Abstract
1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ) are clinically promising biologically active chemicals that have been shown to stimulate the aryl hydrocarbon receptor (AhR) signaling pathway, but whether they are direct or indirect ligands or activate the AhR in a ligand-independent manner is unknown. Given the structural diversity of AhR ligands, multiple mechanisms of AhR activation of gene expression, and species differences in AhR ligand binding and response, we examined the ability of 1,2-NQ and 1,4-NQ to bind to and activate the mouse and human AhRs using a series of in vitro AhR-specific bioassays and in silico modeling techniques. Both NQs induced AhR-dependent gene expression in mouse and human hepatoma cells, but were more potent and efficacious in human cells. 1,2-NQ and 1,4-NQ stimulated AhR transformation and DNA binding in vitro and was inhibited by AhR antagonists. Ligand binding analysis confirmed the ability of 1,2-NQ and 1,4-NQ to competitively bind to the AhR ligand binding cavity and the molecular determinants for interactions were predicted by molecular modeling methods. NQs were shown to bind distinctly differently from that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and differences were also observed between species. Mutation of amino acid residues (F289, M334, and M342) involved in critical NQ:AhR binding interactions, decreased NQ- and AhR-dependent gene expression, consistent with a role for these residues in binding and activation of the AhR by NQs. These studies provide insights into the molecular mechanism of action of NQs and contribute to the development of emerging NQ-based therapeutics.
Highlights
Naphthoquinones (NQ) are byproducts of polycyclic aromatic hydrocarbon (PAH) oxidation and photolysis reactions and have been identified in a wide variety of materials [1,2,3,4,5,6]
To demonstrate that 1,2-NQ and 1,4-NQ act as aryl hydrocarbon receptor (AhR) ligands by binding to the AhR ligand binding pocket, we examined their ability to directly compete with [3H]TCDD specific binding to the mAhR and mAhR-hAhRLBD
The results revealed that both 1,2-NQ and 1,4-NQ fit within the mAhR ligand binding cavity but with distinctly different orientations and residue interactions compared to that predicted for the binding of TCDD (Figure 6A,B)
Summary
Naphthoquinones (NQ) are byproducts of polycyclic aromatic hydrocarbon (PAH) oxidation and photolysis reactions and have been identified in a wide variety of materials (i.e., botanicals, air pollutants, and biologics) [1,2,3,4,5,6]. Recent evidence has shown that the addition of electrophilic (reactive oxygen species (ROS)-generating) chemicals such as methylated pentavalent arsenic metabolites [31] or a quinone derivative of a non-dioxin-like polychlorinated biphenyl (4-chlorobiphenyl [32]) to cells in culture can stimulate AhR nuclear translocation and induction of cyp1a1 and DRE-dependent reporter gene expression, and these responses can be inhibited by an AhR antagonist. Chemical-dependent inhibition of CYP1A1/cyp1a1 activity reportedly can lead to activation of the AhR and AhR-dependent gene expression by the low levels of metabolically labile, but highly potent AhR agonists (i.e., FICZ) that can be found in cell culture media [38] Whether this CYP1A1/cyp1a1 inhibitory mechanism plays any role in the induction of AhR signaling by 1,2-NQ, 1,4-NQ or other reactive quinones is currently unknown. We have confirmed that 1,2-NQ and 1,4-NQ can stimulate AhR-dependent gene expression in a species-specific manner and have extended these analyses to examine their ability to directly bind to and activate the AhR in vitro, using a combination of ligand and DNA binding assays and in silico ligand-docking analysis
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