International Journal of

Molecular Sciences
ArticleThe Cellular and Molecular Determinants ofNaphthoquinone-Dependent Activation of the
Aryl Hydrocarbon Receptor
Samantha C. Faber 1,† , Sara Giani Tagliabue 2 , Laura Bonati 2
1
2

*
†

and Michael S. Denison 1, *Department of Environmental Toxicology, University of California, Davis, CA 95616, USA;scfaber@ucdavis.edu
Department of Earth and Environmental Sciences, University of Milano-Bicocca, 20126 Milan, Italy;s.gianitagliabue@campus.unimib.it (S.G.T.); laura.bonati@unimib.it (L.B.)
Correspondence: msdenison@ucdavis.edu; Tel.: +1-(530)-752-3879
Current address: Investigative Toxicology, Drug Safety and Research Evaluation,
Takeda Pharmaceutical Company, Cambridge, MA 02139, USA.



Received: 3 May 2020; Accepted: 5 June 2020; Published: 9 June 2020

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 ina 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 andhuman 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 weremore potent and efficacious in human cells. 1,2-NQ and 1,4-NQ stimulated AhR transformation andDNA 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 themolecular determinants for interactions were predicted by molecular modeling methods. NQs wereshown to bind distinctly differently from that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) anddifferences 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 thedevelopment of emerging NQ-based therapeutics.
Keywords: aryl hydrocarbon receptor; naphthoquinones; ligand binding; docking analysis

1. Introduction
Naphthoquinones (NQ) are byproducts of polycyclic aromatic hydrocarbon (PAH) oxidation andphotolysis reactions and have been identified in a wide variety of materials (i.e., botanicals, air pollutants,
and biologics) [1–6]. Mechanistically, NQ act as redox cyclers to stimulate the generation of free radicals
involved in diverse physiological and pathophysiological outcomes [1,2,7], such as DNA damage andalterations of vital metabolic pathways (i.e., cellular respiration). The ability of NQs to act as potent
cytotoxic, antiproliferative, and antimicrobial agents has led to an interest in designing NQ-based
anticancer agents [1–3,8–11] and 1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ) areconsidered particularly promising in this regard. Although 1,2-NQ and 1,4-NQ are structurally similar(Figure 1), differences in the position of the carbonyl groups have been shown to result in diverse
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and 1,4-NQ are structurally similar (Figure 1), differences in the position of the carbonyl groups
have been shown to result in diverse biological activity [1,9,10,12,13], and development of thesecompounds
for practicalapplications
require of
improved
understandingof theapplications
molecular
biologicalactivity[1,9,10,12,13],and development
these compounds
for practicalmechanisms
underlying
their modes
ofmolecularaction. mechanisms underlying their modes of action.
requireimproved
understandingof theFigurestructuresof 1,2-naphthoquinone(1,2-NQ)
and 1,4-naphthoquinone(1,4-NQ).
Figure1.1.The
Thechemicalchemicalstructuresof 1,2-naphthoquinone(1,2-NQ)
and 1,4-naphthoquinone(1,4NQ).

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates
the expression of a wide variety of genes and it plays a pivotal role in a broad spectrum of biological,
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates
physiological and toxicological effects [14–19]. While the best studied high affinity ligands (agonists) forthe expression of a wide variety of genes and it plays a pivotal role in a broad spectrum of
the AhR include polycyclic and halogenated aromatic hydrocarbons (PAHs and HAHs, respectively),
biological, physiological and toxicological effects [14–19]. While the best studied high affinity
the AhR can bind and be activated by structurally diverse ligands and cross species differences in AhR
ligands (agonists) for the AhR include polycyclic and halogenated aromatic hydrocarbons (PAHs
ligand specificity have been observed [15,20–24]. Localized in the cytosol as part of a multiprotein
and HAHs, respectively), the AhR can bind and be activated by structurally diverse ligands andcomplex, agonist binding to the AhR stimulates translocation of the AhR protein complex into thecross species differences in AhR ligand specificity have been observed [15,20–24]. Localized in the
nucleus, where dimerization of the AhR with its binding partner, the aryl hydrocarbon nuclear
cytosol as part of a multiprotein complex, agonist binding to the AhR stimulates translocation of thetranslocator (ARNT), releases the ligand-bound AhR from its associated proteins and converts the
AhR protein complex into the nucleus, where dimerization of the AhR with its binding partner, theAhR into its DNA binding form [15,24,25]. Binding of the ligand: AhR:ARNT complex to its specificaryl hydrocarbon nuclear translocator (ARNT), releases the ligand-bound AhR from its associatedDNA binding site, the dioxin response element (DRE), results in transcriptional activation of adjacentproteins and converts the AhR into its DNA binding form [15,24,25]. Binding of the ligand:genes [15,21,24–26]. Notably, while naphthalene (NA) (the precursor to both 1,2-NQ and 1,4-NQ)
AhR:ARNT complex to its specific DNA binding site, the dioxin response element (DRE), results inreportedly fails to bind to and/or activate the AhR, 1,4-dihydroxy-2-naphthoic acid and several relatedtranscriptional activation of adjacent genes [15,21,24–26]. Notably, while naphthalene (NA) (theNA derivatives were shown to be able to act as AhR agonists/antagonists [27]. Additionally, numerous
precursor to both 1,2-NQ and 1,4-NQ) reportedly fails to bind to and/or activate the AhR, 1,4polychlorinated NAs (PCNs) have been shown to be AhR agonists and some higher chlorinateddihydroxy-2-naphthoic acid and several related NA derivatives were shown to be able to act ascongeners (those with five or more chlorines) have physicochemical characteristics similar to the
AhR agonists/antagonists [27]. Additionally, numerous polychlorinated NAs (PCNs) have beenhigh affinity and prototypical HAH AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin),
shown to be AhR agonists and some higher chlorinated congeners (those with five or moreand can stimulate both AhR-dependent gene expression and produce TCDD-like toxic effects [28].
chlorines) have physicochemical characteristics similar to the high affinity and prototypical HAH
Highly reactive quinone metabolites are known to stimulate electrophilic signal transduction
AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and can stimulate both AhRpathways by their ability to covalently modify and activate transcription factors such as nuclear factor
dependent gene expression and produce TCDD-like toxic effects [28].
erythroid 2-related factor 2 (NRF2) [1,2]. Recently, quinone metabolites of NA (1,2-NQ and 1,4-NQ) as
Highly reactive quinone metabolites are known to stimulate electrophilic signal transduction
well as 1,4-benzoquinone, tert-butyl-1,4-benzoquinone, and other electrophilic mono- and di-cyclic
pathways by their ability to covalently modify and activate transcription factors such as nuclear
quinones
with covalentbinding
upregulate
CYP1A1/cyp1a1
mRNAandinfactor erythroid
2-relatedfactor capacity
2 (NRF2)were[1,2].observed
Recently,toquinone
metabolites
of NA (1,2-NQ
human
HepG2
and
mouse
hepa1c1c7
cells,
whiletheparentcompounds
hadlittleeffect[29,30].
Further,
1,4-NQ) as well as 1,4-benzoquinone, tert-butyl-1,4-benzoquinone, and other electrophilic monosome
quinones,
including
1,2-NQ
andbinding
1,4-NQ,capacity
simulatewere
AhRobserved
nuclear translocationinduction ofand di-cyclic
quinoneswithcovalent
to upregulateand
CYP1A1/cyp1a1
cyp1a1inmouse
hepatoma
cells
in
an
AhR-dependent
mannerandtheinduction
could
blockedmRNA in human HepG2 and mouse hepa1c1c7 cells, while the parent compounds hadbe
littleeffectbyanAhR
antagonist.
Interestingly,
1,2-NQwasobserved
tocovalently
bind
to
the
AhR,
stimulate[29,30]. Further, some quinones, including 1,2-NQ and 1,4-NQ, simulate AhR nuclear translocation
AhR
binding toofthe
cyp1a1
promoterregion and
DRE-dependentmannerreportergene
and induction
cyp1a1
in mouse
hepatomacellsinducein an AhR-dependent
andtheinduction.
induction
This
led
to
the
hypothesisthat
1,2-NQandotherquinones
activated
the
AhR
via
covalent
modification
could be blocked by an AhR antagonist. Interestingly, 1,2-NQ was observed to covalently bind toofin the AhR
AhR, binding
presumably
withcyp1a1
one residue(C327)region
contained
theDRE-dependent
ligand binding
thea thiol
AhR,group
stimulateto thepromoterandwithin
inducepocket[29].
Nevertheless,the
distinctmechanism(s)ofAhR
activation
andwhetherthese
compounds
reporter gene induction. This led to the hypothesis that 1,2-NQ and other quinones activatedthebindwithinthe
AhR
ligandbinding
domain(LBD)
isunknown.
Consideringthesignificant
differences
AhR via covalent modification of a thiol group in the AhR, presumably with one residue (C327)
inphysicochemical
characteristics
of 1,2-NQ
and 1,4-NQ
from known
high affinity
AhR agonists,
contained
within theligand binding
pocket [29].
Nevertheless,the distinct
mechanism(s)of AhR
coupled
with
the
lack
of
results
demonstrating
directcompetitive
bindingofthesechemicals
to theactivation and whether these compounds bind within the AhR ligand binding domain (LBD)
is
AhR
LBD,
raisesquestionsastowhetherthesequinonesaredirectorindirectligandsfortheAhR
unknown. Considering the significant differences in physicochemical characteristics of 1,2-NQ andor if they activate the AhR in a ligand-independent manner. In addition, given the role of 1,2-NQ

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and 1,4-NQ as active functional groups in promising therapeutics, investigation of the cross-species
similarities and differences in AhR binding is also warranted.
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. However, since none of
the tested arsenicals or 4-chlorobiphenyl metabolites were shown to be competitive AhR ligands,
these compounds appeared to activate the AhR and AhR signaling pathway in a ligand-independent
manner. Ligand-independent activation of the AhR and AhR-dependent induction of cyp1a1 by
a variety of chemicals and signal transduction pathways has been previously reported [33–35].
Additionally, since the oxidant H2 O2 has been shown to stimulate the formation of tryptophan
oxidation products (6-formylindolo[3,2-b]carbazole (FICZ) and others) that are extremely high affinity
AhR agonists [36,37], indirect activation of the AhR by chemicals that can generate reactive oxygen
species could explain or partially explain AhR activation by quinones as well as the methylated arsenicalmetabolites. Additionally, chemical-dependent inhibition of CYP1A1/cyp1a1 activity reportedly canlead to activation of the AhR and AhR-dependent gene expression by the low levels of metabolicallylabile, 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. Here we have confirmed that
1,2-NQ and 1,4-NQ can stimulate AhR-dependent gene expression in a species-specific manner andhave 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.
2. Results
2.1. 1,2-NQ and 1,4-NQ Induce AhR-Dependent Gene Expression in Mouse and Human Cells
1,2-NQ and 1,4-NQ were previously reported by Akibo et al. [29,30] to activate the AhR and induce
AhR-dependent gene transcription in mice. Prior to more extensive analysis of the specific interactionsof these compounds with the AhR, we first confirmed that 1,2-NQ and 1,4-NQ stimulated inductionof the AhR-responsive gene CYP1A1/cyp1a1 in human and mouse hepatoma cells, respectively,
in a concentration-dependent manner (Figure 2). In human (HG2L7.5c1) cells, 1,2-NQ and 1,4-NQ
exposure induced CYP1A1 mRNA levels by ~20-fold, whereas these compounds only induced cyp1a1
mRNA levels by ~5-fold in mouse (H1L7.5c3) cells. These results not only showed that 1,2-NQ and
1,4-NQ were more potent inducers of CYP1A1 in human cells relative to cyp1a1 in mouse cells, but that
1,4-NQ was somewhat more potent than 1,2-NQ. Thus, these studies confirm the ability of 1,2-NQ and
1,4-NQ to simulate induction of the AhR-responsive CYP1A1/cyp1a1 gene in human and mouse cells.
2.2. NQ-Mediated Induction of AhR-Dependent Gene Expression Requires ARNTThe canonical AhR gene induction pathway requires AhR heterodimerization with ARNT
for DNA binding and downstream gene transcription, although alternate AhR-dependent and
ARNT-independent mechanisms of gene expression have been reported [39]. While previous
cell-free experiments reported the ability of 1,2-NQ to stimulate binding of his-tagged ARNT withluciferase-tagged AhR [29,30], the direct involvement of ARNT in NQ-mediated gene induction in intact
cells was not examined. To demonstrate a requirement for ARNT in 1,2-NQ- and 1,4-NQ-dependent
induction of AhR-responsive gene expression, we examined the ability of 1,2-NQ and 1,4-NQ to
induce AhR-dependent reporter gene expression in wild-type (Hepa1c1c7) and ARNT-deficient (BPR c1)
mouse hepatoma cells [40]. Cells were transiently transfected with the AhR-responsive luciferase
reporter construct pGudLuc6.1 and either an empty vector (pcDNA3.1) or an ARNT expression
vector and TCDD-, 1,2-NQ, or 1,4-NQ induction of luciferase reporter gene activity determined.

Int. J. Mol. Sci. 2020, 21, 4111

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As expected, while 1,2-NQ, 1,4-NQ, and TCDD induced luciferase activity in wild-type mouse cells
that contain endogenous levels of ARNT, no induction was observed in the ARNT-deficient mouse
cells (Figure 3). However, contransfection of ARNT-deficient cells with an ARNT expression vector,
restored the ability of TCDD, 1,2-NQ, and 1,4-NQ to stimulate luciferase reporter gene activity to levels
comparable to that of wild-type mouse hepatoma cells. Together, these results confirm that ARNT isrequired for the ability of 1,2-NQ and 1,4-NQ to stimulate AhR-dependent gene expression and areconsistent with NQs activating AhR-dependent gene expression through the canonical AhR:ARNT:DRE
Int.
J. Mol. Sci.
2020, 21, x
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signalingmechanism.

Mouse

*

*

20

*

15
10

*
5

50
0n
M
10
00
nM
50
00
nM

50
nM

0

30

50
00
nM

10
00
nM

1,4 NQ

25
20
15
10

*

5

*

*

*

0

50
00
nM

25

*

*

*

0

50
00
nM

10
00
nM

50
0n
M

1,4 NQ

5n
M

Fold Change (cyp1A1 Relative to ACTB)

1,4-NQ

30

50
nM

0

*

5

10
00
nM

5

10

50
0n
M

*

15

50
0n
M

*
10

20

50
nM

*

25

50
nM

15

1,2 NQ

5n
M

*
20

30

5n
M

25

Fold Change (cyp1a1 Relative to ACTB)

1,2 NQ

Fold Change (cyp1a1 Relative to ACTB)

30

5n
M

Fold Change (cyp1A1 Relative to ACTB)

1,2-NQ

Human

Figure 2. 1,2-NQ and 1,4-NQ induce CYP1A1/cyp1a1 mRNA in mouse and human hepatoma cells.
Figure 2. 1,2-NQ and 1,4-NQ induce CYP1A1/cyp1a1 mRNA in mouse and human hepatoma cells.
Human HG2L7.5c.1 and mouse H1L7.5c.3 hepatoma cells were incubated with DMSO (1% v/v),
Human HG2L7.5c.1 and mouse H1L7.5c.3 hepatoma cells were incubated with DMSO (1% v/v), 1,21,2-NQ (5 nM–5 µM) or 1,4-NQ (5 nM–5 µM). After 4h incubation, cells were harvested with TRIzol
NQ (5 nM–5 μM) or 1,4-NQ (5 nM–5 μM). After 4h incubation, cells were harvested with TRIzol
reagent, RNA isolated, cDNA reverse transcribed and CYP1A1/1a1 mRNA transcripts quantitated byreagent, RNA isolated, cDNA reverse transcribed and CYP1A1/1a1 mRNA transcripts quantitated
qPCR as described under Materials and methods. CYP1A1 and cyp1a1 transcript was normalized toby qPCR as described under Materials and methods. CYP1A1 and cyp1a1 transcript was normalized
beta-actin reference gene control and background DMSO was subtracted. Values were expressed as theto beta-actin reference gene control and background DMSO was subtracted. Values were expressedmean ± standard deviation of triplicates of three biological replicates cells and an asterisk (*) indicatesas the mean ± standard deviation of triplicates of three biological replicates cells and an asterisk (*)
those values that were significantly different from DMSO treated cells at p < 0.05 as determined byindicates those values that were significantly different from DMSO treated cells at p < 0.05 as
Two-Way ANOVA.
determined by Two-Way ANOVA.

2.2. NQ-Mediated Induction of AhR-Dependent Gene Expression Requires ARNTThe canonical AhR gene induction pathway requires AhR heterodimerization with ARNT for
DNA binding and downstream gene transcription, although alternate AhR-dependent and ARNTindependent mechanisms of gene expression have been reported [39]. While previous cell-freewild-type mouse cells that contain endogenous levels of ARNT, no induction was observed in the
ARNT-deficient mouse cells (Figure 3). However, contransfection of ARNT-deficient cells with an
ARNT expression vector, restored the ability of TCDD, 1,2-NQ, and 1,4-NQ to stimulate luciferase
reporter gene activity to levels comparable to that of wild-type mouse hepatoma cells. Together,
these results confirm that ARNT is required for the ability of 1,2-NQ and 1,4-NQ to stimulate AhRInt. J. Mol. Sci.
2020, 21, 4111
of 18
dependent
gene expression and are consistent with NQs activating AhR-dependent gene 5expression
through the canonical AhR:ARNT:DRE signaling mechanism.

200

Luciferase Activity (RLU x 103)

TCDD
1,2-NQ
1,4-NQ
150

100

50

*
0

*

*

(+)pcDNA3.1(+) (+)pcDNA3.1(+)
Hepa1c1c7(+)ARNT

BpRc1

Figure 3. Aryl hydrocarbon nuclear translocator (ARNT) is required for aryl hydrocarbon receptor
Figure 3. Aryl hydrocarbon nuclear translocator (ARNT) is required for aryl hydrocarbon
receptor(AhR)-dependent
gene induction by 1,2-NQ and 1,4-NQ. Wild-type and ARNT-defective (BpR c1) mouse(AhR)-dependent gene induction by 1,2-NQ and 1,4-NQ. Wild-type and ARNT-defective (BpRc1)
hepatoma (Hepa1c1c7) cells were transiently transfected with control pcDNA3.1(+) or ARNT expression
mouse hepatoma (Hepa1c1c7) cells were transiently transfected with control pcDNA3.1(+) or ARNT
vector and the AhR-responsive luciferase reporter plasmid pGudLuc6.1. After 24 h, cells were incubatedexpression vector and the AhR-responsive luciferase reporter plasmid pGudLuc6.1. After 24h, cellswith DMSO (0.1%, v/v), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10 nM), 1,2-NQ (5 µM), or 1,4-NQ
were incubated with DMSO (0.1%, v/v), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10 nM), 1,2-NQ(5 µM) for 18 to 22 h. Luciferase activity was measured and corrected for background luciferase activity(5 μM), or 1,4-NQ (5 μM) for 18 to 22 h. Luciferase activity was measured and corrected for(DMSO). Values were expressed as the mean ± standard deviation of triplicates of three biologicalbackground luciferase activity (DMSO). Values were expressed as the mean ± standard deviation of
replicates and an asterisk (*) indicates those values that were significantly different from wild-type
triplicates of three biological replicates and an asterisk (*) indicates those values that wereHepa1c1c7 cells at p < 0.05 as determined by Two-Way ANOVA.
significantly different from wild-type Hepa1c1c7 cells at p < 0.05 as determined by Two-Way
2.3. 1,2-NQ andANOVA.
1,4-NQ Directly Stimulate AhR Transformation and DNA Binding

To assessdirect andactivationof the AhR
by 1,2-NQ
and 1,4-NQ,
examined
2.3. 1,2-NQ
1,4-NQ Directly
Stimulate
AhR Transformation
andwe
DNA
Binding their ability tostimulate AhR transformation and DNA binding. In vitro synthesized mouse AhR (mAhR), human
To assess direct activation of the AhR by 1,2-NQ and 1,4-NQ, we examined their ability to
AhR (hAhR), or the chimeric mAhR-hAhRLBD (a mAhR that contains the hAhR ligand binding
stimulate AhR transformation and DNA binding. In vitro synthesized mouse AhR (mAhR), humandomain [23]) and human or mouse ARNT (hARNT or mARNT, respectively) were incubated with
AhR (hAhR), or the chimeric mAhR-hAhRLBD (a mAhR that contains the hAhR ligand binding
DMSO, TCDD,
andincreasing
concentrationsof 1,2-NQ
or 1,4-NQ.
Therespectively)
mAhR-hAhRLBD
chimera withdomain[23])
and humanor mouse ARNT(hARNT
or mARNT,
were incubated
was used intheseexperiments
todeterminewhetherthe
LBD
was
responsiblefor,orcontributes
to,
DMSO, TCDD, and increasing concentrations of 1,2-NQ or 1,4-NQ. The mAhR-hAhRLBD chimera
the enhancedof theexperiments
NQs of thetohAhR.
In addition,
results
will be ablefor,
to or
becontributes
directly to,
was activity
used in these
determinewhether these
the LBDwas responsible
compared to the subsequent [3 H]TCDD competitive ligand binding analysis experiments whichmust carried out using the mAhR-hAhRLBD chimera instead of the wild-type hAhR because of theextremely lability of the hAhR which makes ligand binding analysis of wild-type hAhR problematic [41].
AhR:ARNT:[32 P]DRE complex formation determination (gel retardation analysis) revealed that 1,2-NQ
and 1,4-NQ stimulated transformation and DNA binding of the human, mouse and mAhR-hAhRLBD
in a concentration-dependent manner (Figure 4). In all cases, 1,4-NQ was a more potent and efficacious
activator of AhR transformation and DNA binding than 1,2-NQ. The enhanced efficacy of NQ-induced
transformation and DNA binding observed with the mAhR-hAhRLBD, compared to the mAhR,
indicates that the hAhR LBD is responsible, at least in part, for the enhanced DNA binding response
observed with NQ activated hAhR. Additionally, co-incubation with the species- and ligand-selective
AhR antagonists CH223191, Stem Reginin 1, or 6,20 ,40 ,-trimethoxyflavone [14,42,43] resulted inhAhRLBD in a concentration-dependent manner (Figure 4). In all cases, 1,4-NQ was a more potent
and efficacious activator of AhR transformation and DNA binding than 1,2-NQ. The enhanced
efficacy of NQ-induced transformation and DNA binding observed with the mAhR-hAhRLBD,
compared to the mAhR, indicates that the hAhR LBD is responsible, at least in part, for theInt.enhanced
J. Mol. Sci. 2020,
21,binding4111
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DNA
response observed with NQ activated hAhR. Additionally, co-incubation
with the species- and ligand-selective AhR antagonists CH223191, Stem Reginin 1, or 6,2′,4′,trimethoxyflavone [14,42,43] resulted in a significant reduction in 1,2-NQ- and 1,4-NQ-dependent
a significant reduction in 1,2-NQ- and 1,4-NQ-dependent AhR transformation and DNA binding of all
AhR transformation and DNA binding of all three AhRs (Supplemental Figure S1). Together, thesethree AhRs (Supplemental Figure S1). Together, these in vitro transformation and DNA binding resultsin vitro transformation and DNA binding results not only are consistent with 1,2-NQ and 1,4-NQ
not only are consistent with 1,2-NQ and 1,4-NQ being direct activators/ligands of the AhR ligands,
being direct activators/ligands of the AhR ligands, but they also suggest that they interact withbutresidues
they alsosuggestinteractwithresidues
withinthan
the AhRligandbinding
cavitydifferently
withinthe that
AhRtheyligandbinding
cavitydifferently
that ofthe high
affinity
AhR
agonist,
than
that
of
the
high
affinity
AhR
agonist,
TCDD.
TCDD.

200

hAhR

1,2 NQ

1,4 NQmAhR-hAhRLBD

200

1,4 NQ

1,2 NQ

150
125
100
75
50

150
125
100
75
50

25

25

0
-10

0
-10

-9

-8

-7

-6

Chemical Concentration [M]

-5

mAhR

1,2 NQ

1,4 NQ

190

175

Specific protein-DNA complex(Percent of TCDD)

Specific protein-DNA complex(Percent of TCDD)

175

Specific protein-DNA complex(Percent of TCDD)

200

180
170
160
80
60
40
20

-9

-8

-7

-6

Chemical Concentration [M]

-5

0
-10

-9

-8

-7

-6

-5

Chemical Concentration [M]Figure 4. 1,2-NQ and 1,4-NQ directly stimulate mouse and human AhR transformation and DNA
Figure 4. 1,2-NQ and 1,4-NQ directly stimulate mouse and human AhR transformation and DNA
binding in vitro. In vitro synthesized hAhR, mAhR-hAhRLBD, or mAhR and hARNT or mARNT
binding in vitro. In vitro synthesized hAhR, mAhR-hAhRLBD, or mAhR and hARNT or mARNT
were incubated in the presence of DMSO (1%, v/v), TCDD (20 nM), 1,2-NQ (0.5 nM–1 µM), or 1,4-NQ
were incubated in the presence of DMSO (1%, v/v), TCDD (20 nM), 1,2-NQ (0.5 nM − 1 μM), or 1,4(0.5 nM–1 µM) for 2 h and protein-DNA complexes were detected by gel retardation analysis. Inducible
NQ (0.5 nM – 1 μM) for 2h and protein-DNA complexes were detected by gel retardation analysis.
AhR:ARNT:[32 P]dioxin response
element (DRE) complexes (indicated by the arrows) were quantified
Inducible AhR:ARNT:[32P]dioxin response element (DRE) complexes (indicated by the arrows) wereusing
Fujifilm
MultiGauge
softwareand values corrected for the amount of protein: DNA complex inquantified using Fujifilm MultiGauge software and values corrected for the amount of protein:
theDNA
presenceof DMSOandthen normalized
to thenthe amountof complex
inducedofby
TCDD.
complexin the (control),presence of
DMSO(control), andnormalized
to the amountcomplex
Values represent the mean ± standard deviation of three exposure replicates and three experiments andrepresentative gels are shown.

2.4. 1,2-NQ and 1,4-NQ Compete with [3H]TCDD for Binding to the Mouse and Human AhR
To demonstrate that 1,2-NQ and 1,4-NQ act as AhR ligands by binding to the AhR ligand binding
pocket, we examined their ability to directly compete with [3 H]TCDD specific binding to the mAhR andmAhR-hAhRLBD. The human AhR is extremely labile and degrades quickly, making ligand binding
analysis of wild-type hAhR difficult [41]. The enhanced stability of the hAhR LBD relative to wild-type
hAhR in the mAhR-hAhRLBD chimera allows quantification of [3 H]TCDD specific binding to the hAhR
LBD [23]. The results in Figure 4 demonstrate that the human LBD present in the mAhR-hAhRLBD
chimera was responsible for enhanced activity of NQs similar to that of wild-type hAhR and thus the
chimera is an appropriate surrogate AhR for hAhR ligand binding analysis of NQs. These analyses

the mAhR and mAhR-hAhRLBD. The human AhR is extremely labile and degrades quickly,
making ligand binding analysis of wild-type hAhR difficult [41]. The enhanced stability of thehAhR LBD relative to wild-type hAhR in the mAhR-hAhRLBD chimera allows quantification of[3H]TCDD specific binding to the hAhR LBD [23]. The results in Figure 4 demonstrate that theInt. J. Mol.
Sci.present
2020, 21, in
4111
human
LBD
the mAhR-hAhRLBD chimera was responsible for enhanced activity of NQs7 of 18
similar to that of wild-type hAhR and thus the chimera is an appropriate surrogate AhR for hAhR
ligand binding analysis of NQs. These analyses demonstrated thatboth 1,2-NQ and 1,4-NQ
demonstrated that both 1,2-NQ and 1,4-NQ competitively inhibit [3 H]TCDD specific binding to thecompetitively inhibit [3H]TCDD specific binding to the mAhR and mAhR-hAhRLBD in a
mAhR and mAhR-hAhRLBD
in a(Figure
concentration-dependent
manner5). Bothwere also
concentration-dependent
manner5). Both NQs werealso (Figure
somewhat
moreNQs
effective3
somewhat ofmore
effectivebinding
competitors[ H]TCDD binding
to thetomAhR-hAhRLBD
comparedcompetitors[3H]TCDD
to the ofmAhR-hAhRLBD
comparedthe mAhR, indicatingthat tothemAhR,
indicating
that
these
NQs
have
a
higherrelativeaffinity
for
the
hAhR
LBD.
these NQs have a higher relative affinity for the hAhR LBD. Overall, these results confirm that Overall,
1,2these
confirm
thatcompetitive
1,2-NQ andAhR
1,4-NQ
are fordirectAhR ligands
for themouse
NQ
andresults
1,4-NQare direct
ligands
the competitive
mouse and human
AhR LBDs
andthese andhuman
AhR
LBDs andthese
areincreased
consistentrelativewith theobserved
increased
relative response of
resultsareconsistentwiththe results
observed
responseof NQson hAhR-dependent
NQs
onhAhR-dependent
gene
expression
andDNAbinding(Figures3
and4).
gene expression and DNA binding (Figures 3 and 4).

Figure 5. 1,2-NQ and 1,4-NQ competitively bind to the human and mouse AhR ligand binding domain.
3 H]TCDD
Figure5. synthesized
1,2-NQ and mAhR
1,4-NQorcompetitively
bind wasto thehuman withand mouseligandinbinding
In vitromAhR-hAhRLBD
incubated
20 nM [AhR
the absence3
domain.
In vitro
synthesizedmAhRor (red)mAhR-hAhRLBD
incubatedwith[ H]TCDD
inor presenceof 1,2-NQ(50 nM–1µM)
or 1,4-NQ (50wasnM–1µM) (blue)
for2030nM
min,
and [3 H]TCDD
theabsenceor presenceof 1,2-NQ(50measurednM–1 μM)
or 1,4-NQ (50 nM–1μM)assay.
(blue)Unprogrammed
for 30 min,boundto theprotein fractionwasby(red)the hydroxyapatite
bindingand[3H]TCDD
bound
to the protein
fraction
was measured
bybindingthe hydroxyapatite
bindinglysatewas used
as a nonspecificbinding
control,and specific
was calculated
as theassay.
difference
Unprogrammed
lysateof wasused asa nonspecificbinding
control,reactions.
and specificwasbetween the amount[3 H]TCDD
boundin the total andnonspecific
Valuesbinding
were normalized
3H]TCDD bound in the total and nonspecific3calculatedasthedifference
betweentheamountof[to the total specific binding of [ H]TCDD in the absence of competitor (set at 100%)
values represent
reactions.
weredeviation
normalized
to the total
specificbindingof [3H]TCDD
the absence ofofthree
the meanValues± standardof triplicate
analysesof threeincubations
and are in
representativecompetitor
at 100%) and values represent the mean ± standard deviation of triplicate analyses ofindividual(setexperiments.
three incubations and are representative of three individual experiments.

2.5. 1,2-NQ and 1,4-NQ Bind within the mAhR and hAhR Ligand Binding Cavity Differently Than TCDD
2.5. 1,2-NQ and 1,4-NQ Bind within the mAhR and hAhR Ligand Binding Cavity Differently than TCDD
The above results demonstrate that 1,2-NQ and 1,4-NQ are structurally unique AhR ligands that candirectlybind toresults
and activate
the mouse
human
and areAhR-dependent
gene expression
through
The above
demonstratethat and
1,2-NQ
andAhR
1,4-NQ
structurally unique
AhR ligands
that
directlybind
to and pathway.
activate themouse differences
and humaninAhR
and AhR-dependent
gene andthe cancanonical
AhR
signaling
However,
the abilityof the three ligandexpression
throughtheantagonists
canonical AhR
signaling
pathway.
However,differencesin the ability Figureof the S1),
species-specific
AhR
to inhibit
the agonist
activityof theseNQs (Supplemental
three
ligandand interact
species-specific
AhR within
antagonists
to ligandinhibit binding
the agonistof these
NQs orsuggestthat they
with residues
the AhRcavityactivity
differently
from TCDD(Supplemental
FigureS1), suggest
thatinteract
residues
within the
AhR ligand
binding [29]other known AhR
agonists.
At first,
wetheyexaminedthewithhypothesis
proposedby Abikoand coworkers
cavityTCDD orbond
otherbetween
known AhR
agonists.
At thiolfirst, group
we examined
aboutdifferently
formationfrom
of a covalent
1,2-NQand theof C327,thebyhypothesis
predicting thepKa of this residue in the 3D model of the mAhR LBD. The obtained pKa value of about 8.9 suggested
that both the orientation of this side-chain into the buried binding cavity and the lack of proximal
basic amino acids do not facilitate the thiol deprotonation at pH = 7. On this basis, we hypothesized
non-covalent interactions between the residues lining the AhR internal cavity and the NQ ligands
drive ligand binding and AhR activation, similar to that of TCDD and other aromatic compounds.
Therefore, to verify our hypothesis of differential binding, we predicted the binding geometries
of NQs by molecular docking to the mAhR LBD generated by homology modeling [44]. 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). TCDD is predicted to occupy the central part of the mAhR cavity and interact withthe most internal hydrophobic region of the ligand binding cavity via the 2-, 3-, or 7-, and 8-position
chlorine atoms on TCDD, which reach the L302 and L309 residues [44]. In stark contrast, both NQs areresidues lining the AhR internal cavity and the NQ ligands drive ligand binding and AhR
activation, similar to that of TCDD and other aromatic compounds.
Therefore, to verify our hypothesis of differential binding, we predicted the binding
geometries of NQs by molecular docking to the mAhR LBD generated by homology modeling [44].
The results revealed that both 1,2-NQ and 1,4-NQ fit within the mAhR ligand binding cavity but
Int. J. Mol. Sci. 2020, 21, 4111
8 of 18
with distinctly different orientations and residue interactions compared to that predicted for thebinding of TCDD (Figure 6A,B). TCDD is predicted to occupy the central part of the mAhR cavity
and interact with the most internal hydrophobic region of the ligand binding cavity via the 2-, 3-, orpredicted to bind at the entrance of the cavity and primarily interact with F289, M334 and M342 residues
7-, and 8-position chlorine atoms on TCDD, which reach the L302 and L309 residues [44]. In stark
for stabilization(Figure 6B). While F289 is known to play a role in stability of TCDD within the mAhR
contrast, both NQs are predicted to bind at the entrance of the cavity and primarily interact withcavity[45],
the two
identified
methionine
residues
(M334
M342)
fromtothe
TCDD-binding
F289,
M334
and M342
residues
for stabilization(Figure6B).andWhile
F289lieis farknown
play
a role inregion,suggesting
that
NQs
and
TCDD
have
distinctly
differentmAhR
bindingfeatures.
tostability of TCDD within the mAhR cavity [45], the two identified methionine residues (M334Similarandthe mAhR,
NQ
molecular
dockingwithinhAhRhomology
models[44]
predicted
that
F295,
M340,
M342) lie far from the TCDD-binding region, suggesting that NQs and TCDD have distinctly
differentmAhR
bindingfeatures.
Similarto alsothe mAhR,
NQ tomoleculardocking within
within thehAhR
and M348(F289,
M334,
and M342
in themAhR)
contribute
NQ stabilization
binding
homology[44] primarypredicted stabilizationthat F295, M340,
and M348 residues(F289, M334,
the mAhR)
pocket(Figuremodels
6D). Themethionine
for andthe M342
NQs in(M340
and M348)
contribute
NQ stabilization
thewithinbindingthe
pocket(Figure6D).(Figure
The primarystabilization
werealso
predicted
totointeract
also withwithin
TCDD
hAhR
cavity6C,D).
The results inmethionine residues for the NQs (M340 and M348) were predicted to interact also with TCDD
Figure 5 indicate that NQs have a somewhat higher relative affinity for the hAhR compared to thewithin the hAhR cavity (Figure 6C,D). The results in Figure 5 indicate that NQs have a somewhat
mAhR, which is also reflective of the NQ-dependent AhR activation and gene expression results.
higher relative affinity for the hAhR compared to the mAhR, which is also reflective of the NQAdditional
stabilization and enhanced affinity of NQs within the hAhR LBD was predicted to be due
dependent AhR activation and gene expression results. Additional stabilization and enhanced
to hydrophobic
interactions
V381
in the mAhR)
thehydrophobichydrogen-bondingof awithcarbonyl
affinity of NQs
within thewith
hAhR
LBD(A375
was predicted
to be anddue tointeractions
oxygen
atom
with
S365
(S359
in
the
mAhR)
(Figure6D).
Molecular
dockingalongwithfunctional
V381 (A375 in the mAhR) and the hydrogen-bonding of a carbonyl oxygen atom with S365 (S359 inligandDNA binding,and gene
expression
analysisconfirmthat
1,2-NQDNA
andbinding,
1,4-NQ bindthebinding,mAhR) (Figure
6D). Moleculardockingalong withfunctional
ligandbinding,gene both
expression
analysisconfirm
1,4-NQbind
and activate bothgene
the mAhR
and andactivate
the mAhR
andhAhRthat
and1,2-NQ
induceandmAhRandhAhR-dependent
expression
and
hAhR
and
inducemAhRandhAhR-dependent
gene
expression
followinginteractions
withM340,
following interactions with key stabilizing residues within the AhR LBD. Stabilization by F295,
keystabilizing
residues
withinthe
AhR
LBD.
Stabilization
by
F295,
M340,
and
M348,
along
and M348, along with additional interactions with V381 and S365 are likely responsible forwith
apparent
additional interactions with V381 and S365 are likely responsible for apparent increased affinity ofincreasedaffinity of NQs for the hAhR.
NQs for the hAhR.

E

150

TCDD

1,2-NQ

1,4-NQ

Relative Luciferase Units(Percent of mAhR TCDD)

125
100
75

* *

25
0

*

*

50

mAhR

*

*

F289A

M334A

*
M342A

Figure 6. NQ interact with specific residues within the AhR binding cavity that are required for AhR
functional activity. The models are shown as cartoons (mAhR in gray, hAhR in orange), and TCDD
and NQs as sticks. The residues that mainly contribute to ∆Gbind are also shown as sticks (green for
TCDD and dark gray for NQs). (A) TCDD pose in the mAhR LBD, taken as reference from previous
analyses [44–46]: (B) NQs bind differently in the mAhR LBD compared to TCDD: the main interactions
are with M334, M342, and F289. (C) TCDD pose in the hAhR: due to the steric hindrance of V381
both the position and the laying plane of the molecule within the cavity are different compared tothe pose in mAhR. (D) The NQs poses in the hAhR are in the same relative position in the bindingsite as in the mAhR LBD, but additional stabilization provided by ligand interactions with V381 andS365 result in differences in NQ orientations. (E) COS-1 cells transiently transfected with wild-type ormutant mAhR pGudLuc6.1 were incubated with DMSO (0.1%, v/v), TCDD (10 nM), 1,2-NQ (5 µM),
or 1,4-NQ (5 µM) for 18 to 22 h. Luciferase activity was measured, corrected for background activity(DMSO), and normalized to TCDD activity induced in mAhR transfected cells. Values represent themean ± standard deviation of three independent experiments. Asterisks (*) indicate those values thatare significantly reduced relative to that of mAhR at p < 0.05 as determined by Two-Way ANOVA.

Int. J. Mol. Sci. 2020, 21, 4111

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2.6. Mutation of F289, M334, and M342 Attenuates NQ-Mediated Induction of
AhR-Dependent Gene Expression
To examine the functional role of the identified NQ ligand binding stabilizing residues in themAhR LBD, F289, M334, and M342, each residue was mutated to alanine (A) and the impact of themutations on NQ-stimulated AhR-dependent gene expression assessed. Mutation of these residues toalanine had no effect on expression levels relative to wild-type mAhR as previously determined via[35 S]methionine-labeling and western blot analysis [22,46]. COS-1 cells were transiently transfectedwith pGudLuc6.1 and wild-type mAhR or mutant mAhR (with F289A, M334A, or M342A substitution)and incubated with TCDD, 1,2-NQ, or 1,4-NQ. As expected, cells transfected with wild-type mAhR
demonstrated induction of AhR-dependent luciferase gene expression following incubation with
TCDD, 1,2-NQ, or 1,4-NQ. In contrast, transfection with F289A mutant mAhR resulted in a ~75%
decrease in TCDD-stimulated AhR-dependent luciferase gene activity and a decrease in 1,2-NQ and
1,4-NQ-stimulated reporter gene activity from 50% of TCDD to less than 15% (Figure 6E). While mutationof M334 and M342 to alanine had no significant effect on TCDD-stimulated AhR-dependent luciferase
gene expression, both mutations decreased induction by 1,2-NQ by ~20% and that of 1,4-NQ by~30–40% compared to wild-type mAhR. 1,4-NQ-stimulated AhR-dependent luciferase gene expression
was more affected by the F289A, M334A, and M342A mutations compared to 1,2-NQ, suggesting
differences in specific NQ binding interactions within the cavity. These results support the AhR
homology model and molecular docking predictions for 1,2-NQ and 1,4-NQ and confirm that mutationof key stabilizing residues (F289, M334, and M342) within the mAhR alter NQ- and AhR-dependent
gene expression.
3. Discussion
The emerging role of naphthoquinones in human disease and potential therapeutic applications
have led to renewed interest in understanding the molecular mechanisms underlying NQ-dependent
physiological and pathophysiological functions. In addition to its role in enhancing xenobiotic
metabolism, the AhR mediates a wide variety of biological and toxicological pathways [14–19]
and the molecular mechanism(s) of NQ-dependent AhR activation (i.e., direct, indirect, and/or
ligand-independent) will aid in the development of novel therapeutics [11,31–35,37,38].
Utilizing a combination of gene expression, reporter gene analysis, gel retardation and ligand
binding analysis, we demonstrate that 1,2-NQ and 1,4-NQ are full AhR ligands/agonists that cancompete with [3 H]TCDD for specific binding to the AhR, stimulate AhR transformation and DNA(DRE) binding in vitro and induce AhR-dependent induction of CYP1A1/cyp1a1 and DRE-driven
reporter gene activity in human and mouse cell lines. Gene expression analysis not only revealed that
1,2-NQ and 1,4-NQ were more potent and efficacious AhR agonists in human cells compared to mouse
cells, but that 1,4-NQ was slightly more potent than 1,2-NQ, consistent with its relative binding to the
AhR. In addition, NQ-stimulated AhR transformation and DNA binding was greater with the hAhR
compared to the mAhR and 1,4-NQ was a more efficacious activator of AhR transformation and DNA
binding than 1,2-NQ in both species. Interestingly, the NQ-stimulated DNA binding results using
the mAhR-hAhRLBD chimera, which contains the hAhR LBD in place of the mAhR LBD, were morecomparable to DNA binding results with the hAhR than that with the mAhR, suggesting that thehAhR LBD is responsible for increased affinity, potency, and efficacy of AhR transformation and DNA
binding observed in human cells. These species-specific differences in potency and efficacy of 1,2-NQ
and 1,4-NQ likely result from their differential interactions with residues within the LBD of the AhR ineach species.
The AhR is a promiscuous transcription factor that has been shown to bind and be activated bya wide range of structurally diverse ligands, and more recently, our group has described ligand-selective
and species-specific AhR activation by the endogenous AhR ligand indirubin [23]. In the case ofindirubin, differences in species-specificity and ligand-selectivity were shown to result from alteredinteractions between the ligand and residues within the human or mouse AhR ligand binding

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domain that resulted in altered AhR-dependent functional changes. This species-selectivity in ligand
binding has also been observed with other structurally diverse AhR ligands [15,21,23]. To assess
whether differences in binding interactions of NQs with residues within the mouse and human AhR
LBDs account for their differential AhR response, we employed molecular docking simulations withestablished hAhR and mAhR homology models [44]. Simulation of the ligand binding event bymolecular docking has previously allowed prediction of the TCDD binding geometry in both themAhR and hAhR LBDs, and the main residues that contribute to the ligand:AhR complex stabilization
were identified and confirmed by site-directed mutagenesis [44]. The same analysis was performed to
predict the binding modes of 1,2-NQ and 1,4-NQ in both the mAhR and hAhR LBD in order to compare
their arrangements within the binding cavity with that of TCDD, as well as to gain insights into
possible species differences in NQ-dependent response. Despite similarities of NQs with TCDD and
TCDD-like chemicals in some molecular features (e.g., coplanarity), NQs show smaller dimensions andhigher polarity. Consequently, docking analysis indicated NQ binding geometries within the mAhR
and hAhR binding cavity that were distinctly different from that of TCDD (Figure 5). TCDD binds
deep within the cavity exhibiting stabilizing interactions both with residues in the middle of the cavity(i.e., the “TCDD-binding fingerprint”, that includes T283, H285, F289, Y316, I319, F345, and A375) [45]and with some hydrophobic residues (L302 and L309) lining the inner part of the cavity [44]. In contrast,
both NQs bind at the entrance of the cavity, thanks to stabilizing interactions with M334 and M342,
as well as some of the TCDD binding residues. Mutation of F289, M334, and M342 to alanine confirmed
this prediction; only 1,2-NQ and 1,4-NQ binding was significantly decreased with the M334A and
M342A mutants, while the F289A mutant AhR failed to respond to NQs or TCDD (Figure 6E). F289A has
been previously shown to negatively affect ligand-dependent AhR transformation and DNA bindingby structurally diverse ligands [22]. Other Authors recently published a docking study, including
the treatment of protein flexibility, of a compound similar to 1,4-NQ, 1,4-dihydroxy-2-naphthoic acid
(1,4-DHNA) [27], and predicted that this ligand binds the AhR in a more internal region of the binding
cavity compared to 1,4-NQ. However, the difference in binding of 1,4-DHNA might be due to theadditional salt-bridge and H-bonds formed by the 2-carboxyl group, which are absent with 1,4-NQ.
Although docking analysis revealed some significant differences in the specific orientation of the NQ
molecules at the entrance of the cavities of the mouse and human AhRs, it remains to be determined
whether these differences in orientation contribute to the observed species differences in AhR response.
Previous TCDD docking simulations within the hAhR homology model have helped provide
explanation as to the role of the internal hAhR residue, V381 (A375 in the mouse), in the reduced
affinity of TCDD for the hAhR as compared to the mAhR. These studies indicated that the larger
valine side-chain reduced the internal space required for accommodating the TCDD molecule [44].
Conversely, both 1,2-NQ and 1,4-NQ ligands were predicted to bind effectively into the modeled hAhR
binding cavity, maintaining relative binding geometries similar to the mAhR (Figure 6D). Not only
does V381 facilitate efficient binding by smaller ligands, such as the NQs, that occupy the entranceof the cavity, but the NQ:hAhR complexes also show enhanced stability compared to the mAhR,
in agreement with the higher relative binding affinity for hAhR determined experimentally. Further,
additional stabilization within the hAhR ligand binding cavity via hydrophobic interaction with V381
is consistent with quantitative structure-activity relationship (QSAR) studies identifying enhanced
antiproliferative and cytotoxic capabilities of naphthoquinones with increased hydrophobicity [8,47].
Compared to 1,2-NQ, 1,4-NQ demonstrated a somewhat higher AhR binding affinity anda greater biological activity in terms of magnitude of AhR-dependent gene expression. Interestingly,
examination of 1,2-NQ and 1,4-NQ as inhibitors of the ATPase domain demonstrated similar resultswith 1,4-NQ binding and acting as a more potent inhibitor of ATPase activity [8]. Further, simple
structural changes in the location of functional groups along the benzene ring appear to alter theability of quinone-based compounds to activate or suppress AhR activation. For example, Fukuda
and coworkers [48] reported that the anthraquinone (AQ) compounds, juglone, 1,4-AQ, and anthralin,suppressed the TCDD-dependent rat cytosolic AhR transformation and DNA binding. Although AQs

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are structurally similar to NQs, AQs contain additional hydroxyl groups that are believed to confer
suppressive AhR effects. In fact, levels of AQ inhibition are suggested to be enhanced by hydroxyl
groups at the C1 or C4 position, which in conjunction with enhanced biological activity observedwith 1,4-NQ, suggests that the C1 and C4 position increase stability through specific interactions
with internal AhR residues for increased/decreased AhR transformation. Flavonoid compounds,
which are structurally similar to NQ compounds but contain an additional heterocyclic ring at theC2 position (i.e., resveratrol, flavone, flavonol, and flavanone), also suppress DNA-binding activityof the AhR; however, flavonoids demonstrate an inverse correlation between number of hydroxyl
groups and inhibitory effects [49]. Moreover, differential binding of NQ-based compounds can resultin varied functional effects. Recently, the microbiota-derived hydroxylated quinoline derivative,
2,8-hydroxyquinoline (2,8-DHQ), was shown to modulate intestinal immune function through thecanonical AhR signaling pathway and the hydroxyl groups were identified as critical structural
determinants for AhR activation [50]. The diversity in AhR ligand binding has also been shown toresult from species-specific differences in the amino acids within the LBD of the AhRs that may alter the
structure of the binding cavity and the specific residues available for ligand binding [23,51,52]. Together,
these studies may help explain why naphthalene, which does not contain additional functional groups(i.e., quinone or hydroxyl groups), fails to bind or activate the AhR, while naphthalene-like structurescontaining one or more functional groups (i.e., quinone, hydroxyl, or halogen groups) can interact withkey amino acid residues to enable AhR binding and activation. The specific differences in AQ, NQ,
and halogenated naphthalene compounds binding within the AhR is beyond the scope of this study,
but future studies comparing the molecular dynamics of these compounds within the AhR binding
cavity will aid in elucidating the mechanisms of binding of diverse naphthalene-based compounds
within the AhR.
1,2-NQ and 1,4-NQ have been shown to influence oxidative status in rodent models and cells in
culture, and have been linked to toxicity [1,2,6,53,54]. Their potential to activate the AhR signaling
pathway in vivo could lead to increased production of reactive metabolites as a result of the inductionof expression of AhR-responsive CYPs (e.g., CYP1A1, CYP1B1, CYP1A2, CYP2A5, etc.) as well as otherenzymes known to be important in the metabolism and activation of naphthalene [2,55,56]; therefore,
we investigated whether AhR antagonists can inhibit NQ-dependent activation of the AhR. Due to theextreme promiscuity of the AhR and diversity of ligands that can bind within the AhR ligand binding
domain, known AhR antagonists have been shown to exhibit ligand-selective and species-specificinhibition of AhR function. CH223191 and TMF reportedly preferentially antagonize the ability of
TCDD and TCDD-like HAHs, but not that of beta-naphthoflavone (BNF) and other flavonoid-like
ligands, from binding to and activating the AhR and AhR-dependent gene expression [43]. In addition,
the AhR antagonist SR1 is reported to be a human-selective AhR antagonist [14]. Interestingly,
co-treatment of CH223191, SR1, or TMF resulted in repression of both 1,2-NQ- and 1,4-NQ-dependent
AhR transformation and DNA binding of the mAhR, hAhR and the chimeric mAhR-hAhRLBD(Supplemental Figure S1). This suggests that each antagonist interacts with distinct residues withinboth the human and mouse AhR ligand binding cavity that effectively inhibits NQ-dependent AhR
binding and/or transformation and is also consistent with the NQs binding differently within the LBD
compared to that of TCDD.
Overall, the ability of 1,2-NQ and 1,4-NQ to directly bind to and activate the AhR and AhR
signal transduction pathways have important implications for their biological, pharmacological,
and toxicological effects. The findings from this study support preferential activation of the human
AhR relative to the mouse, and the propensity of 1,2-NQ and 1,4-NQ to exhibit species-specific AhR
activation. Future studies should investigate whether these species-specific effects can translate into
diverse downstream AhR-dependent effects in order to aid in the development of novel risk mitigation
strategies and therapeutic interventions.

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4. Materials and Methods
4.1. Chemicals
TCDD was a gift from Dr. Stephen Safe (Texas A&M University). [3 H]TCDD (13 Ci/mmol)
was obtained from ChemSyn Laboratories (Lenexa, KS, USA) and [32 P]-ATP (~6000 Ci/mmol)from Perkin-Elmer Life & Analytical Sciences (Waltham, MA, USA). Dimethylsulfoxide (DMSO)(≥ 99.9%), 1,2-NQ (97%) and 1,4-NQ (97%) were from Sigma-Aldrich Chemicals (St. Louis, MO, USA),
CH223191 was from Chembridge Corporation (San Diego, CA, USA), StemRegenin (SR1) from Cayman
hemicals (Ann Arbor, MI, USA), and 6,20 ,40 -trimethoxyflavone (TMF) was a gift from Dr. Gary Perdew(Pennsylvania State University, State College, PA, USA). All compounds were dissolved in DMSO.
4.2. Plasmids
mAhR and mouse ARNT (mARNT) expression plasmids, mβAhR/pcDNA3 andmβARNT/pcDNA3, respectively, have been previously described [22]. The chimeric mouse/human AhR
expression plasmid, mAhR/hAhRLBD/pcDNA3, contains a full length mAhR cDNA in which the ligand
binding domain (LBD) was replaced with the corresponding region of the hAhR LBD [23]. Human AhR
and ARNT cDNAs were obtained from Christopher Bradfield (University of Wisconsin, Madison,
WI, USA) and Oliver Hankinson (University of California, Los Angeles, LA, USA), respectively, and wereinserted in pcDNA3β [57] to generate hβAhR/pcDNA3 and hβARNT/pcDNA3. Point mutations
within the mβAhR/pcDNA3 mutant expression plasmids were generated with the QuikChangeLightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and confirmedby sequencing.
4.3. AhR-Dependent Reporter Gene Induction in Transiently and Stably Transfected Cells
In transient transfection experiments, COS-1 (African green monkey kidney), Hepa1c1c7(mouse hepatoma), and or BPr c1 (ARNT-deficient Hepa1c1c7 [40]) cells maintained in Minimal
Essential Medium (MEM) (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (FBS)(Atlanta Biologicals, Flowery Branch, GA, USA) and 5% Penicillin/Streptomycin (P/S) (Gibco, Carlsbad,
CA, USA) (10,000 U/mL) were plated in a 24-well culture plate at a density of 375,000 cells/well andallowed to attach overnight. Cells were transiently transfected (per well) with 20 ng wild-type
mAhR (mAhR/pcDNA3.1(+)) or mutant AhR expression plasmids (in pcDNA3.1(+)), 20 ng ofmARNT/pcDNA3.1(+) or pcDNA3.1(+) and 200 ng of the AhR-responsive luciferase reporter plasmid
pGudLuc6.1 [58] using Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA, USA) [23].
Twenty-four hours after transfection, cells were incubated with DMSO (0.1%, v/v), or the indicatedconcentration of TCDD, 1,2-NQ, or 1,4-NQ for 18 to 22 h, followed by visual inspection of thecells for toxicity, washing of the cells with phosphate-buffered saline, addition of 50 µL of passive
lysis buffer (Promega, Madison, WI, USA), and lysis of cells for 20 min at room temperature withshaking [59]. Luciferase activity in 50 µL aliquots was measured in an Orion microplate luminometer(Berthold Technologies, Baden-Württemberg, Germany) following automatic injection of stabilized
luciferase reagent as described [59]. In stably transfected cell experiments, recombinant mouse(H1L7.5c3) and human (HG2L7.5c.1) hepatoma cells, which contain the stably transfected enhanced
AhR-responsive reporter gene plasmid pGudLuc7.5 [60,61], were plated (75,000 cells/well) in 96-wellplates and incubated at 37 ◦ C for 24 h prior to chemical treatment. Cells were incubated with DMSO
(1% (v/v)) or the indicated concentration of test chemical in DMSO for 4 h, followed by visual inspection
for toxicity, lysis and measurement of luciferase activity in each well as previously described indetail [59]. Luciferase activity in treated cells was corrected for background luciferase activity present
in DMSO-treated cells and values expressed as relative light units (RLUs) or as a percent of theluciferase activity obtained with the maximally inducing concentration of TCDD.

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4.4. AhR DNA Binding (Gel Retardation) Assay
Wild-type and mutant AhRs and ARNT were synthesized in vitro in the presence of
unlabeled L-methionine using rabbit reticulocyte lysate (Promega, Madison, WI, USA) as previously
described [22,23]. The resulting AhR and ARNT translation mixtures and MEDGK buffer (25 mM MOPS
(3-(N-morpholino)propanesulfonic acid; pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol,
150 mM KCl) were mixed in a 1:1:8 (v/v/v) ratio and incubated with DMSO (1% final concentration)or the indicated concentration of TCDD, 1,2-NQ, or 1,4-NQ in DMSO, in the absence or presence of
10 µM of the desired AhR antagonist (CH223191, SR1, or TMF) (Sigma Aldrich, St. Louis, MO, USA)for the indicated period of time at room temperature. An aliquot of each incubation was mixed with
[32 P]-labeled double-stranded DRE3 containing oligonucleotide and protein-DNA complexes resolved
by gel retardation analysis as described in detail [62]. Gels were visualized using a FLA-9000 Fujifilm
Imager (Fujifilm Life Sciences, Cambridge, MA, USA) and protein-DNA complexes quantitated with
Fujifilm MultiGauge software v3.0.
4.5. Hydroxyapatite [3 H]TCDD Ligand Binding Assay[3 H]TCDD specific binding to wild-type mAhR and the chimeric mouse/human mAhR/hAhRLBD
synthesized in vitro using rabbit reticulocyte lysate (Promega, Madison, WI, USA) was carried out aspreviously described [22). [3 H]TCDD specific binding was determined by subtracting the amount of[3 H]TCDD bound to unprogrammed lysate (nonspecific binding) from the total amount of [3 H]TCDD
binding to lysate containing in vitro expressed AhR. The amount of [3 H]TCDD specific binding
remaining in the presence of competitor chemical was expressed as a percent of the total [3 H]TCDD
specific binding.
4.6. Molecular Modeling
The mAhR and hAhR PASB LBD structures have been previously generated by homology modelingstarting from the sequences: UniProt id P30561 and residues 278−384 for mAhR; UniProt id P35869
and residues 284−390 for hAhR. Three ligand-bound X-ray structures of the HIF-2α protein (PDB ID:
3F1O, 3H7W, and 3H82) were used as templates [44]. Four out of the one hundred models with the best
DOPE score obtained by MODELLER (v. 9v7, University of California, San Francisco, CA, USA) [63]for each species were selected as representatives of the conformational variety of the modeled LBD bycluster analysis. Each model was refined by Molecular Mechanics (MM) energy minimization using
MacroModel (Schrödinger Release 2015-4, Schrödinger, New York, NY, USA) [64] and maintaining
a template ligand inside the binding cavity, as previously described [44]. The NQs molecular structures
were downloaded from PubChem (1,4-NQ CID: 8530; 12-NQ CID: 10667), prepared with LigPrep(Schrödinger Release 2015-4, Schrödinger, New York, NY, USA) [65], and optimized by MM energy
minimization. Molecular docking was carried out using Glide extra precision (XP) (Schrödinger
Release 2015-4, Schrödinger, New York, NY, USA) [66,67]. An ensemble docking approach, basedon ligand docking to the four representative conformations of the modeled receptor, was used toinclude receptor flexibility. To take into account the induced-fit effects of the NQs on the protein
domain, a further optimization of the docking poses was performed using MacroModel [64]. Finally,
the binding free energy (∆Gbind ) of the obtained ligand-receptor complexes was calculated with the
MM Generalized Born Surface Area (MM-GBSA) method [68] implemented in Prime MM-GBSA(Schrodinger Release 2015-4, Schrodinger, New York, NY, USA) [69] that includes evaluation of thesolvation contributions by an implicit solvent model. Visualization of the models was accomplishedusing PyMOL (DeLano Scientific, San Francisco, CA, USA) [70].
4.7. Quantitative Real-Time Polymerase Chain Reaction
Recombinant mouse hepatoma H1L7.5c3 and HG2L7.5c.1 cells, maintained in MEM with 10%
FBS and 5% P/S, were plated in 6-well plates (750,000 cells/mL) and allowed to attach for 24 h.

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DMSO (1% (v/v)) or the indicated concentration of TCDD, 1,2-NQ, or 1,4-NQ was added and the cells
incubated for 4 h followed by isolation of total RNA using TRIzol reagent (ThermoFisher Scientific,
Waltham, MA, USA). cDNA concentration was adjusted to 2 µg/µL and synthesis was performed
using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA)with a Bio-Rad T100 Thermal Cycler (thermal cycler conditions were 10 min 25 ◦ C, 120 min 37 ◦ C,
5 min 85 ◦ C) (Bio-Rad, Hercules, CA, USA). qPCR was performed using the TaqMan Gene Expression
Assay (Applied Biosystems, Foster City, CA, USA) with the TaqMan Fast Universal PCR Master Mix(ThermoFisher Scientific, Waltham, MA, USA), β-actin control (ID: Hs01054797_g1 (Human β-actin;
catalog number 4331182) and Mm02619580_g1 (Mouse β-actin; catalog number 4331182)), and mouse
Cyp1a1 (ID: Mm00487218_m1; catalog number 4331182) or human CYP1A1 (ID: Hs01054796_g1;
catalog number 4331182) TaqMan Gene Expression Probes (Applied Biosystems, Foster City, CA,
USA). The assays were performed in an Applied Biosystems 7500 Fast Sequence Detection System(ThermoFisher Scientific, Waltham, MA, USA), mRNA levels were normalized to β-actin and values
expressed relative to β-actin mRNA levels in untreated cells (set to a value of 1.0) as per the delta-deltaCt (2−∆∆CT ) method [59].
4.8. Statistical Analysis
Analysis of statistical significance of differences between experimental values was conducted using
Two-Way ANOVA. EC50 and IC50 values were calculated using nonlinear regression (three-parameter)
analysis in GraphPad Prism v. 8.1 (La Jolla, CA, USA).
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/11/
4111/s1. Figure S1. NQ-stimulated AhR DNA binding is inhibited by AhR antagonists.
Author Contributions: S.C.F., M.S.D., S.G.T. and L.B. contributed to the conception and design of the study, to theinterpretation of the data, and wrote the manuscript. S.C.F. and S.G.T. performed the experiments. S.G.T. and L.B.
contributed to the modeling and docking studies, while S.C.F. and M.S.D. contributed to the biochemical andmolecular analysis studies. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the National Institute of Environmental Health Sciences (NIEHS)(R01ES007685, Superfund Research Grant P42ES004699, and a Predoctoral Fellowship T32ES007059), the California
Agriculture Experiment Station and the American taxpayers.
Acknowledgments: We gratefully acknowledge Stephen Safe and Gary Perdew for providing reagents and Laura
Van Winkle for helpful discussion on naphthoquinones.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of thestudy; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

Abbreviations
2:8-DHQ
1,2-NQ
1,4-NQ
AhR
AQ
ARNT
BNF
CYP
DMSO
DRE
FICZ
HAH
hAhR
LBD
MM
MM-GBSA

2,8-hydroxyquinoline
1,2-naphthoquinone
1,4-naphthoquinone
aryl hydrocarbon receptor
anthraquinone
aryl hydrocarbon nuclear translocatorbeta-naphthoflavone
cytochrome P450
dimethylsulfoxide
dioxin response element
6-formylindolo[3,2-b]carbazole
halogenated aromatic hydrocarbons
human AhR
ligand binding domain
Molecular Mechanics
MM Generalized Born Surface Area

Int. J. Mol. Sci. 2020, 21, 4111

NQ
mAhR
mAhR-hAhRLBD
NRF2
PAH
PCN
QSAR
TCDD
TMF

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naphthoquinone
mouse AhR
mouse AhR containing the human AhR ligand binding domain
nuclear factor erythroid 2-related factor 2polycyclic aromatic hydrocarbons
polychlorinated naphthalene
quantitative structure-activity relationship
2,3,7,8-tetrachlorodibenzo-p-dioxin
6,20 ,40 -trimethoxyflavone

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