The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates a wide range of biological and toxicological effects that result from exposure to a structurally diverse variety of ...synthetic and naturally occurring chemicals. Although the overall mechanism of action of the AhR has been extensively studied and involves a classical nuclear receptor mechanism of action (i.e., ligand-dependent nuclear localization, protein heterodimerization, binding of liganded receptor as a protein complex to its specific DNA recognition sequence and activation of gene expression), details of the exact molecular events that result in most AhR-dependent biochemical, physiological, and toxicological effects are generally lacking. Ongoing research efforts continue to describe an ever-expanding list of ligand-, species-, and tissue-specific spectrum of AhR-dependent biological and toxicological effects that seemingly add even more complexity to the mechanism. However, at the same time, these studies are also identifying and characterizing new pathways and molecular mechanisms by which the AhR exerts its actions and plays key modulatory roles in both endogenous developmental and physiological pathways and response to exogenous chemicals. Here we provide an overview of the classical and nonclassical mechanisms that can contribute to the differential sensitivity and diversity in responses observed in humans and other species following ligand-dependent activation of the AhR signal transduction pathway.
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse chemicals. To examine the mechanisms responsible for the promiscuity in ...AhR ligand binding, we determined the effects of mutations within the AhR ligand-binding domain (LBD) on the activity of diverse AhR ligands. Site-directed mutagenesis identified Ile319 of the mouse AhR and, to a lesser extent, Phe318 as residues involved in ligand-selective modulation of AhR transformation using a panel of 12 AhR ligands. These ligands could be categorized into four distinct structurally related groups based on their ability to activate AhR mutants at position 319 in vitro. The mutation I319K was selectively activated by FICZ and not by other examined ligands in vitro and in cell culture. F318L and F318A mutations resulted in the conversion of AhR agonists β-naphthoflavone and 3-methylcholanthrene, respectively, into partial agonists/antagonists. Hsp90 binding to the AhR was decreased with several mutations and was inversely correlated with AhR ligand-binding promiscuity. Together, these data define overlapping amino acid residues within the AhR LBD involved in the selectivity of ligand binding, the agonist or antagonist mode of ligand binding, and hsp90 binding and provide insights into the ligand diversity of AhR activators.
Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with ...primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34⁺ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.
The induction of expression of genes for xenobiotic metabolizing enzymes in response to chemical insult is an adaptive response found in most organisms. In vertebrates, the AhR is one of several ...chemical/ligand-dependent intracellular receptors that can stimulate gene transcription in response to xenobiotics. The ability of the AhR to bind and be activated by a range of structurally divergent chemicals suggests that the AhR contains a rather promiscuous ligand binding site. In addition to synthetic and environmental chemicals, numerous naturally occurring dietary and endogenous AhR ligands have also been identified. In this review, we describe evidence for the structural promiscuity of AhR ligand binding and discuss the current state of knowledge with regards to the activation of the AhR signaling pathway by naturally occurring exogenous and endogenous ligands.
In 2011, a joint World Health Organization (WHO) and United Nations Environment Programme (UNEP) expert consultation took place, during which the possible inclusion of brominated analogues of the ...dioxin-like compounds in the WHO Toxicity Equivalency Factor (TEF) scheme was evaluated. The expert panel concluded that polybrominated dibenzo-p-dioxins (PBDDs), dibenzofurans (PBDFs), and some dioxin-like biphenyls (dl-PBBs) may contribute significantly in daily human background exposure to the total dioxin toxic equivalencies (TEQs). These compounds are also commonly found in the aquatic environment. Available data for fish toxicity were evaluated for possible inclusion in the WHO-UNEP TEF scheme (van den Berg et al., 1998). Because of the limited database, it was decided not to derive specific WHO-UNEP TEFs for fish, but for ecotoxicological risk assessment, the use of specific relative effect potencies (REPs) from fish embryo assays is recommended. Based on the limited mammalian REP database for these brominated compounds, it was concluded that sufficient differentiation from the present TEF values of the chlorinated analogues (van den Berg et al., 2006) was not possible. However, the REPs for PBDDs, PBDFs, and non-ortho dl-PBBs in mammals closely follow those of the chlorinated analogues, at least within one order of magnitude. Therefore, the use of similar interim TEF values for brominated and chlorinated congeners for human risk assessment is recommended, pending more detailed information in the future.
Surface water can contain countless organic micropollutants, and targeted chemical analysis alone may only detect a small fraction of the chemicals present. Consequently, bioanalytical tools can be ...applied complementary to chemical analysis to detect the effects of complex chemical mixtures. In this study, bioassays indicative of activation of the aryl hydrocarbon receptor (AhR), activation of the pregnane X receptor (PXR), activation of the estrogen receptor (ER), adaptive stress responses to oxidative stress (Nrf2), genotoxicity (p53) and inflammation (NF-κB) and the fish embryo toxicity test were applied along with chemical analysis to water extracts from the Danube River. Mixture-toxicity modeling was applied to determine the contribution of detected chemicals to the biological effect. Effect concentrations for between 0 to 13 detected chemicals could be found in the literature for the different bioassays. Detected chemicals explained less than 0.2% of the biological effect in the PXR activation, adaptive stress response, and fish embryo toxicity assays, while five chemicals explained up to 80% of ER activation, and three chemicals explained up to 71% of AhR activation. This study highlights the importance of fingerprinting the effects of detected chemicals.
The aryl hydrocarbon (dioxin) receptor (AhR) is a ligand-dependent transcription factor that produces a wide range of biological and toxic effects in many species and tissues. Whereas the ...best-characterized high-affinity ligands include structurally related halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs), the AhR is promiscuous and can also be activated by structurally diverse exogenous and endogenous chemicals. However, little is known about how these diverse ligands actually bind to and activate the AhR. Utilizing AhR ligand binding, DNA binding, and reporter gene expression assays, we have identified a novel ligand-selective antagonist (CH223191) that preferentially inhibits the ability of some classes of AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin and related HAHs), but not others (PAHs, flavonoids, or indirubin), to bind to and/or activate the AhR and AhR signal transduction. HAH-specific antagonism of AhR-dependent reporter gene expression by CH223191 was observed with mouse, rat, human, and guinea pig cell lines. Ligand- and species-selective antagonism was also observed with the AhR antagonists 3′-methoxy-4′-nitroflavone and 6,2′,4′,-trimethoxyflavone. Our results suggest that the differences in the binding by various ligands to the AhR contribute to the observed structural diversity of AhR ligands and could contribute in ligand-specific variation in AhR functionality and the toxic and biological effects of various classes of AhR agonists.
Diverse organic compounds, many derived from consumer products, are found in sewage sludge worldwide. Understanding which of these poses the most significant environmental threat following land ...application can be investigated through a variety of predictive and cell-based toxicological techniques. Nontargeted analysis using high-resolution mass spectrometry with predictive estrogenic activity modeling was performed on sewage sludge samples from 12 wastewater treatment plants in California. Diisobutyl phthalate and dextrorphan were predicted to exhibit estrogenic activity and identified in >75% of sludge samples, signifying their universal presence and persistence. Additionally, the application of an estrogen-responsive cell bioassay revealed reductions in agonistic activity during mesophilic and thermophilic treatment but significant increases in antagonism during thermophilic treatment, which warrants further research. Ten nontarget features were identified (metoprolol, fenofibric acid, erythrohydrobupropion, oleic acid, mestranol, 4′-chlorobiphenyl-2,3-diol, medrysone, scillarenin, sudan I, and N,O-didesmethyltramadol) in treatment set samples and are considered to have influenced the in vitro estrogenic activity observed. The combination of predictive and in vitro estrogenicity with nontargeted analysis has led to confirmation of 12 estrogen-active contaminants in California sewage sludge and has highlighted the importance of evaluating both agonistic and antagonistic responses when evaluating the bioactivity of complex samples.
Patients with chronic kidney disease (CKD) are exposed to uremic toxins and have an increased risk of cardiovascular disease. Some uremic toxins, like indoxyl sulfate, are agonists of the ...transcription factor aryl hydrocarbon receptor (AHR). These toxins induce a vascular procoagulant phenotype. Here we investigated AHR activation in patients with CKD and in a murine model of CKD. We performed a prospective study in 116 patients with CKD stage 3 to 5D and measured the AHR-Activating Potential of serum by bioassay. Compared to sera from healthy controls, sera from CKD patients displayed a strong AHR-Activating Potential; strongly correlated with eGFR and with the indoxyl sulfate concentration. The expression of the AHR target genes Cyp1A1 and AHRR was up-regulated in whole blood from patients with CKD. Survival analyses revealed that cardiovascular events were more frequent in CKD patients with an AHR-Activating Potential above the median. In mice with 5/6 nephrectomy, there was an increased serum AHR-Activating Potential, and an induction of Cyp1a1 mRNA in the aorta and heart, absent in AhR–/– CKD mice. After serial indoxyl sulfate injections, we observed an increase in serum AHR-AP and in expression of Cyp1a1 mRNA in aorta and heart in WT mice, but not in AhR–/– mice. Thus, the AHR pathway is activated both in patients and mice with CKD. Hence, AHR activation could be a key mechanism involved in the deleterious cardiovascular effects observed in CKD.
Quaternary ammonium salts (QUATS), such as cetylpyridinium chloride (CPC) and benzalkonium chloride (BAK), are frequently used in antiseptic formulations, including toothpastes, mouthwashes, ...lozenges, throat and nasal sprays, and as biocides. Although in a recent ruling, the U.S. Food and Drug Administration (FDA) banned CPC from certain products and requested more data on BAK's efficacy and safety profile, QUATS, in general, and CPC and BAK, in particular, continue to be used in personal health care, food, and pharmaceutical and cleaning industries.
We aimed to assess CPC's effects on mitochondrial toxicity and endocrine disruption
.
Mitochondrial O
consumption and adenosine triphosphate (ATP) synthesis rates of osteosarcoma cybrid cells were measured before and after CPC and BAK treatment. Antiestrogenic effects of the compounds were measured by a luciferase-based assay using recombinant human breast carcinoma cells (VM7Luc4E2, ERalpha-positive).
CPC inhibited both mitochondrial O
consumption half maximal inhibitory concentration (IC
): 3.8μM and ATP synthesis (IC
: 0.9μM), and additional findings supported inhibition of mitochondrial complex 1 as the underlying mechanism for these effects. In addition, CPC showed concentration-dependent antiestrogenic activity half maximal effective concentration (EC
): 4.5μM). BAK, another antimicrobial QUATS that is structurally similar to CPC, and the pesticide rotenone, a known complex 1 inhibitor, also showed mitochondrial inhibitory and antiestrogenic effects. In all three cases, there was overlap of the antiestrogenic activity with the mitochondrial inhibitory activity.
Mitochondrial inhibition
occurred at a CPC concentration that may be relevant to human exposures. The antiestrogenic activity of CPC, BAK, rotenone, and triclosan may be related to their mitochondrial inhibitory activity. Our findings support the need for additional research on the mitochondrial inhibitory and antiestrogenic effects of QUATS, including CPC and BAK. https://doi.org/10.1289/EHP1404.
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