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 (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.

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The US EPA's ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to false positive (chemical is detoxified in vivo) as well as false negative results (chemical is bioactivated in vivo) and thus potential mischaracterization of chemical hazard. To address this challenge, the ten most prevalent human liver cytochrome P450 (CYP) enzymes were introduced into a human cell line (HEK293T) with low endogenous metabolic capacity. The CYP enzymes were introduced via transfection of modified mRNAs as either singlets or as a mixture in relative proportions as expressed in human liver. Initial experiments using luminogenic substrates demonstrate that CYP enzyme activities are significantly increased when co-transfected with an mRNA encoding a CYP accessory protein, P450 oxidoreductase (POR). Transfected HEK293T cells demonstrate the ability to produce predicted metabolites following treatment with well-studied CYP substrates for at least 18 h post-treatment. As a demonstration of how this method can be used to retrofit existing HTS assays, a proof-of-concept screen for cytotoxicity in HEK293T cells was conducted using 56 test compounds. The results demonstrate that the xenobiotic metabolism conferred by transfection of CYP-encoding mRNAs shifts the dose-response relationship for some of the tested chemicals such as aflatoxin B1 (bioactivation) and fenazaquin (detoxification). Overall, transfection of CYP-encoding mRNAs is an effective and portable solution for retrofitting existing cell-based HTS assays with metabolic competence.

The U.S. EPA Endocrine Disruptor Screening Program utilizes data across the ToxCast/Tox21 high-throughput screening (HTS) programs to evaluate the biological effects of potential endocrine active substances (EAS). A potential limitation to the use of in vitro assay data in regulatory decision-making is the lack of coverage for xenobiotic metabolic processes. Both hepatic- and peripheral-tissue metabolism can yield metabolites that exhibit greater activity than the parent compound (bioactivation) or are inactive (bioinactivation) for a given biological target. Interpretation of biological effect data for both putative EAS, as well as other chemicals, screened in HTS assays may benefit from the addition of xenobiotic metabolic capabilities to decrease the uncertainty in predicting potential hazards to human health. The objective of this study was to develop an approach to retrofit existing HTS assays with hepatic metabolism. The Alginate Immobilization of Metabolic Enzymes (AIME) platform encapsulates hepatic S9 fractions in alginate microspheres attached to 96-well peg lids. Functional characterization across a panel of reference substrates for phase I cytochrome P450 enzymes revealed substrate depletion with expected metabolite accumulation. Performance of the AIME method in the VM7Luc estrogen receptor (ER) transactivation assay was evaluated across 15 reference chemicals and 48 test chemicals that yield metabolites previously identified as ER active or inactive. The results demonstrate the utility of applying the AIME method for identification of false positive and false negative target assay effects, reprioritization of hazard based on metabolism-dependent bioactivity, and enhanced in vivo concordance with the rodent uterotrophic bioassay. Integration of the AIME metabolism method may prove useful for future biochemical and cell-based HTS applications.