno-Histoquímica , Lesão Pulmonar/complicações , Lesão Pulmonar/imunologia , Lesão Pulmonar/patologia , Camundongos , Camundongos Knockout , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/patologia , Eosinofilia Pulmonar/etiologia , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/patologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologiaRESUMO
The aryl-hydrocarbon receptor (AHR), a ligand activated PAS superfamily transcription factor, mediates most, if not all, of the toxicity induced upon exposure to various dioxins, dibenzofurans, and planar polyhalogenated biphenyls. While AHR-mediated gene regulation plays a central role in the toxic response to dioxin exposure, a comprehensive understanding of AHR biology remains elusive. AHR-mediated signaling starts in the cytoplasm, where the receptor can be found in a complex with the heat shock protein of 90 kDa (Hsp90) and the immunophilin-like protein, aryl-hydrocarbon receptor-interacting protein (AIP). The role these chaperones and other putative interactors of the AHR play in the toxic response is not known. To more comprehensively define the AHR-protein interaction network (AHR-PIN) and identify other potential pathways involved in the toxic response, a proteomic approach was undertaken. Using tandem affinity purification (TAP) and mass spectrometry we have identified several novel protein interactions with the AHR. These interactions physically link the AHR to proteins involved in the immune and cellular stress responses, gene regulation not mediated directly via the traditional AHR:ARNT heterodimer, and mitochondrial function. This new insight into the AHR signaling network identifies possible secondary signaling pathways involved in xenobiotic-induced toxicity.
RESUMO
Allergic airway disease is characterized by a T helper type 2 cell-mediated airway inflammation and airway hyperresponsiveness. Little is known about the role of hypoxia-mediated signaling in the progression of the disease. To address this knowledge gap, a mouse model was created in which doxycycline exposure induces the functional deletion of hypoxia inducible factor-1α from alveolar type II and Clara cells of the lung. When hypoxia inducible factor-1α deletion was induced during the early postnatal development period of the lung, the mice displayed an enhanced response to the ovalbumin model of allergic airway disease. These hypoxia inducible factor-1α-deficient mice exhibit increased cellular infiltrates, eosinophilia in the lavage fluid and parenchyma, and T helper type 2 cytokines, as compared with ovalbumin-treated control mice. Moreover, these hypoxia inducible factor-1α-deficient mice display increased airway resistance when compared with their control counterparts. Interestingly, if the loss of hypoxia inducible factor-1α was induced in early adulthood, the exacerbated phenotype was not observed. Taken together, these results suggest that epithelial hypoxia inducible factor-1α plays an important role in establishing the innate immunity of the lung and epithelial-specific deficiency in the transcription factor, during early postnatal development, increases the severity of inflammation and functional airway resistance, following ovalbumin challenge. Finally, these results might explain some of the chronic respiratory pathology observed in premature infants, especially those that receive supplemental oxygen. This early hyperoxic exposure, from normal ambient and supplemental oxygen, would presumably inhibit normal hypoxia inducible factor-1α signaling, mimicking the functional deletion described.
Assuntos
Asma/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pulmão/metabolismo , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Asma/induzido quimicamente , Asma/imunologia , Líquido da Lavagem Broncoalveolar/química , Broncoconstritores/farmacologia , Contagem de Células , Citocinas/metabolismo , Proteína Básica Maior de Eosinófilos/metabolismo , Eosinófilos/metabolismo , Eosinófilos/patologia , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imunidade Inata , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/patologia , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Transgênicos , Ovalbumina , Distribuição AleatóriaRESUMO
Wild-type and HIF1α -/- MEF cells were used to determine the role of HIF1α in cadmium-induced toxicity. Cadmium treatment did not affect HIF1-mediated transcription but led to caspase activation and apoptotic cell death in wild-type and HIF1α -/- cells. Cadmium-induced cell death, however, was significantly higher in HIF1α -/- cells as compared to their wild-type counterparts. Increased cell death in the HIF1α -/- cells was correlated with lower metallothionein protein, elevated levels of reactive oxygen species, and decreased superoxide dismutase enzyme activity. The total and oxidized glutathione levels, and, correspondingly, lipid peroxidation levels were elevated in the null cells compared to wild-type cells, indicating increased antioxidant demand and greater oxidative stress. Overall, the results suggest that basal levels of HIF1α play a protective role against cadmium-induced cytotoxicity in mouse embryonic fibroblasts by maintaining metallothionein and antioxidant activity levels.
RESUMO
Hypoxia-inducible factor-1α (HIF-1α) is a critical transcription factor that controls oxygen homeostasis in response to hypoxia, inflammation, and oxidative stress. HIF has been implicated in the pathogenesis of liver injury in which these events play a role, including acetaminophen (APAP) overdose, which is the leading cause of acute liver failure in the United States. APAP overdose has been reported to activate HIF-1α in mouse livers and isolated hepatocytes downstream of oxidative stress. HIF-1α signaling controls many factors that contribute to APAP hepatotoxicity, including mitochondrial cell death, inflammation, and hemostasis. Therefore, we tested the hypothesis that HIF-1α contributes to APAP hepatotoxicity. Conditional HIF-1α deletion was generated in mice using an inducible Cre-lox system. Control (HIF-1α-sufficient) mice developed severe liver injury 6 and 24 h after APAP overdose (400 mg/kg). HIF-1α-deficient mice were protected from APAP hepatotoxicity at 6 h, but developed severe liver injury by 24 h, suggesting that HIF-1α is involved in the early stage of APAP toxicity. In further studies, HIF-1α-deficient mice had attenuated thrombin generation and reduced plasminogen activator inhibitor-1 production compared with control mice, indicating that HIF-1α signaling contributes to hemostasis in APAP hepatotoxicity. Finally, HIF-1α-deficient animals had decreased hepatic neutrophil accumulation and plasma concentrations of interleukin-6, keratinocyte chemoattractant, and regulated upon activation normal T cell expressed and secreted compared with control mice, suggesting an altered inflammatory response. HIF-1α contributes to hemostasis, sterile inflammation, and early hepatocellular necrosis during the pathogenesis of APAP toxicity.