RESUMO
The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biologic and toxicologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we utilized two AhR-dependent bioassay systems as screening tools to identify novel AhR agonists and to detect the presence of AhR agonists in sample extracts. These assays measure the ability of a chemical to activate AhR DNA binding in vitro (GRAB bioassay) or AhR-dependent (luciferase) gene expression in cultured cells (CALUX bioassay). Known AhR agonists (halogenated and nonhalogenated aromatic hydrocarbons) were positive in both assays, whereas the AhR antagonist alpha-naphthoflavone exhibited agonist activity only in the GRAB assay. In vitro GRAB analysis has identified several imidazoline receptor ligands and beta-carbolines as AhR agonists and also revealed the presence of AhR agonist activity in crude DMSO extracts of commercial newspapers. In contrast to their positive activity in the GRAB assay, the majority of these chemicals/extracts were only weakly active or inactive in the cell-based CALUX assay. Our results not only reveal that the ability of a chemical to activate the AhR in vitro does not necessarily correlate with its ability to induce gene expression in intact cells, but the high level of false positives obtained with the GRAB assay clearly demonstrates its inability to accurately identify AhR agonists or agonist activity. Screening of unknown chemicals, chemical classes, and samples for AhR agonist activity will require the use of intact cell bioassays.
Assuntos
Receptores de Hidrocarboneto Arílico/agonistas , Animais , Bioensaio , Carcinógenos/toxicidade , Células Cultivadas , Cromatografia em Gel , Citosol/efeitos dos fármacos , Citosol/metabolismo , Cobaias , Imidazóis/toxicidade , Luciferases/biossíntese , Luciferases/genética , Masculino , Oligonucleotídeos/farmacologia , Papel , Dibenzodioxinas Policloradas/farmacologia , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Proteínas Recombinantes/química , Fatores de TempoRESUMO
The metabolism and distribution of newly synthesized aggrecan present in the extracellular matrix of intact explant cultures of mature articular cartilage was investigated with respect to type VI collagen-stained chondrons. Using biochemical, autoradiographical and novel confocal immunohistochemical techniques it was shown that aggrecan exists as a number of distinct pools that are located within the extracellular matrix of the tissue. The first was identified as a pool of high specific radioactivity, much of which appeared in the medium one day after incubation with radiolabeled sulfate. Of the radiolabeled aggrecan remaining within the extracellular matrix, three pools were differentiated on the basis of time and location within the extracellular matrix. One pool was resident within the pericellular microenvironment associated with the chondron, one migrated into the territorial matrix adjacent to the chondron and one was sequestered long term in the interterritorial matrix. Analysis of the kinetics of loss of radiolabeled aggrecan macromolecules present in the region of matrix defined by the chondron suggests that this pool rapidly turns over and is a precursor to the pools of aggrecan present in the territorial and interterritorial matrix. There were marked differences in the distribution of newly synthesized aggrecan present in these regions of the extracellular matrix in explant cultures maintained with or without fetal calf serum. In the absence of serum, more of the newly synthesized aggrecan moved into the territorial and interterritorial matrix indicating that the presence of serum in the culture medium influenced the tissue distribution of aggrecan. This work indicates that the pericellular microenvironment of the chondron plays an important role in the retention and maturation of aggrecan prior to the sequestration of aggrecan complexes into the functional load bearing matrices of adult articular cartilage.
Assuntos
Cartilagem Articular/metabolismo , Colágeno/metabolismo , Proteínas da Matriz Extracelular , Proteoglicanas/metabolismo , Agrecanas , Animais , Autorradiografia , Cartilagem Articular/citologia , Cartilagem Articular/cirurgia , Bovinos , Meios de Cultura , Matriz Extracelular/metabolismo , Lectinas Tipo C , Soroalbumina Bovina , Radioisótopos de Enxofre , Fatores de TempoRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a variety of hydrophobic natural and synthetic chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Induction of CYP1A1 gene expression is one such response that is known to be regulated by the AhR complex. It was recently reported (Ledirac et al., Toxicol. Appl. Pharmacol. 144, 177-182, 1997) that, although carbaryl, a carbamate insecticide, can induce AhR-dependent expression of CYP1A1, it was not an AhR ligand. Since this apparent ligand-independent activation of the AhR is difficult to reconcile given what is known about the mechanism of AhR action, we have examined the ability of carbaryl to stimulate the AhR signaling pathway. Not only was dioxin responsive element-driven luciferase gene expression induced by carbaryl in stably transfected mouse, rat, guinea pig, and human cells, gel retardation analysis revealed that carbaryl stimulated AhR transformation and DNA binding in vitro and in cells in culture. Dose-response experiments revealed that carbaryl was 300,000-fold less potent that the prototypical inducer, TCDD, in both inducing luciferase gene expression and stimulating AhR transformation and DNA binding in vitro, suggesting that carbaryl itself was the inducing agent. The identification of carbaryl as an AhR ligand was demonstrated by its ability to competitively inhibit [3H]-TCDD to the guinea pig hepatic cytosolic AhR. Our results confirm that carbaryl is both a weak AhR ligand and inducer of AhR-dependent gene expression and argue against its proposed ligand-independent mechanism of AhR activation.
Assuntos
Carbaril/metabolismo , Expressão Gênica/efeitos dos fármacos , Inseticidas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Ligação Competitiva , Carbaril/farmacologia , Proteínas de Ligação a DNA/metabolismo , Dioxinas/farmacologia , Genes Reporter , Cobaias , Humanos , Inseticidas/farmacologia , Ligantes , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Ratos , Receptores de Hidrocarboneto Arílico/genética , Células Tumorais CultivadasRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Although no endogenous physiological ligand for the AhR has yet been described, persistent expression of hepatic CYP1A1 gene expression (an AhR-dependent response) in congenitally jaundiced Gunn rats indirectly supports the existence of such a ligand(s) in these animals. High plasma levels of the heme degradation product bilirubin (BR) in these animals prompted us to evaluate whether BR is an endogenous AhR agonist. Expression of dioxin responsive element (DRE)-driven luciferase gene expression in stably transfected mouse, guinea pig, rat, and human cells was induced by treatment with physiological concentrations of BR. Biliverdin (BV), the metabolic precursor of bilirubin, also induced luciferase activity in all species. BR and BV not only stimulated AhR transformation and DRE binding in vitro andin cells in culture, but competitive inhibition of [3H]TCDD-specific binding to the cytosolic AhR revealed that these chemicals are AhR ligands. The significantly greater inducing potency of these chemicals in intact cells, compared to their ligand binding and AhR transformation potency in vitro, suggests that BR and BV may also be converted within the cell to a more potent activator(s). Our results demonstrate that the heme degradation products BR and BV are AhR ligands which can regulate the AhR-dependent gene expression pathway.