TOXICOLOGICAL SCIENCES, 155(2), 2017, 458–473
doi: 10.1093/toxsci/kfw230
Advance Access Publication Date: November 11, 2016
Research article

Microbial-Derived 1,4-Dihydroxy-2-naphthoic Acidand Related Compounds as Aryl Hydrocarbon Receptor
Agonists/Antagonists: Structure–Activity Relationships
and Receptor Modeling
Yating Cheng,*,2 Un-Ho Jin,*,2 Laurie A. Davidson,† Robert S. Chapkin,†
Arul Jayaraman,‡ Phanourios Tamamis,‡ Asuka Orr,‡ Clint Allred,†Michael S. Denison,§ Anatoly Soshilov,§ Evelyn Weaver,¶ and Stephen Safe*,1*Department of Veterinary Physiology and Pharmacology; †Department of Nutrition and Food Science; ‡Artie
McFerrin Department of Chemical Engineering, Texas A&M University, College Station, Texas 77843;§
Department of Environmental Toxicology, University of California, Davis, California 95616; and ¶Departmentof Animal Science, Texas A&M University, College Station, Texas 77843
1

To whom correspondence should be addressed at Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, College
Station, TX 77843-4466. Fax: 979-862-4929. E-mail: ssafe@cvm.tamu.edu.

2

These authors contributed equally to this study.

ABSTRACT
1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) is a bacterial-derived metabolite that binds the aryl hydrocarbon receptor (AhR)
and exhibits anti-inflammatory activity in the gut. The structure-dependent AhR activity of hydroxyl/carboxy-substituted
naphthoic acids (NAs) was determined in young adult mouse colonic (YAMC) cells and human Caco2 colon cancer cells using
CYP1A1/CYP1B1 mRNAs as Ah-responsive genes. Compounds used in this study include 1,4-, 3,5-, and 3,7-DHNA, 1,4dimethoxy-2-naphthoic acid (1,4-DMNA), 1- and 4-hydroxy-2-naphthoic acid (1-HNA, 4-HNA), 1- and 2-naphthoic acid (1-NA, 2NA), and 1- and 2-naphthol (1-NOH, 2-NOH). 1,4-DHNA was the most potent compound among hydroxyl/carboxy naphthalene
derivatives, and the fold induction response for CYP1A1 and CYP1B1 was similar to that observed for 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) in YAMC and Caco2 cells. 1- and 4-HNA were less potent than 1,4-DHNA but induced maximal (TCDD-like)
response for CYP1B1 (both cell lines) and CYP1A1 (Caco2 cells). With the exception of 1- and 2-NA, all compounds significantly
induced Cyp1b1 in YAMC cells and these responses were not observed in AhR-deficient YAMC cells generated using CRISPR/
Cas9 technology. In addition, we also observed that 1- and 2-NOH (and 1,4-DHNA) were weak AhR agonists, and 1- and 2-NOH
also exhibited partial AhR antagonist activity. Structure–activity relationship studies for CYP1A1 but not CYP1B1 were similar inboth cell lines, and CYP1A1 induction required one or both 1,4-dihydroxy substituents and activity was significantly enhancedby the 2-carboxyl group. We also used computational analysis to show that 1,4-DHNA and TCDD share similar interactions
within the AhR binding pocket and differ primarily due to the negatively charged group of 1,4-DHNA.
Key words: 1,4-DHNA; structure-activity; Ah receptor; agonists; antagonists.

Bifidobacteria are prominent in the gastrointestinal tract, andthese bacteria and their metabolites have been associated withpromotion of good health and are used as probiotic agents (Caniand Delzenne, 2011; Picard et al., 2005). For example, bifidobacteria alone or in combination with other bacterial species (eg,Lactobacillus) result in decreased inflammation associated withC The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology.
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Crohn’s disease and ulcerative colitis (Furrie et al., 2005;
Ishikawa et al., 2011; Kato et al., 2004; Wildt et al., 2011). It was reported that cell-free filtrate from the bifidobacteria
Propionibacterium freudenreichii stimulates bifodobacterial growth
and this is attributed primarily to 2 microbial metabolites,
namely 2-amino-3-carboxy-1,4-naphthoquinone (minor) and
1,4-dihydroxy-2-naphthoic acid (1,4-DHNA) (major) (Isawa et al.,
2002; Mori et al., 1997). 1,4-DHNA is an intermediate in the biosynthesis of menaquinone (vitamin K2) (Bentley andMeganathan, 1982), and 1,4-DHNA has also been identified inlactic acid-producing bacterial Lactobacillus casei LP1 and in
Korea traditional rice wine (Eom et al., 2012; Kang et al., 2015).
Subsequent studies showed that 1,4-DHNA inhibits dextran sodium sulfate (DSS)-induced colitis in mice and also decreases
induced inflammation and colitis in interleukin 10-deficient
mice by suppressing macrophage-derived pro-inflammatory cytokines (Okada et al., 2006, 2013). These results suggest that 1,4DHNA contributes to the health-promoting effects of bifidobacteria. It was also reported that 1,4-DHNA inhibits growth of
Helicobacter pylori and induces apoptosis in human keratinocytes, indicating a potential application for treating psoriasis(Mok et al., 2013; Nagata et al., 2010).
Several studies show that ligands for aryl hydrocarbon receptor (AhR) including TCDD and 1,4-DHNA are inhibitors of colitis
and development of colorectal cancer in rodent models (Benson
and Shepherd, 2011; Furumatsu et al., 2011; Monteleone et al.,2011; Singh et al., 2011). In vitro studies in human Caco2 colon cancer cells showed that 1,4-DHNA induces CYP1A1 gene expression,a marker of Ah responsiveness, and similar results were observedin the small intestine of wild type (wt) but not AhR knockout mice
(Fukumoto et al., 2014). 1,4-DHNA also inhibits DSS-induced colitis
and this response is attenuated after cotreatment with 1,4-DHNA
plus CH-223191, an AhR antagonist (Fukumoto, et al., 2014). Thus,
the health-promoting effects of 1,4-DHNA in the gut are due, inpart, to its activity as an AhR agonist.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototypicaland highly potent AhR agonist and environmental toxicant, andseveral halogenated aromatic industrial compounds and byproducts and polynuclear aromatic hydrocarbons also act
through the AhR (Van den Berg et al., 2006). In addition, otherclasses of AhR ligands include endogenous biochemicals suchas indolo-2,3[b]-carbazole, kynurenine and microbiota-derived
tryptophan metabolites, health promoting phytochemicals, andpharmaceuticals (Cheng et al., 2015; Denison et al., 2011;
Hubbard et al., 2015; Jin et al., 2014). The structure-dependent effects of TCDD and related halogenated aromatics have been extensively investigated; however, less is known about otherstructural classes of AhR ligands. Therefore, in this study wefurther elucidated the structure–activity relationships (SARs) of
1,4-DHNA and related naphthalene analogs as AhR ligands anddemonstrate the important roles of both the hydroxyl- and carboxyl substituents and their location in the naphthalene ring.
Moreover, since the structures of TCDD and 1,4-DHNA are different, we have also used computational modeling approaches
to investigate differences in their interactions with the AhR.

MATERIALS AND METHODS
Cell lines, antibodies, and reagents. The young adult mousecolonic (YAMC) cell line was initially generated from the
Immorto mouse (Whitehead et al., 1993) and has been previously used in our studies (Turk et al., 2011; Weige et al., 2009).
Cells were maintained in RPMI 1640 medium with 5% fetal
bovine serum (FBS), 5 U/ml mouse interferon-c (IF005) (EMD|

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Millipore, Massachusetts), 1% ITS “” minus (insulin, transferrin, selenium) (41-400-045) (Life Technologies, Grand
Island, New York) at 33  C (permissive conditions). In preparation for experiments, cells were transferred to 37  C (nonpermissive conditions). Caco2 human colon cancer cell line wasobtained from the American Type Culture Collection (ATCC,
Manassas, Virginia). Caco2 cells were maintained in
Dulbecco’s modified Eagle’s medium nutrient mixture supplemented with 20% FBS, 10 ml/l 100 MEM nonessential aminoacid solution (Gibco), and 10 ml/l 100 antibiotic/antimycotic
solution (Sigma-Aldrich). Caco2 cells were maintained at 37  C
in the presence of 5% CO2, and the solvent (dimethyl sulfoxide,
DMSO) used in the experiments was  0.2%. Mouse AhR antibody (BML-SA210) was purchased by Enzo (Enzo Life Sciences
Inc., Farmingdale, New York). b-Actin (A1978) was purchasedfrom Sigma-Aldrich (St Louis, Missouri), and mouse Cyp1a1
antibody was kindly provided by the late Dr Paul Thomas(Rutgers University) and Dr B. Moorthy (Baylor College ofMedicine, Houston). Human CYP1A1, AHR, and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa
Cruz, California). 1,4-DHNA, 3,5-DHNA, 3,7-DHNA, 1,4-dimethoxy-2-naphthoic acid (1,4-DMNA), 1-naphthoic acid (1-NA),2-naphthoic acid (2-NA), 1-naphthol (1-NOH), and 2-naphthol(2-NOH) used in this study were purchased from SigmaAldrich (St Louis, Missouri). 1-Hydroxy-2-naphthoic acid
(1-HNA) was purchased from Alfa Aesar (Ward Hill,
Massachusetts, USA) and 4-HNA was purchased from Chem
Scene (www.chemscene.com).
Chromatin immunoprecipitation assay. The chromatin immunoprecipitation (ChIP) assay was performed using the ChIP-IT Express
Magnetic Chromatin Immunoprecipitation kit (Active Motif,
Carlsbad, California) according to the manufacturer’s protocol.
YAMC cells (1.2  107 cells) were treated with TCDD and/or compounds for 2 or 24 h. Caco2 cells (5  106 cells) were treated with
TCDD and/or compounds for 2 h. The cells were then fixed with
1% formaldehyde, and the cross-linking reaction was stopped byaddition of 0.125 M glycine. After washing with phosphatebuffered saline, cells were scraped and pelleted. The collected
cells were hypotonically lysed, and nuclei were collected andthen sonicated to the desired chromatin length (200–1500 bp).
The sonicated chromatin was immunoprecipitated with normal
rabbit IgG or AhR antibodies and protein A-conjugated magnetic
beads at 4  C for overnight. After the magnetic beads were extensively washed, protein-DNA crosslinks were reversed and eluted.
DNA was prepared by proteinase K digestion followed by polymerase chain reaction (PCR) amplification. The mouse Cyp1a1
primers were 50 -CAG GAG AGC TGG CCC TTT A-30 (sense) and 50 TAA GCC TGC TC ATC CTG TG-30 (antisense), and subsequently
amplified by targeting a 215-bp region of mouse Cyp1a1 promoter, which contained the AhR-binding sequences. The human
CYP1A1 primers were 50 -TCA GGG CTG GGG TCG CAG CGC TTC
T-30 (sense) and 50 -GCT ACA GCC TAC CAG GAC TCG GCA G-30
(antisense) which amplified a 112-bp region of the human
Cyp1A1 promoter which containing the AhR-binding sequences.
PCR products were resolved on a 2% agarose gel in the presenceof ETBR.
Quantitative real-time PCR. Total RNA was isolated using Zymo
Quick RNA MiniPrep Kit (Zymo Research, Irvine, California)
according to the manufacturer’s protocol. RNA was eluted with
RNase-free water and stored at 80  C. Real-time (RT)-PCR was
carried out using iTaq Universal SYBR Green One-step Kit (BioRad, Hercules, California). The following primers were used.

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Mouse
Name

Forward Primer

Reverse Primer

TBP
GAACAATCCAGACTAGCAGCA GGGAACTTCACATCACAGCTC
Cyp1a1 ATCCAAGGCAGAATACGGTG TCCACTCCATCTTCCGACTT
Cyp1b1 GGATATCAGCCACGACGAAT ATTATCTGGGCAAAGCAACG

Human
Name

Forward Primer

Reverse Primer

TBP
GATCAGAACAACAGCCTGCC TTCTGAATAGGCTGTGGGGT
CYP1A1 GACCACAACCACCAAGAAC AGCGAAGAATAGGGATGAAGCYP1B1 CACTGACATCTTCGGCG
ACCTGATCCAATTCTGCCTG

Western blot analysis. Cells were treated with different concentrations of the compounds for 18 h and then collected using
high-salt buffer (50 mM HEPES, 0.5 M NaCl, 1.5 mM MgCl2, 1 mM
EGTA, 10% glycerol, and 1% Triton-X-100) and 10 mM Protease
Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO). Protein lysates
were incubated for 5 min at 95  C before electrophoresis andthen separated on 10% SDS-polyacrylamide gel electrophoresis
120 V for 2–3 h. Proteins were transferred onto polyvinylidene
difluoride membranes by wet electroblotting in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol for 1.5 h
at 180 mA. Membranes were then blocked for 30 min with specific antibodies. Detection of specific proteins was performed
using Chemiluminescence and then exposed to Kodak image
station 4000 mm Pro (Carestream Health, Rochester, New York).
Gel retardation. Gel retardation experiments were performed
using guinea pig hepatic cytosol according to a previously published standard protocol (Soshilov and Denison, 2014a).
Generation of AhR-deficient YAMC cells. Two AhR CRISPR guideRNAs, in a Cas9 vector which also expresses GFP, were purchased from GenScript (Piscataway, New Jersey). Sequences of
the guide RNAs were CGGTCTCTGTGTCGCTTAGA andGAACACAGAGTTAGACCGCC. YAMC cells were cotransfectedwith both plasmids and 48 h later, cells were FACS sorted to collect the 5% highest GFP expressing cells into individual wells ofa 96-well plate. Clonal cells were grown into larger cultures andtested for knock-out of AhR protein.
Statistical analysis. Statistical significance of differences between
the treatment groups was determined by an analysis of variance and/or Student’s t test, and levels of probability werenoted. At least 3 repeated experiments were determined foreach data point, and results are expressed as means 6 SD.
Computational homology modeling of AhR. Residues 241 through 400(sequence HGQNKKGKDG-ALLPPQLALF-AIATPLQPPS-ILEIRTKNFIFRTKHKLDFT-PIGCDAKGQL-ILGYTEVELC-TRGSGYQFIH-AADMLH
CAES-HIRMIKTGES-GMTVFRLFAK-HSRWRWVQSN-ARLIYRNGRPDYIIATQRPL-TDEEGREHLQ-KRSTSLPFMF) of the mouse AhR were
investigated through homology modeling. The homology modelof the AhR was derived using I-TASSER (Yang and Zhang, 2015).
All binding site residues characterized by mutagenesis studies
and known to be critical or influence TCDD binding (Motto et al.,2011; Pandini et al., 2009; Xing et al., 2012), which were also

investigated in a previous study (Bisson et al., 2009), are included
in our model. The homology model was built using the crystal
structure of the hypoxia-inducible factor-2a:AhR nuclear translocator complex (PDB ID: PZP4 [Wu et al., 2015], chain B) as an initial
template. The N- and C- terminal ends of the modeled protein
were acetylated and amidated to avoid any artifacts which could
occur due to the artificial placement of positively and negatively
charged groups at the backbone termini of the truncated ends ofthe modeled systems under investigation.
Generation of docking poses. TCDD and 1,4-DHNA were independently positioned into the binding sites of AhR using AutoDock
Vina (Trott and Olson, 2010). The structures for both TCDD and
1,4-DHNA were obtained from the ZINC database (Irwin et al.,2012). The search space used was 20  24  20 Å so as to include
in the binding pocket AhR residues involved in TCDD binding
according to mutagenesis studies (Motto et al., 2011; Pandini
et al., 2009; Xing et al., 2012). During the initial AutoDock Vina
(Trott and Olson, 2010) docking, the side-chains of residues
identified as binding pocket residues for TCDD binding to AhR
according to mutagenesis studies (Motto et al., 2011; Pandini
et al., 2009; Xing et al., 2012) were treated as flexible. The produced complex conformations of TCDD and 1,4-DHNA in complex with AhR with the lowest binding free energy according to
AutoDock Vina (Trott and Olson, 2010) were used as initialstructures for docking simulation runs performed in CHARMM(Brooks et al., 2009). Six separate docking simulation protocols
were introduced independently to investigate the binding of
TCDD and 1,4-DHNA to AhR. In summary, during the docking
simulations each ligand independently was constrained using
harmonic or quartic potential energy functions to the dockedbinding site through the MMFP module of CHARMM [42]; aquartic potential energy function was used so as to avoid bias
toward the initial positioning of the molecules performed by
Autodock Vina (Trott and Olson, 2010). In each 20 independentruns comprising 200 of short 2 ps simulations were performed.
In each step, prior to the short MD simulation run, the ligands
were independently rotated about a randomly generated axis
and posterior to the short MD simulation run, the complex conformation was minimized and was saved for evaluation. This
procedure resulted in the generation of 4000 binding conformations for each ligand in complex with AhR per protocol.
Additional information on the protocols used in the docking
simulation runs are provided in the Supplemental Materials. As
an initial screening, from each docking simulation protocol, outof the 4000 complex structures produced, we extracted the 3complex structures with the lowest interaction energy for further analysis. This translated to 18 docking conformations of
TCDD in complex with AhR and 18 docking conformations of
1,4-DHNA in complex with AhR were extracted in total.
Molecular dynamics simulations of selected TCDD:AhR and 1,4DHNA:AhR complexes. In order to refine the ligand:receptor structures, optimize intermolecular interactions, determine thestructural stability of the selected binding modes, and assess
the most energetically favored binding modes of the TCDD:AhR
and 1,4-DHNA:AhR complexes, we performed 36 independent
MD simulation runs of which the initial structures corresponded to the 18 selected TCDD:AhR docking conformations,and the 18 selected 1,4-DHNA: AhR docking conformations. All
MD simulations of the 36 systems under investigation were performed in explicit solvent using CHARMM (Brooks, et al., 2009)
and CHARMM36 topology and parameters (Best et al., 2012) with

CHENG ET AL.

periodic boundary conditions. Additional information on the
MD simulations is provided in the Supplemental Materials.
MM GBSA association free energy calculations. To identify the mostenergetically favorable conformation of TCDD and 1,4-DHNA incomplex with AhR, we calculated the association free energy of
the 18 complexes per ligand over the 10 ns production runs
using the Molecular Mechanics Generalized Born Surface Area
(MM GBSA approximation [Carney et al., 2008; Sambuy et al.,2005; Tamamis et al., 2012]), by extracting snapshots from thesimulations every 20 ps. Additional information on the MM
GBSA calculations is provided in the Supplemental Materials.
Selection and analysis of the binding modes with lowest association
free energy. The simulations of the TCDD:AhR and 1,4DHNA:AhR binding modes with the most favorable MM GBSA
association free energies were selected as the ones representing
the most likely naturally occurring binding conformations of
the 2 ligands in complex with AhR; the selection was performed
similarly to the studies as previously described (Tamamis andFloudas, 2014a,b,c; Tamamis et al., 2014). The selected simulations of both the TCDD:AhR and 1,4-DHNA:AhR binding modes
were extended for an additional 20 ns, for a total production runof 30 ns each. To determine the stability of the ligands in the
AhR binding pockets, the entire complexes were structurallyaligned by the backbone atoms of the pocket residues and the
RMSD of the ligand heavy atoms was calculated with respect tothe average conformation over the entire 30 ns production run
duration using VMD (Eargle et al., 2006; Humphrey et al., 1996).
To determine the key interactions occurring in the lowest association free energy binding modes, the average per AhR residue
interaction free energies between the AhR protein and eachligand of the structures with the lowest MM GBSA association
free energies were calculated for the entire 30 ns production
runs (Tamamis et al., 2010, 2011, 2012). Additional informationon the per AhR residue interaction free energy calculations isprovided in the Supplementary Materials.

RESULTS
Induction of Cyp1a1 and Cyp1b1 in Mouse YAMC Cells by TCDD andNaphthalene Compounds
Both TCDD and 1,4-DHNA induce Cyp1a1 and inhibit DSSinduced colitis; however, 1,4-DHNA is approximately 3 orders ofmagnitude lower in potency (Benson and Shepherd, 2011;
Fukumoto et al., 2014; Furumatsu et al., 2011). In contrast to thewell-known SARs for dioxin-like compounds, the contributionsof the hydroxyl and carboxylic acid substituents and their positions on the naphthalene ring has not previously been reported.
In this study, we used mouse YAMC cells as a model for investigating the Ah-responsiveness of naphthalene derivatives on
normal colon, and also human Caco2 colon cancer cells which
are frequently used as an in vitro model for colonic responses
and 1,4-DHNA induced AhR-dependent Cyp1a1 gene expression
in this cell line has previously been reported (Fukumoto, et al.,2014). The substituted naphthalene derivatives used in thisstudy included 1,4-DHNA, 3,5- and 3,7-DHNA, 1,4DMNA, 1-HNA,
4-HNA, 2-NA, 1-NA, 2-naphthol (2-NOH), and 1-NOH(Supplementary Figure S1). The effects of these compounds on
YAMC and Caco2 cell viability are summarized in
Supplementary Figure S2. In YAMC cells, 1,4-DHNA was themost cytotoxic of the substituted naphthalenes, and only 500
and 1000 lM 1- and 2-NOH were more cytotoxic than DHNA.

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Cyp1a1 induction is widely used as a marker of Ahresponsiveness, and 5-50 lM DHNA induced a concentrationdependent increase in Cyp1a1 mRNA levels (Figure 1A). TCDD
(10 nM) induced approximately a 600-fold increase in Cyp1a1
mRNA levels, and the maximal induction by 1,4-DHNA (50 lM)
was approximately 450-fold. In contrast, 3,5- and 3,7-DHNA
exhibited minimal activity as Cyp1a1 inducers (15- to 40-foldlower induction than TCDD) (Figs. 1B and C), and even lower
inducibility was observed for 1,4-DMNA (Figure 1D). Thus, maximal activity was observed for the 1,4-dihydroxy substitution
and methylation of these hydroxyl groups resulted in loss ofactivity. Both 1-HNA and 4-HNA contain a single hydroxyl substituent and these compounds induced Cyp1a1 mRNA (1-HNA >4-HNA) (Figs. 1E and F), whereas 1- and 2-NA (containing nohydroxyl groups) exhibited low to nondetectable induction(Figs. 1G and H). 1- and 2-NOH maximally induced a 43- and 50fold enhancement of Cyp1a1 mRNA compared with the approximately 600-fold induction response observed for TCDD (Figs. 1I
and J), indicating that loss of the 2-carboxyl substituent resulted
in decreased activity. 1,4-Dihydroxynaphthalene is unstable insolution and is oxidized to the 1,4-quinone; however, inductionof Cyp1a1 by this compound was also observed (Supplementary
Figure S4). Western blot analysis showed that TCDD induced
Cyp1a1 protein in YAMC cells with only minimal changes inprotein levels by 1,4-DHNA, 1- and 4-HNA; however, TCDD, 1,4DHNA, 1- and 4-HNA, 1- and 2- NOH decreased expression of
the AhR protein (Figure 1K). TCDD, 1,4-DHNA, 1- and 4-HNA, 1and 2-NOH treatment downregulated AhR expression and minimal effects were observed for the other analogs. Thus, theinduction response (Cyp1a1) for 1,4-DHNA and related compounds was maximal for 1,4-DHNA, and the loss of one or bothhydroxyl or carboxyl groups decreased potency.
Basal levels of Cyp1b1 mRNA in YAMC cells were higher thanobserved for Cyp1a1, and 10 nM TCDD induced a 10-fold increase
in Cyp1b1 mRNA in this cell line and 50 lM DHNA induced asimilar fold induction response (Figure 2A). 3,5- and 3,7-DHNA,
1,4-DMNA, 1- and 4-HNA induced Cyp1b1 mRNA (Figs. 2B–F),
and with the exception of 3,7-DHNA, the maximal induction
response for the naphthalene derivatives was similar to thatobserved for TCDD. Both NAs (1-NA and 2-NA) exhibited minimal induction of Cyp1b1 mRNA (Figs. 2G and H), whereas 1- and
2-NOH induced Cyp1b1 levels > 50% of that observed for TCDD
(Figs. 2I and 2J). Thus, the fold induction of Cyp1b1 by TCDD wasmuch lower than observed for Cyp1a1 in YAMC cells andalthough the SARs for the naphthalene compounds were similar for both responses, their fold induction responses comparedwith TCDD were significantly higher for Cyp1b1 compared with
Cyp1a1. The most striking difference in compound-induced
gene expression was observed for 1,4-DMNA which did not
induce Cyp1a1 but induced levels of Cyp1b1 mRNA >80% of thelevel observed for 10 nM TCDD (Figure 2D).

Induction of CYP1A1 and CYP1B1 in Human Caco2 Cells
by TCDD and Naphthalene Compounds
The SARs for 1,4-DHNA and structurally-related analogs werealso carried out in human Caco2 cells, a colon cancer cell line
used extensively as a model for investigating colonic effects ofvarious drugs and dietary factors (Engle et al., 1998; Sambuy,
et al., 2005). 1,4-DHNA (Figure 3A) but not 3,5-DHNA, 3,7-DHNA
or 1,4-DMNA (Figs. 3B–D) induced CYP1A1 mRNA levels >50%of that observed for TCDD (140-fold induction). In contrast,
both 1-HNA and 4-HNA maximally induced CYP1A1 mRNA
(Figs. 3E and F); 1- and 2-NA (Figs. 3G and H) were relatively

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FIG. 1. Induction of Cyp1a1 in mouse YAMC cells. YAMC cells were treated with different concentrations of 1,4-DHNA (A), 3,5-DHNA (B), 3,7-DHNA (C), 1,4-DMNA (D), 1HNA (E), 4-HNA (F), 1-NA (G), 2-NA (H), 1-NOH (I), and 2-NOH (J) for 18 h, and Cyp1a1 mRNA levels were determined (in triplicate) by real time PCR as outlined in the
Materials and Methods. (K) Western blot analysis. YAMC cells were treated with a single concentration of the naphthalene compounds for 24 h, and whole cell lysates
were then analyzed by western blots. TCDD (10 nM) was used a positive control. Significant (P < .05) induction is indicated (*).

inactive, and both 1- and 2-NOH (Figs. 3I and J) induced <15%of the maximal response observed for TCDD. 1,4Dihydroxynaphthalene also induced CYP1A1 in Caco2 cells(Supplementary Figure S4B). Western blot analysis showedthat TCDD and 1,4-DHNA induced CYP1A1 protein, and boththe 1- and 4-HNA compounds also induced these responses
and all 4 compounds decreased AhR protein levels. TCDD, 1,4DHNA, 1- and 4-HNA induced AhR downregulation and theseCHENG ET AL.

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FIG. 2. Induction of Cyp1b1 in mouse YAMC cells. YAMC cells were treated with different concentrations of 1,4-DHNA (A), 3,5-DHNA (B), 3,7-DHNA (C), 1,4-DMNA (D), 1HNA (E), 4-HNA (F), 1-NA (G), 2-NA (H), 1-NOH (I), and 2-NOH (J) for 18 h and Cyp1b1 mRNA levels were determined (in triplicate) by real time PCR as outlined in the“Materials and Methods” section. Significant (P < .05) induction is indicated (*).

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FIG. 3. Induction of CYP1A1 in human Caco2 cells. Caco2 cells were treated with different concentrations of 1,4-DHNA (A), 3,5-DHNA (B), 3,7-DHNA (C), 1,4-DMNA (D), 1HNA (E), 4-HNA (F), 1-NA (G), 2-NA (H), 1-NOH (I), and 2-NOH (J) for 18 h, and CYP1A1 mRNA levels were determined (in triplicate) by real time PCR as outlined in the“Materials and Methods” section. (K) Western blot analysis. Caco2 cells were treated with the various compounds for 24 h, and whole cell lysates were then analyzed bywestern blots. TCDD (10 nM) was used a positive control. Significant (P < .05) induction is indicated (*).

results were different than their effects on the AhR in YAMC
cells (Figure 1).
In Caco2 cells, TCDD induced a 31-fold induction of CYP1B1
compared with controls (Figure A), and only minimal tonondetectable induction was observed for 3,5-DHNA, 3,7-DHNA
or 1,4-DMNA (Figs. 4B–D), whereas maximal induction
responses were observed for 1- and 2- HNA (Figs. 4E and F).
Minimal induction was observed for 1- and 2-NA (Figs. 4G and H),

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FIG. 4. Induction of CYP1B1 in human Caco2 cells. Caco2 cells were treated with different concentrations of 1,4-DHNA (A), 3,5-DHNA (B), 3,7-DHNA (C), 1,4-DMNA (D), 1HNA (E), 4-HNA (F), 1-NA (G), 2-NA (H), 1-NOH (I), and 2-NOH (J) for 18 h, and CYP1B1 mRNA levels were determined (in triplicate) by real time PCR as outlined in the“Materials and Methods” section. Significant (P < .05) induction is indicated (*).

whereas both 1-NOH and 2-NOH induced CYP1B1 (50% of TCDDinduced response) (Figs. 4I and J) (2-NOH > 1-NOH).
Supplementary Figure S5 compares the induction of CYP1A1/

CYP1B1 in YAMC and Caco2 cells after treatment for 6 or 18 h.
Induction responses for CYP1A1 (YAMC and Caco2) and CYP1B1(Caco2) were significantly higher at the latter time point and this

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is consistent with previous studies on induction of CYP1A1mRNA in this cell line (de Waard et al., 2008). In contrast, comparable compound-dependent induction of Cyp1b1 mRNA levels
was observed YAMC cells after treatment for 6 and 18 h. The
SARs for induction of CYP1A1 by 1,4-DHNA and related compounds (compared with TCDD) were similar in Caco2 and YAMC
cells; however, both 1- and 4-HNA induction responses werelower in YAMC vs. Caco2 cells, suggestive of species differencesin the AhR and/or cell-specific differences in metabolism of thesecompounds. In contrast, SARs for induction of Cyp1b1/CYP1B1
were highly variable and cell context-dependent for the naphthalene compounds.
Effects of Naphthalene Compounds as AhR Antagonists and inTransformation of Guinea Pig Cytosol and AhR Dioxin-responsive
Element (DRE) Localization in ChIP Assays
In order to confirm the application of mouse YAMC cells forinvestigating the Ah-responsiveness of compounds (Chenget al., 2015), we used the CRISPR/Cas9 technology to generate
AhR knockout cells and expression of AhR in one of these celllines used in this study is illustrated in Figure 5A. Treatment of
the knockout cells with TCDD, 1,4-DHNA and related compounds did not induce Cyp1a1 (Figure 5B) or Cyp1b1 (Figure 5C),
confirming the AhR-dependence of the induction response in
wt YAMC cells (Figs. 1 and 2). We also investigated the potential
AhR antagonist activities of 1,4-DHNA and related compounds
in mouse YAMC and human Caco2 cells by determining theirinhibition of TCDD-induced Cyp1a1 gene expression (Figure 5D).
In YAMC cells, all compounds, with the exception of 1,4- and3,7-DHNA, inhibited TCDD-induced Cyp1a1 mRNA expression at
one or more of the higher concentrations. However, AhR antagonist activity for these substituted naphthalenes was observedat concentrations that induced some level of cytotoxicity(Supplementary Figure S2). The only compound that did not act
as an AhR antagonist (3,7-DHNA) was not cytotoxic. In contrast,with the exception of 1- and 2-NOH, this series of substituted
naphthalenes exhibited minimal cytotoxicity in Caco2 cells(Supplementary Figure S3), suggesting that this cell line may be
more suitable for determining AhR antagonist activity. The
results (Figure 5E) show that 1,4-DHNA (20–100 lM) significantly
inhibited TCDD-induced CYP1A1 gene expression in Caco2 cells,
providing an explanation for the reduced reduction response at
higher 1,4-DHNA concentrations (Figure 3A). Noncytotoxic concentrations of both 1- and 2-NOH (eg, 250 lM) also exhibitedAhR antagonist activity with 1-NOH being highly active (>80%
inhibition).
Figures 6A and B summarize the effects of 1,4-DHNA andrelated compounds on transformation of hepatic cytosol(guinea pig) to its DNA binding form in a gel mobility shift assay.
1,4-DHNA (10 and 100 lM) and 100 lM 3,5-DHNA and 1- and 4HNA, 1- and 2-NOH alone significantly stimulated AhR transformation/DNA binding of guinea pig hepatic cytosol, whereas
AhR transformation/DNA binding was not observed for 3,7DHNA, 1,4-DMNA, 1- and 2-NA. In combination studies with
TCDD, only 1- and 2-NOH inhibited TCDD-induced transformation and this correlated with the AhR antagonist activity
observed in the transactivation assays (Figs. 5D and E). As a positive control, we show that the AhR antagonist CH229131 did
not stimulate AhR transformation/DNA binding of guinea pig
cytosol but inhibited TCDD-induced transformation/DNA binding. These AhR transformation/DNA binding assays confirmedthat 1,4-DHNA, 1- and 4-HNA which induced CYP1A1 andCYP1B1 also induced AhR transformation. The results obtainedfor 3,5-DHNA were somewhat surprising based on the lack of

CYP1A1 induction; however, this compound induced Cyp1b1 in
YAMC cells (Figure 2). The effects of 1- and 2-NOH (100 lM) alone
and in combination with TCDD show that both compounds
alone induced transformation/DNA binding but also inhibited
TCDD-induced transformation/DNA binding to an extent similar to that observed for the well-characterized AhR antagonist
CH229131. These data further confirm the partial AhR agonist/
antagonist activities observed for 1- and 2-NA for induction of
Cyp1a1 in colon cells.
We also examined the effects of TCDD, 1,4-DHNA and 1NOH alone and in combination with TCDD in a ChIP assay.
Treatment with TCDD for 2 h resulted in the recruitment of the
AhR and pol II to the DRE region of the CYP1A1 promoter inCaco2 and YAMC cells (Figure 6C). Similar effects were observedfor 1-NA and 1,4-DHNA in YAMC and for 1-NOH (but not 1,4DHNA) in Caco2 cells. Surprisingly, results observed in cells
treated with 1-NOH plus TCDD for 2 h appeared to be additive
with respect to AhR interactions with the Cyp1a1 promoter.
These unexpected ligand-induced effects after treatment for 2 h
were further investigated in YAMC cells after treatment for 24 h.
TCDD and to a lesser extent 1-NOH recruited the AhR and pol II
to the Cyp1a1 promoter, and in the combination treatment (1NOH plus TCDD), the 1-NOH compound decreased the effects of
TCDD on AhR and pol II recruitment. 1-NOH alone which
induced minimal expression of Cyp1a1 mRNA (Figure 1I)
recruited relatively high levels of the AhR to the Cyp1a1 promoter. Currently, we are further investigating both the timeand compound-dependent recruitment of the AhR, pol II andother nuclear cofactors, including coactivators and corepressors, to the DRE region of the Cyp1a1 and other Ah-responsive
genes.
Modeling of TCDD and 1,4-DHNA Interactions with the AhR in
Order to Identifying the Most Energetically Favored Binding
Conformations
We introduced the MM GBSA approximation and identified the
TCDD:AhR and 1,4-DHNA:AhR-binding modes which acquire
the lowest MM GBSA association free energy across all 18 simulated TCDD:AhR and 1,4-DHNA:AhR-binding modes, respectively. The average association free energies of the simulated
TCDD:AhR and 1,4-DHNA:AhR-binding modes are tabulated in
Supplementary Tables S1 and S2, respectively. The simulations
encompassing the most energetically favored binding conformations of TCDD:AhR and 1,4-DHNA:AhR according to MM
GBSA were both derived from the docking protocols using
quartic potential energy functions and are the most likely tocorrespond to the naturally occurring binding conformations.
Structural Stability of Binding Modes
The stability of lowest association free energy binding modes of
TCDD and 1,4-DHNA in complex with AhR was confirmed
through RMSD calculations over the 30 ns simulation production runs. The RMSD of the heavy atoms of TCDD in complex
with AhR is 0.9 6 0.5 Å with respect to the average structure of
TCDD in complex with AhR, and the RMSD of heavy atoms of
1,4-DHNA in complex with AhR is 0.9 6 0.5 Å with respect to theaverage structure of 1,4 in complex with AhR.
Interactions between TCDD and AhR
The average per-residue interaction free energy between AhR
residues and TCDD are decomposed into polar and nonpolar
contributions, and selected interactions are presented in
Figure 7. Residues contributing the most interaction free energies are presented in Figure 8A. In the TCDD:AhR-binding mode,

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FIG. 5. 1,4-DHNA and related compounds do not activate AhR-deficient YAMC cells and their partial AhR antagonist activity. (A) AhR knockout YAMC cells. The AhR
was knocked out (ko) in YAMC cells using CRISPR/Cas9, and expression of the AhR in wt and ko cells was determined by western blots as outlined in the Materials andMethods. ko-YAMC cells were treated with 10 nM TCDD, 10 lM 1,4-DHNA, 500 lM 3,5-DHNA, 3,7-DHNA, 1,4-DMNA, and 1-HNA, 4-HNA, 1-NA, 2-NA, and 250 lM 1-NOH
and 2-NOH for 18 h and analyzed for expression of Cyp1a1 (B) and Cyp1b1 (C) mRNA (in triplicate) by real time PCR. YAMC (D) and Caco2 (E) cells were treated with 10 nM
TCDD alone and in combination with the hydroxyl/NAs for 18 h, and Cyp1a1/CYP1A1 mRNA levels were determined (in triplicate) by real time PCR. Significant (P < .05)
antagonist activity is indicated (*). In the YAMC knockout cells, 3,5-DHNA, 1,4-DMNA and 1-NA slightly induced (<50%) Cyp1a1 mRNA levels.

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FIG. 6. Effects of 1,4-DHNA and related compounds on transformation and DNA binding of guinea pig cytosol and ChIP analysis of the Cyp1a1 promoter. A,
Transformation and DNA binding of guinea pig hepatic cytosolis AhR. DMSO (solvent control), 10 lM CH229131, 10 and 100 lM 1,4-DHNA, and 100 lM concentrations of
the remaining compounds alone and in combination with TCDD were incubated with guinea pig cytosol and analyzed by gel mobility shift assays as outlined in the
Materials and Methods. B, A representative gel showing the ligand-induced transformed AhR-DRE complex is illustrated in this panel. C, ChIP assay. YAMC and Caco2
cells were treated with various compounds and analysis of recruitment of pol II and the AhR to the Cyp1a1/CYP1A1 promoters was determined in a ChIP assay as outlined in the “Materials and Methods” section.

the medial oxygen atom of TCDD forms a hydrogen bond with
the NE group of Gln377, indicated with a black dotted line in
Figure 8A. As predicted by previous studies (Motto et al., 2011;
Pandini et al., 2009; Xing et al., 2012), the TCDD-binding pocket of
AhR is highly hydrophobic, and the binding of TCDD in AhR is
primarily stabilized by nonpolar interactions (Figure 8A).

Residues Phe289, Cys327, Met342, Ile319, Phe345, and Leu347
form hydrophobic walls around the left side of the ligand, in theperspective of Figure 8A. The aromatic rings residues Phe289,
Phe345, and, less frequently, Phe318 form p-p interactions withthe aromatic rings of TCDD. Van der Waals interactions are also
formed between TCDD and the side-chain atoms of residues

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FIG. 7. Average interaction free energies (kcal/mol) decomposed into polar (red or orange) and nonpolar (blue or purple) contributions for AhR interacting residues incomplex with TCDD (first bar per residue) and in complex with DHNA (second bar per residue). The sum of polar and nonpolar contributions corresponds to the total
average per AhR residue interaction free energy. Only residues with at < 0.5 kcal/mol average interaction free energy are presented. Results were calculated using theensemble of snapshots extracted from simulation trajectories of the most energetically favored binding conformations. The average and standard deviation values forthe polar and nonpolar components of the interaction free energies were calculated over 4 “measurements”, where the first, second, third, and fourth measurement
corresponds to the individual average interaction free energy components of the first, second, third, and fourth 7.5 ns segment of the 30 ns MD simulation productionrun.

Phe281, Thr283, His285, Pro291, Cys294, Leu302, Leu309, Ile319,
Cys327, Ser330, Met342, Leu347, Ser359, Ala361, and Ala375 aswell as the backbone atoms of residues Gly315 and Tyr316 dueto their close proximity to the bound TCDD molecule.
Interactions between 1,4-DHNA and AhR
As with the TCDD:AhR complex, the average per residue interaction free energy between AhR residues and 1,4-DHNA aredecomposed into polar and nonpolar contributions, andselected interactions are presented in Figure 7. Residues withthe largest associated interaction free energy contributions arepresented in Figure 8B. The hydroxyl group of 1,4-DHNA farthest from its carboxylic acid group forms a hydrogen bondwith the NE group of Gln377; the hydroxyl group of 1,4-DHNA
closest to its carboxylic acid group forms a hydrogen bond with
the NE group of Arg312, and the oxygen atoms of the carboxylic
acid group of 1,4-DHNA forms hydrogen bonds with the backbone amino groups of Tyr316 and Gln317 as well as a low interacting salt-bridge with the NH group of Arg312. These hydrogenbonds are indicated using black dotted lines in Figure 8B.
Hydrophobic residues Phe281, Phe289, Pro291, Cys294, Leu302,
Leu309, Phe318, Ile319, Phe345, and Leu347 predominantly
form the 1,4-DHNA binding pocket of AhR (Figure 8B). The aromatic rings of residues Phe318 and, occasionally, Phe289
participate in p–p interactions with the aromatic rings of 1,4DHNA. Due to their close proximity, strong van der Waals
interactions are formed between 1,4-DHNA and the sidechains of residues Phe281, Thr283, Pro291, Cys294, Leu302,
Leu309, Ile319, Phe345, and Leu347 as well as the backbone
atoms of Gly315 and Tyr316.

DISCUSSION
TCDD and structurally-related halogenated aromatic compounds have been characterized as widespread and persistentenvironmental contaminants, and risk assessment of dioxinlike compounds have been developed and are based on wellknown SARs (Denison et al., 2011; Van den Berg et al., 2006). In
contrast, SARs for other structural classes of AhR ligands
including pharmaceuticals, microbiota-derived compounds
such as 1,4-DHNA, endogenous AhR ligands, and food-derived
compounds have not been determined and despite someinsights on their intake and levels of exposure, it is difficult topredict their potencies and also their interactions with “dioxinlike” compounds.
Among PAHs, naphthalene is not an AhR ligand (Till et al.,1999) and therefore the 1- and 4-hydroxyl and carboxylic acidgroups are responsible for the activity of 1,4-DHNA which induces near maximal expression (compared with 10 nM TCDD) of
Cyp1a1 and Cyp1b1 in YAMC and Caco2 cells. The loss of bothhydroxyl groups to give 2-NA (or 1-NA) or replacement withmethoxyl substituent (1,4-DMNA) resulted in significant loss of
Cyp1a1/Cyp1b1 inducibility in both cell lines. Both 3,5- and 3,7DHNA exhibited minimal activity as Cyp1a1 inducers in bothcell lines; however, in YAMC but not Caco2 cells, these compounds induced Cyp1b1 expression, indicating that the requirement of the dihydroxy groups was gene-dependent and specificto the cell context of YAMC and Caco2 cell lines. Differences in
SARs for induction of CYP1B1 may also be species-dependentand due not only to differences between the human and mouse
AhR but also in expression of cofactors. Hydroxy NAs are

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FIG. 8. Molecular graphics images of TCDD (A) and 1,4-DHNA (B) in complex with AhR, which correspond to snapshots extracted from the most energetically favoredbinding conformations. The ligand molecules are shown in licorice representation in both panels. Interacting AhR protein residues are shown in thin licorice representation, and the entire AhR protein is shown in transparent, gray new cartoon representation. Hydrogen bonds are indicated using black dotted lines. Q377 forms hydrogen bonds with both TCDD and 1,4-DHNA.

bacterial metabolites of PAHs, and previous studies on theirdevelopmental toxicity in medaka fish embryos show that 1HNA was not only the most toxic compound but also induced
Cyp1a1 expression in medaka and mouse Hepa1c1c7 liver cancer cell lines (Carney et al., 2008). We also observed that both 1and 4-HNA induced Cyp1a1/Cyp1b1 in YAMC and Caco2 cells
and with the exception of relative low induction of Cyp1a1 in
YAMC cells, their maximal induction responses were comparable to 1,4-DHNA and TCDD but 5-20 fold less potent than the former compounds. We further confirmed the role of the AhR inmediating compound-induced Cyp1a1 and Cyp1b1 using AhR
knockout YAMC cells obtained using the CRISPR/Cas9 technology (Figure 5). It was interesting to note that compounds such
as 3,5-DHNA, 3,7-DHNA and 1,4-DMNA that exhibited minimal
to nondetectable induction of Cyp1a1 were effective inducers of
Cyp1b1 (>50% of the response induced by 10 nM TCDD). The differential induction of 2 AhR-responsive genes in the same cellline is typical of selective AhR modulators and due, in part, togene/promoter histone and chromatin differences and is currently being investigated.
1,4-Dihydroxynaphthalene is readily oxidized to the quinone; however, our results show that although this dihydroxy/quinone mixture is cytotoxic, we also observed induction of
CYP1A1 in Caco2 and YAMC cells (Supplementary Figure S4).
Thus, the loss of the carboxyl group from 1,4-DHNA to give 1,4dihydroxynaphthalene decreases but does not abrogate AhR
activity of this compound. 1- and 2-NOH are biomarkers ofhuman exposure to PAH and smoking (Jain, 2016; Nethery et al.,2012; Sudakin et al., 2013; Sul et al., 2012; Wilhelm et al., 2008),
and both compounds exhibited weak induction of Cyp1a1/
CYP1A1 (YAMC and Caco2) and CYP1B1 (Caco2) mRNA; however,Cyp1b1 was induced to >50% of the maximal TCDD-induced
response in YAMC cells. Both 1- and 2-NOH inhibited TCDDinduced Cyp1a1 gene expression in YAMC and Caco2 cells (Figs.
5D and E), and their AhR antagonist activity was also observedin the gel mobility shift assay (Figure 6A), suggesting that 1- and
2-NOH represent a new class of partial AhR antagonists. We
also observed some inconsistencies in the effects of 1-NOH in
ChIP assays in YAMC and Caco2 cells (Figure 6C) with respect torecruitment of the AhR to the DRE region of the Cyp1a1 promoter, and this is currently being investigated.
The most energetically favored binding mode of TCDD incomplex with AhR according to our computational studies is instrong accordance with previous experimental and computational studies examining TCDD binding to AhR. Recent experiments suggest that residues Phe318, Ile319, and Ala375 are key
to ligand selectivity (Soshilov and Denison, 2014b). Other mutagenesis studies revealed that substitutions in residues Thr283,
His285, Phe289, Pro291, Leu302, Leu309, Cys327, Phe345, andLeu347 result in a reduction in TCDD binding (Motto et al., 2011;
Pandini et al., 2009; Soshilov and Denison, 2011). Finally, bothcomputational methods and further mutagenesis studies performed on Gln377 support the contention that the polar sidechain atoms of Gln377 form hydrogen bonds with the medialoxygen of TCDD (Xing et al., 2012). The excellent agreement of
our work in comparison to previous studies support the validityof the computational protocol introduced here, and suggest thatthe in silico identified binding modes most likely represent thenaturally occurring binding modes of TCDD and 1,4-DHNA withresidues in the AhR ligand binding pocket
The presence of more polar groups which include the negatively charged carboxylic group in 1,4-DHNA compared with
TCDD contributes in general to stronger polar interactions
between 1,4-DHNA and AhR compared with TCDD and AhR.
Although in the TCDD:AhR complex, only one hydrogen bond isformed with the NE group of Gln377 and the medial oxygen of
TCDD, 1,4-DHNA forms a hydrogen bond with the NE group of
Gln377, the NE group of Arg312, and the backbone amino groupsof Tyr316 and Gln317. Additionally, the negatively charged
group of 1,4-DHNA forms a low interacting salt-bridge with the
NH group of Arg312. The binding of both TCDD and 1,4-DHNA in
AhR is also stabilized by nonpolar interactions. Both the aromatic rings of TCDD and 1,4-DHNA form p-p interactions withthe aromatic rings of Phe289 and Phe318. Both TCDD and 1,4DHNA also form strong van der Waals interactions with theside-chains of residues Phe281, Thr283, Pro291, Cys294, Leu302,
Leu309, Ile319, Phe345, and Leu347 as well as the backbone
atoms of Gly315 and Tyr316. Alanine mutagenesis has shownthat Thr283, Phe289, Leu302, Leu309 Phe318, Ile319, Phe345, andLeu347 are critical for TCDD binding (Motto et al., 2011; Soshilov
and Denison, 2011). These similarities are largely due to TCDD
and 1,4-DHNA sharing the same binding site and both ligands
containing aromatic rings. Owing to the smaller size of DHNA,
and thus a lesser amount of possible van der Waals interaction
and p–p interaction sites, interactions between 1,4-DHNA andthe side-chains of residues His285, Cys327, Ser330, Met342,

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Ser359, Ala361, and Ala375 are weaker compared with the
TCDD:AhR complex. Thus, the modeling studies show that both
1,4-DHNA and TCDD interact within the same binding pocket of
the AhR but 1,4-DHNA binds with lower affinity. This observation is consistent with similar efficacies of 1,4-DHNA and TCDD
for induction of Cyp1a1 but differences in their potencies. To
further understand the differences between both 1,4-DHNA and
TCDD in comparison to a minimally active compound, 3,7DHNA, we performed a preliminary study of the latter in complex with AhR using a similar strategy to the one used in the
study of TCDD and 1,4-DHNA, with the only difference that thesimulation entailing the binding mode with the lowest association free energy was not extended for an additional 20 ns. In
comparison with both 1,4-DHNA and TCDD, 3,7-DHNA forms
weaker interactions with Thr283, Pro291, Cys294, Leu302,
Leu309, and Phe318. Alanine mutagenesis experiments suggestthat Thr283, Leu302, Leu309 and Phe318 are critical to TCDD
binding (Motto et al., 2011; Soshilov and Denison, 2011), andPhe318 has been shown to be an “agonist/antagonist switch”(Soshilov and Denison, 2014b).
In summary, this study confirms that 1,4-DHNA is a relatively
potent AhR agonist in both YAMC and Caco2 cells; however, thepotential impact of endogenous 1,4-DHNA alone or in combination with other AhR agonists/antagonists on gut health isunknown and in the future, we hope to more accurately quantitate these compounds and determine their combined effects.
Structure activity studies show that both the hydroxyl and carboxyl groups and their positions on the naphthalene ring areimportant for AhR activity. Both TCDD and 1,4-DHNA are ligands
for the AhR and predominantly form strong interactions with thesame AhR residues. More polar interactions occur in the AhR:1,4DHNA complex in comparison to the AhR:TCDD complex due, inpart, to differences in overall charge and substituent interactions
with various amino acid side-chains. Our results demonstratethat for some of the hydroxyl NA analogs, there are differences intheir activation of Cyp1a1 versus Cyp1b1 in the same cell line andalso differences in the mouse (YAMC) versus human (Caco2)
colon-derived cells. However, 1,4-DHNA induced >70% of the
response observed for 10 nM TCDD for Cyp1a1 and Cyp1b1 in boththe mouse and human cell lines. This suggests possible efficacy
for 1,4-DHNA in humans as an AhR agonist that is protective in
the gut; however, this will require more definitive proof from dietary studies and manipulation of gut microorganisms.

SUPPLEMENTARY DATA
Supplementary data are available at Toxicological Sciences online.

FUNDING
This work was supported by National Institutes of Health (R01ES025713, R01-ES007685, P30-ES023512, R35-CA197707, and R01CA202697); Cancer Prevention Research Institute of Texas;
Texas AgriLife Research; the Office of Graduate and Professional
Studies and the Artie McFerrin Department of Chemical
Engineering at Texas A&M University; and the Sid Kyle
Endowment.

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