Bioimmunoassay of Aryl Hydrocarbon (Ah) Receptor Transformation in Vitro by 2,3,7,8- Tetrachlorodibenzo-p-Dioxin(TCDD)

1996 ◽  
Vol 6 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Geoffrey D. Wheelock ◽  
Kelley R. Hurst ◽  
John G. Babish
Keyword(s):  
Ah Receptor ◽  
Aryl Hydrocarbon ◽  
Tetrachlorodibenzo P Dioxin

scholarly journals Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist

2018 ◽  
Vol 19 (9) ◽  
pp. 2692 ◽  
Author(s):  
Samantha Faber ◽  
Anatoly Soshilov ◽  
Sara Giani Tagliabue ◽  
Laura Bonati ◽  
Michael Denison
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Ligand Binding ◽  
Species Differences ◽  
In Silico Analysis ◽  
Site Directed Mutagenesis ◽  
Ah Receptor ◽  
Aryl Hydrocarbon ◽  
Silico Analysis

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

scholarly journals Aryl Hydrocarbon Receptor Activation Impairs Cortisol Response to Stress in Rainbow Trout by Disrupting the Rate-Limiting Steps in Steroidogenesis

Endocrinology ◽  
2006 ◽  
Vol 147 (4) ◽  
pp. 1895-1903 ◽  
Author(s):  
Neelakanteswar Aluru ◽  
Mathilakath M. Vijayan
Keyword(s):  
Regulatory Protein ◽  
Receptor Activation ◽  
Control Fish ◽  
Ah Receptor ◽  
Receptor Signaling ◽  
Aryl Hydrocarbon ◽  
Response To Stress ◽  
Rate Limiting

Anthropogenic stressors activating aryl hydrocarbon (Ah) receptor signaling, including polychlorinated biphenyls, impair the adaptive corticosteroid response to stress, but the mechanisms involved are far from clear. Using Ah receptor agonist (β-naphthoflavone; BNF) and antagonist (resveratrol; RVT), we tested the hypothesis that steroidogenic pathway is a target for endocrine disruption by xenobiotics activating Ah receptor signaling. Trout (Oncorhynchus mykiss) were fed BNF (10 mg/kg·d), RVT (20 mg/kg·d) or a combination of both for 5 d, and subjected to a handling disturbance. BNF induced cytochrome P4501A1 expression in the interrenal tissue and liver, whereas this response was abolished by RVT, confirming Ah receptor activation. In control fish, handling disturbance transiently elevated plasma cortisol and glucose levels and transcript levels of interrenal steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage (P450scc) and 11β-hydroxylase over a 24-h period. BNF treatment attenuated this stressor-induced plasma and interrenal responses; these BNF-mediated responses were reverted back to the control levels in the presence of RVT. We further examined whether these in vivo impacts of BNF on steroidogenesis can be mimicked in vitro using interrenal tissue preparations. BNF depressed ACTH-mediated cortisol production, and this decrease corresponded with lower StAR and P450scc, but not 11β-hydroxylase mRNA abundance. RVT eliminated this BNF-mediated depression of interrenal corticosteroidogenesis in vitro. Altogether, xenobiotics activating Ah receptor signaling are steroidogenic disruptors, and the mode of action includes inhibition of StAR and P450scc, the rate-limiting steps in steroidogenesis.

The aromatic hydrocarbon receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and variable levels of induced aryl hydrocarbon hydroxylase activity in clones of mouse hepatoma cells

1988 ◽  
Vol 66 (12) ◽  
pp. 1278-1286 ◽  
Author(s):  
Cynthia J. Forster-Gibson ◽  
Yu Xiao Fei ◽  
Michael J. Dufresne
Keyword(s):  
Ah Receptor ◽  
Aryl Hydrocarbon Hydroxylase ◽  
Relative Level ◽  
Coordinate Measurement ◽  
Aromatic Hydrocarbon Receptor ◽  
Aryl Hydrocarbon ◽  
Mouse Hepatoma ◽  
Receptor Sites ◽  
Aryl Hydrocarbon Hydroxylase Activity ◽  
Tetrachlorodibenzo P Dioxin

Induction of aryl hydrocarbon hydroxylase (AHH) activity was studied in clones and subclones of mouse hepatoma (Hepa-lcl) cells. When maximally induced, one clone had significantly lower (p < 0.005), two had approximately the same, and two had significantly higher (p < 0.005) levels of AHH activity compared with Hepa-lcl. The maximal level of induced activity, relative to the parent population, in two clones chosen for further analysis was 0.14 ± 0.09 for clone 1 (Hs-1) and 0.94 ± 0.28 for clone 9 (Hs-9). These relative levels were stable over a period of 10 months and were similar when activity was induced either with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz[a]anthracene. Subclones of Hepa-lcl cells, derived from the Hs-9 clone, also demonstrated variation in induced AHH activity. When maximally induced with TCDD, six subclones had significantly lower AHH activity (p < 0.005), two had approximately the same, and one had significantly higher levels (p < 0.005) compared with the progenitor Hs-9 population. Comparative analysis of Ah receptor characteristics in two unselected clones of Hepa-lcl with significantly different levels of AHH activity demonstrated that there was no apparent correlation between relative level of induced AHH activity and (i) total quantity of Ah receptor (cytosol and nuclear), (ii) receptor affinity for TCDD and number of receptor sites in each cell, (iii) subcellular distribution of [3H]TCDD, or (iv) specificity and saturable nature of binding. Coordinate measurement of the concentration of nuclear receptor and absolute induced AHH activity in Hepa-lcl and its clones had a positive correlation (r = 0.79).

scholarly journals Isolation and characterization of revertants from four different classes of aryl hydrocarbon hydroxylase-deficient hepa-1 mutants.

1984 ◽  
Vol 4 (8) ◽  
pp. 1597-1604 ◽  
Author(s):  
J R Van Gurp ◽  
O Hankinson
Keyword(s):  
Enzyme Stability ◽  
Ah Receptor ◽  
Wild Type ◽  
Aryl Hydrocarbon Hydroxylase ◽  
Gene Group ◽  
Aryl Hydrocarbon ◽  
Isolation And Characterization ◽  
Dominant Mutant

Revertants were selected from aryl hydrocarbon hydroxylase (AHH)-deficient recessive mutants belonging to three complementation groups and from a dominant mutant of the Hepa-1 cell line. The recessive mutants had low spontaneous reversion frequencies (less than 4 X 10(-7] that were increased by mutagenesis. The majority of these revertants also had reacquired only partial AHH activity. Revertants of group A mutants were identical to the wild type with respect to both in vivo and in vitro enzyme stability and the Km for the substrate, benzo [alpha]pyrene, and therefore failed to provide evidence that gene A is the AHH structural gene. Group B and group C mutants are defective in the functioning of the Ah receptor required for AHH induction. Revertants of these groups were normal with respect to in vivo temperature sensitivity for AHH induction and for the 50% effective dose for the inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and thus provided no evidence that the B and C genes code for components of the receptor. Two rare group C revertants possessed AHH activity in the absence of induction. The phenotype of one of these was shown to be recessive to the wild type. Spontaneous revertants of the dominant mutant occurred at a frequency 300-fold greater than those of the recessive mutants, and this frequency was not increased by mutagenesis. These revertants all displayed complete restoration of AHH activity to wild type levels. These observations and the results from cell hybridization studies suggest that the dominant revertants arose by a high frequency event leading to functional elimination of the dominant mutation.

scholarly journals Nucleoside triphosphates promote the transformation of Ah receptor to its DNA-binding form

1991 ◽  
Vol 274 (2) ◽  
pp. 401-404 ◽  
Author(s):  
A J Cary ◽  
J J Dougherty
Keyword(s):  
Dna Binding ◽  
Aryl Hydrocarbon Receptor ◽  
Ah Receptor ◽  
Nucleotide Hydrolysis ◽  
Aryl Hydrocarbon ◽  
Low Concentrations ◽  
Nucleoside Triphosphates ◽  
Tetrachlorodibenzo P Dioxin ◽  
Specific Ligand

When it is bound to a specific ligand such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, mild heating can convert the Ah (aryl hydrocarbon) receptor into a form capable of binding DNA. We found that physiological (1-3 mM) levels of ATP substantially increased the transformation of the receptor to its DNA-binding form. GTP, UTP and CTP had similar effects. ADP also promoted this transformation, but was less effective than ATP at low concentrations. Pyrophosphate too promoted transformation, but AMP had little effect. The process did not require nucleotide hydrolysis, since non-hydrolysable analogues of ATP such as adenosine 5′-[beta gamma-imido]triphosphate were nearly as effective as ATP itself. Inhibitors of ATP-stimulated proteases did not significantly affect the ability of ATP to promote receptor transformation, which suggests that the effect of ATP was not mediated by these proteases.

Sign in / Sign up

Export Citation Format

Share Document