RESUMEN
The aryl hydrocarbon receptor is a ligand-activated transcription factor known for mediating the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. TCDD induces nonalcoholic fatty liver disease (NAFLD)-like pathologies including simple steatosis that can progress to steatohepatitis with fibrosis and bile duct proliferation in male mice. Dose-dependent progression of steatosis to steatohepatitis with fibrosis by TCDD has been associated with metabolic reprogramming, including the disruption of amino acid metabolism. Here, we used targeted metabolomic analysis to reveal dose-dependent changes in the level of ten serum and eleven hepatic amino acids in mice upon treatment with TCDD. Bulk RNA-seq and protein analysis showed TCDD repressed CPS1, OTS, ASS1, ASL, and GLUL, all of which are associated with the urea cycle and glutamine biosynthesis. Urea and glutamine are end products of the detoxification and excretion of ammonia, a toxic byproduct of amino acid catabolism. Furthermore, we found that the catalytic activity of OTC, a rate-limiting step in the urea cycle was also dose dependently repressed. These results are consistent with an increase in circulating ammonia. Collectively, the repression of the urea and glutamate-glutamine cycles increased circulating ammonia levels and the toxicity of TCDD.
Asunto(s)
Amoníaco , Redes y Vías Metabólicas , Enfermedad del Hígado Graso no Alcohólico , Dibenzodioxinas Policloradas , Animales , Masculino , Ratones , Amoníaco/sangre , Amoníaco/metabolismo , Fibrosis , Glutamina/metabolismo , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Redes y Vías Metabólicas/efectos de los fármacosRESUMEN
Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.
Asunto(s)
Tejido Adiposo Pardo , Aorta Torácica , Canales Iónicos , Mecanotransducción Celular , Proteínas de la Membrana , Ratas Endogámicas Dahl , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aorta Torácica/fisiopatología , Masculino , Canales Iónicos/metabolismo , Canales Iónicos/genética , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo/metabolismo , Ratas , Hipertensión/metabolismo , Hipertensión/fisiopatología , Hipertensión/genética , Hipertensión/patología , RNA-SeqRESUMEN
Perivascular adipose tissue (PVAT) is known for being anti-contractile in healthy tissues. We discovered a new function of PVAT, the ability to stress relax and maintain a tone in response to a stretch. This is of note because stress relaxation has been attributed to smooth muscle, of which PVAT has none that is organized in a functional layer. We test the hypothesis the interactions of integrins with collagen play a role in stress relaxation. Our model is the thoracic aorta of the male Dahl SS rat. The PVAT and aorta were physically separated for most assays. Results from single nuclei RNA sequencing (snRNAseq) experiments, histochemistry and isometric contractility were also used. Masson Trichrome staining made evident the expression of collagen in PVAT. From snRNA seq experiments of the PVAT, mRNA for multiple collagen and integrin isoforms were detected: the α1 and ß1 integrin were most highly expressed. Pharmacological inhibition of integrin/collagen interaction was effected by the specific α1ß1 distintegrin obtustatin or general integrin inhibitor RGD peptide. RGD peptide but not obtustatin increased the stress relaxation. Cell-cell communication inference identified integrins αv and α5, two major RGD motif containing isoforms, as potential signaling partners of collagens. Collectively, these findings validate that stress relaxation can occur in a non-smooth muscle tissue, doing so in part through integrin-collagen interactions that may not include α1ß1 heterodimers. The importance of this lies in considering PVAT as a vascular layer that possesses mechanical functions.
Asunto(s)
Tejido Adiposo , Aorta Torácica , Colágeno , Integrinas , Ratas Endogámicas Dahl , Animales , Masculino , Tejido Adiposo/metabolismo , Integrinas/metabolismo , Aorta Torácica/metabolismo , Colágeno/metabolismo , RatasRESUMEN
The application of single-cell RNA sequencing (scRNAseq) for the evaluation of chemicals, drugs, and food contaminants presents the opportunity to consider cellular heterogeneity in pharmacological and toxicological responses. Current differential gene expression analysis (DGEA) methods focus primarily on two group comparisons, not multi-group dose-response study designs used in safety assessments. To benchmark DGEA methods for dose-response scRNAseq experiments, we proposed a multiplicity corrected Bayesian testing approach and compare it against 8 other methods including two frequentist fit-for-purpose tests using simulated and experimental data. Our Bayesian test method outperformed all other tests for a broad range of accuracy metrics including control of false positive error rates. Most notable, the fit-for-purpose and standard multiple group DGEA methods were superior to the two group scRNAseq methods for dose-response study designs. Collectively, our benchmarking of DGEA methods demonstrates the importance in considering study design when determining the most appropriate test methods.
Asunto(s)
Benchmarking , Proyectos de Investigación , Teorema de Bayes , Expresión GénicaRESUMEN
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces diverse biological and toxic effects, including reprogramming intermediate metabolism, mediated by the aryl hydrocarbon receptor. However, the specific reprogramming effects of TCDD are unclear. Here, we performed targeted LC-MS analysis of hepatic extracts from mice gavaged with TCDD. We detected an increase in S-(2-carboxyethyl)-L-cysteine, a conjugate from the spontaneous reaction between the cysteine sulfhydryl group and highly reactive acrylyl-CoA, an intermediate in the cobalamin (Cbl)-independent ß-oxidation-like metabolism of propionyl-CoA. TCDD repressed genes in both the canonical Cbl-dependent carboxylase and the alternate Cbl-independent ß-oxidation-like pathways as well as inhibited methylmalonyl-CoA mutase (MUT) at lower doses. Moreover, TCDD decreased serum Cbl levels and hepatic cobalt levels while eliciting negligible effects on gene expression associated with Cbl absorption, transport, trafficking, or derivatization to 5'-deoxy-adenosylcobalamin (AdoCbl), the required MUT cofactor. Additionally, TCDD induced the gene encoding aconitate decarboxylase 1 (Acod1), the enzyme responsible for decarboxylation of cis-aconitate to itaconate, and dose-dependently increased itaconate levels in hepatic extracts. Our results indicate MUT inhibition is consistent with itaconate activation to itaconyl-CoA, a MUT suicide inactivator that forms an adduct with adenosylcobalamin. This adduct in turn inhibits MUT activity and reduces Cbl levels. Collectively, these results suggest the decrease in MUT activity is due to Cbl depletion following TCDD treatment, which redirects propionyl-CoA metabolism to the alternate Cbl-independent ß-oxidation-like pathway. The resulting hepatic accumulation of acrylyl-CoA likely contributes to TCDD-elicited hepatotoxicity and the multihit progression of steatosis to steatohepatitis with fibrosis.
Asunto(s)
Acilcoenzima A , Contaminantes Ambientales , Hígado Graso , Hígado , Dibenzodioxinas Policloradas , Deficiencia de Vitamina B 12 , Vitamina B 12 , Ácido Aconítico/metabolismo , Acilcoenzima A/metabolismo , Animales , Cobalto/metabolismo , Cisteína/metabolismo , Contaminantes Ambientales/toxicidad , Hígado Graso/inducido químicamente , Hígado Graso/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Metilmalonil-CoA Mutasa/genética , Metilmalonil-CoA Mutasa/metabolismo , Ratones , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Succinatos/metabolismo , Vitamina B 12/metabolismo , Deficiencia de Vitamina B 12/inducido químicamente , Deficiencia de Vitamina B 12/complicacionesRESUMEN
Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been associated with the induction of oxidative stress and the progression of steatosis to steatohepatitis with fibrosis. It also disrupts metabolic pathways including one-carbon metabolism (OCM) and the transsulfuration pathway with possible consequences on glutathione (GSH) levels. In this study, complementary RNAseq and metabolomics data were integrated to examine the hepatic transsulfuration pathway and glutathione biosynthesis in mice following treatment with TCDD every 4 days for 28 days. TCDD dose-dependently repressed hepatic cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH) mRNA and protein levels. Reduced CBS and CTH levels are also correlated with dose-dependent decreases in hepatic extract hydrogen sulfide (H2S). In contrast, cysteine levels increased consistent with the induction of Slc7a11, which encodes for the cystine/glutamate Xc- antiporter. Cotreatment of primary hepatocytes with sulfasalazine, a cystine/glutamate Xc- antiporter inhibitor, decreased labeled cysteine incorporation into GSH with a corresponding increase in TCDD cytotoxicity. Although reduced and oxidized GSH levels were unchanged following treatment due to the induction of GSH/GSSG efflux transporter by TCDD, the GSH:GSSG ratio decreased and global protein S-glutathionylation levels in liver extracts increased in response to oxidative stress along with the induction of glutamate-cysteine ligase catalytic subunit (Gclc), glutathione synthetase (Gss), glutathione disulfide reductase (Gsr), and glutathione transferase π (Gstp). Furthermore, levels of ophthalmic acid, a biomarker of oxidative stress indicating GSH consumption, were also increased. Collectively, the data suggest that increased cystine transport due to cystine/glutamate Xc- antiporter induction compensated for decreased cysteine production following repression of the transsulfuration pathway to support GSH synthesis in response to TCDD-induced oxidative stress.
Asunto(s)
Hígado Graso , Dibenzodioxinas Policloradas , Ratones , Animales , Cisteína/metabolismo , Cistina , Disulfuro de Glutatión/metabolismo , Dibenzodioxinas Policloradas/farmacología , Ácido Glutámico , Antiportadores , Glutatión/metabolismoRESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known for mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the canonical mechanism of AhR activation involves heterodimerization with the aryl hydrocarbon receptor nuclear translocator, other transcriptional regulators that interact with AhR have been identified. Enrichment analysis of motifs in AhR-bound genomic regions implicated co-operation with COUP transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF4). The present study investigated AhR, HNF4α and COUP-TFII genomic binding and effects on gene expression associated with liver-specific function and cell differentiation in response to TCDD. Hepatic ChIPseq data from male C57BL/6 mice at 2 h after oral gavage with 30 µg/kg TCDD were integrated with bulk RNA-sequencing (RNAseq) time-course (2-72 h) and dose-response (0.01-30 µg/kg) datasets to assess putative AhR, HNF4α and COUP-TFII interactions associated with differential gene expression. Functional enrichment analysis of differentially expressed genes (DEGs) identified differential binding enrichment for AhR, COUP-TFII, and HNF4α to regions within liver-specific genes, suggesting intersections associated with the loss of liver-specific functions and hepatocyte differentiation. Analysis found that the repression of liver-specific, HNF4α target and hepatocyte differentiation genes, involved increased AhR and HNF4α binding with decreased COUP-TFII binding. Collectively, these results suggested TCDD-elicited loss of liver-specific functions and markers of hepatocyte differentiation involved interactions between AhR, COUP-TFII and HNF4α.
Asunto(s)
Factores de Transcripción COUP/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Genoma , Factor Nuclear 4 del Hepatocito/metabolismo , Hígado/metabolismo , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Secuencia de Bases , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Motivos de Nucleótidos/genética , Unión Proteica , RNA-Seq , Transcripción GenéticaRESUMEN
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent aryl hydrocarbon receptor (AhR) agonist that elicits a broad spectrum of dose-dependent hepatic effects including lipid accumulation, inflammation, and fibrosis. To determine the role of inflammatory lipid mediators in TCDD-mediated hepatotoxicity, eicosanoid metabolism was investigated. Female Sprague-Dawley (SD) rats were orally gavaged with sesame oil vehicle or 0.01-10 µg/kg TCDD every 4 days for 28 days. Hepatic RNA-Seq data was integrated with untargeted metabolomics of liver, serum, and urine, revealing dose-dependent changes in linoleic acid (LA) and arachidonic acid (AA) metabolism. TCDD also elicited dose-dependent differential gene expression associated with the cyclooxygenase, lipoxygenase, and cytochrome P450 epoxidation/hydroxylation pathways with corresponding changes in ω-6 (e.g. AA and LA) and ω-3 polyunsaturated fatty acids (PUFAs), as well as associated eicosanoid metabolites. Overall, TCDD increased the ratio of ω-6 to ω-3 PUFAs. Phospholipase A2 (Pla2g12a) was induced consistent with increased AA metabolism, while AA utilization by induced lipoxygenases Alox5 and Alox15 increased leukotrienes (LTs). More specifically, TCDD increased pro-inflammatory eicosanoids including leukotriene LTB4, and LTB3, known to recruit neutrophils to damaged tissue. Dose-response modeling suggests the cytochrome P450 hydroxylase/epoxygenase and lipoxygenase pathways are more sensitive to TCDD than the cyclooxygenase pathway. Hepatic AhR ChIP-Seq analysis found little enrichment within the regulatory regions of differentially expressed genes (DEGs) involved in eicosanoid biosynthesis, suggesting TCDD-elicited dysregulation of eicosanoid metabolism is a downstream effect of AhR activation. Overall, these results suggest alterations in eicosanoid metabolism may play a key role in TCDD-elicited hepatotoxicity associated with the progression of steatosis to steatohepatitis.
Asunto(s)
Eicosanoides/metabolismo , Ácidos Grasos Insaturados/metabolismo , Hígado/efectos de los fármacos , Dibenzodioxinas Policloradas/farmacología , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos Omega-3/metabolismo , Hígado Graso/metabolismo , Femenino , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Acetamide (CAS 60-35-5) is detected in common foods. Chronic rodent bioassays led to its classification as a group 2B possible human carcinogen due to the induction of liver tumors in rats. We used a toxicogenomics approach in Wistar rats gavaged daily for 7 or 28 days at doses of 300 to 1500 mg/kg/day (mkd) to determine a point of departure (POD) and investigate its mode of action (MoA). Ki67 labeling was increased at doses ≥750 mkd up to 3.3-fold representing the most sensitive apical endpoint. Differential gene expression analysis by RNA-Seq identified 1110 and 1814 differentially expressed genes in male and female rats, respectively, following 28 days of treatment. Down-regulated genes were associated with lipid metabolism while up-regulated genes included cell signaling, immune response, and cell cycle functions. Benchmark dose (BMD) modeling of the Ki67 labeling index determined the BMD10 lower confidence limit (BMDL10) as 190 mkd. Transcriptional BMD modeling revealed excellent concordance between transcriptional POD and apical endpoints. Collectively, these results indicate that acetamide is most likely acting through a mitogenic MoA, though specific key initiating molecular events could not be elucidated. A POD value of 190 mkd determined for cell proliferation is suggested for risk assessment purposes.
Asunto(s)
Acetamidas/toxicidad , Carcinógenos/toxicidad , Contaminación de Alimentos , Neoplasias Hepáticas/genética , Modelos Biológicos , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunidad/efectos de los fármacos , Inmunidad/genética , Antígeno Ki-67/análisis , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/patología , Masculino , RNA-Seq , Ratas , Ratas Wistar , Medición de Riesgo/métodos , Pruebas de Toxicidad Crónica/métodos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Acetamide (CAS 60-35-5) is classified by IARC as a Group 2B, possible human carcinogen, based on the induction of hepatocellular carcinomas in rats following chronic exposure to high doses. Recently, acetamide was found to be present in a variety of human foods, warranting further investigation. The regulatory body JECFA has previously noted conflicting reports on acetamide's ability to induce micronuclei (MN) in mice in vivo. To better understand the potential in vivo genotoxicity of acetamide, we performed acute MN studies in rats and mice, and a subchronic study in rats, the target species for liver cancer. In the acute exposure, animals were gavaged with water vehicle control, 250, 1000, or 2000â¯mg/kg acetamide, or the positive control (1â¯mg/kg mitomycin C). In the subchronic assay, bone marrow of rats gavaged at 1000â¯mg/kg/day (limit dose) for 28 days was evaluated. Both acute and subchronic exposures showed no change in the ratio of polychromatic to total erythrocytes (P/E) at any dose, nor was there any increase in the incidence of micronucleated polychromatic erythrocytes (MN-PCE). Potential mutagenicity of acetamide was evaluated in male rats gavaged with vehicle control or 1500â¯mg/kg/day acetamide using the in vivoPig-a gene mutation assay. There was no increase in mutant red blood cells or reticulocytes in acetamide-treated animals. In both acute and sub-chronic studies, elevated blood plasma acetamide in treated animals provided evidence of systemic exposure. We conclude based on this study that acetamide is not clastogenic, aneugenic, or mutagenic in vivo in rodent hematopoietic tissue warranting a formal regulatory re-evaluation.
Asunto(s)
Acetamidas/toxicidad , Acetamidas/sangre , Acetamidas/farmacocinética , Animales , Eritrocitos/efectos de los fármacos , Femenino , Contaminación de Alimentos , Masculino , Proteínas de la Membrana/genética , Ratones , Pruebas de Micronúcleos , Mutación , Ratas Wistar , Pruebas de Toxicidad SubcrónicaRESUMEN
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces hepatic oxidative stress following activation of the aryl hydrocarbon receptor (AhR). Our recent studies showed TCDD induced pyruvate kinase muscle isoform 2 (Pkm2) as a novel antioxidant response in normal differentiated hepatocytes. To investigate cooperative regulation between nuclear factor, erythroid derived 2, like 2 (Nrf2) and the AhR in the induction of Pkm2, hepatic chromatin immunoprecipitation sequencing (ChIP-seq) analyses were integrated with RNA sequencing (RNA-seq) time-course data from mice treated with TCDD for 2-168 hours. ChIP-seq analysis 2 hours after TCDD treatment identified genome-wide NRF2 enrichment. Approximately 842 NRF2-enriched regions were located in the regulatory region of differentially expressed genes (DEGs), whereas 579 DEGs showed both NRF2 and AhR enrichment. Sequence analysis of regions with overlapping NRF2 and AhR enrichment showed over-representation of either antioxidant or dioxin response elements, although 18 possessed both motifs. NRF2 exhibited negligible enrichment within a closed Pkm chromatin region, whereas the AhR was enriched 29-fold. Furthermore, TCDD induced Pkm2 in primary hepatocytes from wild-type and Nrf2-null mice, indicating NRF2 is not required. Although NRF2 and AhR cooperate to regulate numerous antioxidant gene expression responses, the induction of Pkm2 by TCDD is independent of reactive oxygen species-mediated NRF2 activation.
Asunto(s)
Redes Reguladoras de Genes/efectos de los fármacos , Hígado/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Dibenzodioxinas Policloradas/administración & dosificación , Piruvato Quinasa/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Antioxidantes/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Hepatocitos/citología , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Ratones , Estrés Oxidativo , Dibenzodioxinas Policloradas/farmacología , Unión Proteica , Análisis de Secuencia de ARNRESUMEN
Coagulation cascade activation and fibrin deposits have been implicated or observed in diverse forms of liver damage. Given that fibrin amplifies pathological inflammation in several diseases through the integrin receptor αMß2, we tested the hypothesis that disruption of the fibrin(ogen)-αMß2 interaction in Fibγ(390-396A) mice would reduce hepatic inflammation and fibrosis in an experimental setting of chemical liver injury. Contrary to our hypothesis, α-naphthylisothiocyanate (ANIT)-induced liver fibrosis increased in Fibγ(390-396A) mice, whereas inflammatory cytokine expression and hepatic necrosis were similar to ANIT-challenged wild-type (WT) mice. Increased fibrosis in Fibγ(390-396A) mice appeared to be independent of coagulation factor 13 (FXIII) transglutaminase, as ANIT challenge in FXIII-deficient mice resulted in a distinct pathological phenotype characterized by increased hepatic necrosis. Rather, bile duct proliferation underpinned the increased fibrosis in ANIT-exposed Fibγ(390-396A) mice. The mechanism of fibrin-mediated fibrosis was linked to interferon (IFN)γ induction of inducible nitric oxide synthase (iNOS), a gene linked to bile duct hyperplasia and liver fibrosis. Expression of iNOS messenger RNA was significantly increased in livers of ANIT-exposed Fibγ(390-396A) mice. Fibrin(ogen)-αMß2 interaction inhibited iNOS induction in macrophages stimulated with IFNγ in vitro and ANIT-challenged IFNγ-deficient mice had reduced iNOS induction, bile duct hyperplasia, and liver fibrosis. Further, ANIT-induced iNOS expression, liver fibrosis, and bile duct hyperplasia were significantly reduced in WT mice administered leukadherin-1, a small molecule that allosterically enhances αMß2-dependent cell adhesion to fibrin. These studies characterize a novel mechanism whereby the fibrin(ogen)-integrin-αMß2 interaction reduces biliary fibrosis and suggests a novel putative therapeutic target for this difficult-to-treat fibrotic disease.
Asunto(s)
1-Naftilisotiocianato/toxicidad , Conductos Biliares/metabolismo , Fibrina/metabolismo , Cirrosis Hepática Biliar/metabolismo , Antígeno de Macrófago-1/metabolismo , Animales , Benzoatos/farmacología , Conductos Biliares/patología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Femenino , Fibrina/genética , Humanos , Hiperplasia , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Cirrosis Hepática Biliar/inducido químicamente , Cirrosis Hepática Biliar/genética , Antígeno de Macrófago-1/genética , Masculino , Ratones , Ratones Noqueados , Necrosis , Tiohidantoínas/farmacologíaRESUMEN
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) agonists have been shown to regulate bone development and remodeling in a species-, ligand-, and age-specific manner, however the underlying mechanisms remain poorly understood. In this study, we characterized the effect of 0.01-30⯵g/kg TCDD on the femoral morphology of male and female juvenile mice orally gavaged every 4â¯days for 28â¯days and used RNA-Seq to investigate gene expression changes associated with the resultant phenotype. Micro-computed tomography revealed that TCDD dose-dependently increased trabecular bone volume fraction (BVF) 2.9- and 3.3-fold in male and female femurs, respectively. Decreased serum tartrate-resistant acid phosphatase (TRAP) levels, combined with a reduced osteoclast surface to bone surface ratio and repression of femoral proteases (cathepsin K, matrix metallopeptidase 13), suggests that TCDD impaired bone resorption. Increased osteoblast counts at the trabecular bone surface were consistent with a reciprocal reduction in the number of bone marrow adipocytes, suggesting AhR activation may direct mesenchymal stem cell differentiation towards osteoblasts rather than adipocytes. Notably, femoral expression of transmembrane glycoprotein NMB (Gpnmb; osteoactivin), a positive regulator of osteoblast differentiation and mineralization, was dose-dependently induced up to 18.8-fold by TCDD. Moreover, increased serum levels of 1,25-dihydroxyvitamin D3 were in accordance with the renal induction of 1α-hydroxylase Cyp27b1 and may contribute to impaired bone resorption. Collectively, the data suggest AhR activation tipped the bone remodeling balance towards bone formation, resulting in increased bone mass with reduced marrow adiposity.
Asunto(s)
Adiposidad/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Hueso Esponjoso/efectos de los fármacos , Fémur/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/biosíntesis , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Factores de Edad , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Médula Ósea/metabolismo , Médula Ósea/fisiopatología , Resorción Ósea/inducido químicamente , Resorción Ósea/metabolismo , Resorción Ósea/fisiopatología , Calcitriol/sangre , Hueso Esponjoso/diagnóstico por imagen , Hueso Esponjoso/metabolismo , Hueso Esponjoso/fisiopatología , Catepsina K/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas del Ojo/metabolismo , Femenino , Fémur/diagnóstico por imagen , Fémur/metabolismo , Fémur/fisiopatología , Riñón/enzimología , Masculino , Metaloproteinasa 13 de la Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones Endogámicos C57BL , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/metabolismo , Fosfatasa Ácida Tartratorresistente/sangre , Factores de Tiempo , Microtomografía por Rayos XRESUMEN
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated that B cells are directly impaired by TCDD, and are a sensitive target as evidenced by suppression of antibody responses. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be present within the initial 12h post B cell stimulation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized that TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 515 human, 2371 mouse and 712 rat orthologous genes over the 24-h period. 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with cell-cell signaling in humans, immune response in mice, and oxidation reduction in rats. Overall, these results suggest that despite the conservation of the AhR and its signaling mechanism, TCDD elicits species-specific gene expression changes.
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Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Contaminantes Ambientales/toxicidad , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/biosíntesis , Receptores de Hidrocarburo de Aril/genética , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Persistent aryl hydrocarbon receptor (AhR) agonists elicit dose-dependent hepatic lipid accumulation, oxidative stress, inflammation, and fibrosis in mice. Iron (Fe) promotes AhR-mediated oxidative stress by catalyzing reactive oxygen species (ROS) production. To further characterize the role of Fe in AhR-mediated hepatotoxicity, male C57BL/6 mice were orally gavaged with sesame oil vehicle or 0.01-30µg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4days for 28days. Duodenal epithelial and hepatic RNA-Seq data were integrated with hepatic AhR ChIP-Seq, capillary electrophoresis protein measurements, and clinical chemistry analyses. TCDD dose-dependently repressed hepatic expression of hepcidin (Hamp and Hamp2), the master regulator of systemic Fe homeostasis, resulting in a 2.6-fold increase in serum Fe with accumulating Fe spilling into urine. Total hepatic Fe levels were negligibly increased while transferrin saturation remained unchanged. Furthermore, TCDD elicited dose-dependent gene expression changes in heme biosynthesis including the induction of aminolevulinic acid synthase 1 (Alas1) and repression of uroporphyrinogen decarboxylase (Urod), leading to a 50% increase in hepatic hemin and a 13.2-fold increase in total urinary porphyrins. Consistent with this heme accumulation, differential gene expression suggests that heme activated BACH1 and REV-ERBα/ß, causing induction of heme oxygenase 1 (Hmox1) and repression of fatty acid biosynthesis, respectively. Collectively, these results suggest that Hamp repression, Fe accumulation, and increased heme levels converge to promote oxidative stress and the progression of TCDD-elicited hepatotoxicity.
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Hemo/metabolismo , Hepcidinas/deficiencia , Sobrecarga de Hierro/metabolismo , Hígado/metabolismo , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/toxicidad , Femenino , Hemo/genética , Hepcidinas/genética , Sobrecarga de Hierro/genética , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/genética , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Represoras/genéticaRESUMEN
The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces hepatic steatosis mediated by the aryl hydrocarbon receptor. To further characterize TCDD-elicited hepatic lipid accumulation, mice were gavaged with TCDD every 4 days for 28 days. Liver samples were examined using untargeted lipidomics with structural confirmation of lipid species by targeted high-resolution MS/MS, and data were integrated with complementary RNA-Seq analyses. Approximately 936 unique spectral features were detected, of which 379 were confirmed as unique lipid species. Both male and female samples exhibited similar qualitative changes (lipid species) but differed in quantitative changes. A shift to higher mass lipid species was observed, indicative of increased free fatty acid (FFA) packaging. For example, of the 13 lipid classes examined, triglycerides increased from 46 to 48% of total lipids to 68-83% in TCDD treated animals. Hepatic cholesterol esters increased 11.3-fold in male mice with moieties consisting largely of dietary fatty acids (FAs) (i.e., linolenate, palmitate, and oleate). Phosphatidylserines, phosphatidylethanolamines, phosphatidic acids, and cardiolipins decreased 4.1-, 5.0-, 5.4- and 7.4-fold, respectively, while ceramides increased 6.6-fold. Accordingly, the integration of lipidomic data with differential gene expression associated with lipid metabolism suggests that in addition to the repression of de novo fatty acid synthesis and ß-oxidation, TCDD also increased hepatic uptake and packaging of lipids, while inhibiting VLDL secretion, consistent with hepatic fat accumulation and the progression to steatohepatitis with fibrosis.
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Dibenzodioxinas Policloradas/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Cardiolipinas/metabolismo , Ceramidas/metabolismo , Colesterol/biosíntesis , Ácidos Grasos/análisis , Hígado Graso/metabolismo , Hígado Graso/patología , Femenino , Expresión Génica/efectos de los fármacos , Lipoproteínas VLDL/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ácidos Fosfatidicos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Dibenzodioxinas Policloradas/química , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/genética , Espectrometría de Masas en Tándem , Triglicéridos/análisis , Triglicéridos/metabolismoRESUMEN
Inhalation exposures to ozone commonly encountered in photochemical smog cause airway injury and inflammation. Elevated ambient ozone concentrations have been epidemiologically associated with nasal airway activation of neutrophils and eosinophils. In the present study, we elucidated the temporal onset and lymphoid cell dependency of eosinophilic rhinitis and associated epithelial changes in mice repeatedly exposed to ozone. Lymphoid cell-sufficient C57BL/6 mice were exposed to 0 or 0.5 parts per million (ppm) ozone for 1, 2, 4, or 9 consecutive weekdays (4 h/d). Lymphoid cell-deficient, Rag2(-/-)Il2rg(-/-) mice were similarly exposed for 9 weekdays. Nasal tissues were taken at 2 or 24 hours after exposure for morphometric and gene expression analyses. C57BL/6 mice exposed to ozone for 1 day had acute neutrophilic rhinitis, with airway epithelial necrosis and overexpression of mucosal Ccl2 (MCP-1), Ccl11 (eotaxin), Cxcl1 (KC), Cxcl2 (MIP-2), Hmox1, Il1b, Il5, Il6, Il13, and Tnf mRNA. In contrast, 9-day ozone exposure elicited type 2 immune responses in C57BL/6 mice, with mucosal mRNA overexpression of Arg1, Ccl8 (MCP-2), Ccl11, Chil4 (Ym2), Clca1 (Gob5), Il5, Il10, and Il13; increased density of mucosal eosinophils; and nasal epithelial remodeling (e.g., hyperplasia/hypertrophy, mucous cell metaplasia, hyalinosis, and increased YM1/YM2 proteins). Rag2(-/-)Il2rg(-/-) mice exposed to ozone for 9 days, however, had no nasal pathology or overexpression of transcripts related to type 2 immunity. These results provide a plausible paradigm for the activation of eosinophilic inflammation and type 2 immunity found in the nasal airways of nonatopic individuals subjected to episodic exposures to high ambient ozone.
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Eosinofilia/inmunología , Inmunidad Mucosa , Linfocitos/inmunología , Mucosa Nasal/inmunología , Ozono , Rinitis/inmunología , Animales , Citocinas/genética , Citocinas/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Eosinofilia/inducido químicamente , Eosinofilia/genética , Eosinofilia/metabolismo , Regulación de la Expresión Génica , Genotipo , Mediadores de Inflamación/metabolismo , Exposición por Inhalación , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Linfocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mucosa Nasal/metabolismo , Fenotipo , ARN Mensajero/metabolismo , Rinitis/inducido químicamente , Rinitis/genética , Rinitis/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
BACKGROUND: Dose-dependent differential gene expression provides critical information required for regulatory decision-making. The lower costs associated with RNA-Seq have made it the preferred technology for transcriptomic analysis. However, concordance between RNA-Seq and microarray analyses in dose response studies has not been adequately vetted. RESULTS: We compared the hepatic transcriptome of C57BL/6 mice following gavage with sesame oil vehicle, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 µg/kg TCDD every 4 days for 28 days using Illumina HiSeq RNA-Sequencing (RNA-Seq) and Agilent 4 × 44 K microarrays using the same normalization and analysis approach. RNA-Seq and microarray analysis identified a total of 18,063 and 16,403 genes, respectively, that were expressed in the liver. RNA-Seq analysis for differentially expressed genes (DEGs) varied dramatically depending on the P1(t) cut-off while microarray results varied more based on the fold change criteria, although responses strongly correlated. Verification by WaferGen SmartChip QRTPCR revealed that RNA-Seq had a false discovery rate of 24% compared to 54% for microarray analysis. Dose-response modeling of RNA-Seq and microarray data demonstrated similar point of departure (POD) and ED50 estimates for common DEGs. CONCLUSIONS: There was a strong correspondence between RNA-Seq and Agilent array transcriptome profiling when using the same samples and analysis strategy. However, RNA-Seq provided superior quantitative data, identifying more genes and DEGs, as well as qualitative information regarding identity and annotation for dose response modeling in support of regulatory decision-making.
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Perfilación de la Expresión Génica/métodos , Hígado/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Dibenzodioxinas Policloradas/administración & dosificación , Análisis de Secuencia de ARN/métodos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Dibenzodioxinas Policloradas/toxicidadRESUMEN
High-resolution digitalizing of histology slides facilitates the development of computational alternatives to manual quantitation of features of interest. We developed a MATLAB-based quantitative histological analysis tool (QuHAnT) for the high-throughput assessment of distinguishable histological features. QuHAnT validation was demonstrated by comparison with manual quantitation using liver sections from mice orally gavaged with sesame oil vehicle or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0.001-30 µg/kg) every 4 days for 28 days, which elicits hepatic steatosis with mild fibrosis. A quality control module of QuHAnT reduced the number of quantifiable Oil Red O (ORO)-stained images from 3,123 to 2,756. Increased ORO staining was measured at 10 and 30 µg/kg TCDD with a high correlation between manual and computational volume densities (Vv ), although the dynamic range of QuHAnT was 10-fold greater. Additionally, QuHAnT determined the size of each ORO vacuole, which could not be accurately quantitated by visual examination or manual point counting. PicroSirius Red quantitation demonstrated superior collagen deposition detection due to the ability to consider all images within each section. QuHAnT dramatically reduced analysis time and facilitated the comprehensive assessment of features improving accuracy and sensitivity and represents a complementary tool for tissue/cellular features that are difficult and tedious to assess via subjective or semiquantitative methods.
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Biología Computacional/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Técnicas Histológicas , Toxicología/métodos , Animales , Compuestos Azo , Colágeno/metabolismo , Colorantes , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/toxicidad , Hígado Graso/inducido químicamente , Hígado Graso/patología , Femenino , Fibrosis , Ratones , Ratones Endogámicos C57BL , Dibenzodioxinas Policloradas/toxicidad , Vacuolas/patologíaRESUMEN
Irritable bowel syndrome and related disorders of gut-brain interaction (DGBI) are common and exhibit a complex, poorly understood etiology that manifests as abnormal gut motility and pain. Risk factors such as biological sex, stressors during critical periods, and inflammation are thought to influence DGBI vulnerability by reprogramming gut-brain circuits, but the specific cells affected are unclear. Here, we used a model of early life stress to understand cellular mechanisms in the gut that produce DGBIs. Our findings identify enteric glia as a key cellular substrate in which stress and biological sex converge to dictate DGBI susceptibility. Enteric glia exhibit sexual dimorphism in genes and functions related to cellular communication, inflammation, and disease susceptibility. Experiencing early life stress has sex-specific effects on enteric glia that cause a phenotypic switch in male glia toward a phenotype normally observed in females. This phenotypic transformation is followed by physiological changes in the gut, mirroring those observed in DGBI in humans. These effects are mediated, in part, by alterations to glial prostaglandin and endocannabinoid signaling. Together, these data identify enteric glia as a cellular integration site through which DGBI risk factors produce changes in gut physiology and suggest that manipulating glial signaling may represent an attractive target for sex-specific therapeutic strategies in DGBIs.