Characterization of the antiallergic drugs 3-[2-(2-phenylethyl) benzoimidazole-4-yl]-3-hydroxypropanoic acid and ethyl 3-hydroxy-3-[2-(2- phenylethyl)benzoimidazol-4-yl]propanoate as full aryl hydrocarbon receptor agonists

Article abstract of DOI:10.1021/tx700350v

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates most of the toxic effects of numerous chlorinated (e.g., TCDD) and nonchlorinated polycyclic aromatic compounds (e.g., benzo[a]pyrene). Studies in AhR null mice suggested that this receptor may also play a role in the modulation of immune responses. Recently, two drugs, namely, M50354 and M50367 (ethyl ester derivative of M50354), were described as AhR ligands with high efficacy toward reducing atopic allergic symptoms in an AhR-dependent manner by skewing T helper cell differentiation toward a T_H1 phenotype [Negishi et al. (2005) J. Immunol. 175 (11), 7348-7356]. Surprisingly, these drugs were shown to have minimal activity toward inducing classical dioxin responsive element-driven AhR-mediated CYP1A1 transcription. We synthesized and reevaluated the ability of these drugs to regulate AhR activity. In contrast to previously published data, both M50354 and M50367 were found to be potent inducers of several AhR target genes, namely, CYP1A1, CYP1B1, and UGT1A2. M50367 was a more effective agonist than M50354, perhaps accounting for its higher bioavailability in vivo. However, M50354 was capable of displacing an AhR-specific radioligand more effectively than M50367. This is consistent with M50354 being the active metabolite of M50367. In conclusion, two selective inhibitors of T_H2 differentiation are full AhR agonists.

Full text of DOI:10.1021/tx700350v

472
Chem. Res. Toxicol. 2008, 21, 472–482
Characterization of the Antiallergic Drugs 3-[2-(2-Phenylethyl)
benzoimidazole-4-yl]-3-hydroxypropanoic Acid and Ethyl
3-Hydroxy-3-[2-(2-phenylethyl)benzoimidazol-4-yl]propanoate as
Full Aryl Hydrocarbon Receptor Agonists
José Luis Morales,† Jacek Krzeminski,‡ Shantu Amin,‡ and Gary H. Perdew*,§
Graduate Program in Biochemistry, Microbiology, and Molecular Biology, Department of Pharmacology,
College of Medicine, and Center for Molecular Toxicology and Carcinogenesis and the Department of
Veterinary and Biomedical Sciences, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802
ReceiVed September 26, 2007
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates most of
the toxic effects of numerous chlorinated (e.g., TCDD) and nonchlorinated polycyclic aromatic compounds
(e.g., benzo[a]pyrene). Studies in AhR null mice suggested that this receptor may also play a role in the
modulation of immune responses. Recently, two drugs, namely, M50354 and M50367 (ethyl ester derivative
of M50354), were described as AhR ligands with high efficacy toward reducing atopic allergic symptoms
in an AhR-dependent manner by skewing T helper cell differentiation toward a TH1 phenotype [Negishi
et al. (2005) J. Immunol. 175 (11), 7348–7356]. Surprisingly, these drugs were shown to have minimal
activity toward inducing classical dioxin responsive element-driven AhR-mediated CYP1A1 transcription.
We synthesized and reevaluated the ability of these drugs to regulate AhR activity. In contrast to previously
published data, both M50354 and M50367 were found to be potent inducers of several AhR target genes,
namely, CYP1A1, CYP1B1, and UGT1A2. M50367 was a more effective agonist than M50354, perhaps
accounting for its higher bioavailability in vivo. However, M50354 was capable of displacing an AhR-
specific radioligand more effectively than M50367. This is consistent with M50354 being the active
metabolite of M50367. In conclusion, two selective inhibitors of TH2 differentiation are full AhR agonists.
based on the expression profiles of numerous cellular markers
and cytokines of CD4+ T helper cells. However, while this
Mosmann paradigm (5) has proven useful and simplifies how
immunological diseases are analyzed and classified, its applica-
tion to other immune cells, namely, CD8+ lymphocytes, is still
occasionally challenged by complex signaling and phenotypic
manifestations that cannot be fit into the TH1-TH2 paradigm
(6, 7). Regardless, TH2-driven conditions such as the acute
relapse symptoms of atopic asthma and airway hyperrespon-
siveness are commonly treated with steroid drugs, such as the
glucocorticoid receptor agonists dexamethasone and prednisone
(8). Unfortunately, these drugs have global immunosuppressive
properties toward both TH1- and TH2-driven clinical conditions
(9, 10) and can also become ineffective during their prolonged
use (11). As a result, the search for drugs capable of specifically
preserving the TH1-driven innate immune system response,
while skewing naïve TH differentiation away from TH2 pheno-
types, may hold promise in the treatment of atopic allergic
diseases and similar TH2-driven conditions.
In 1999, Kato and colleagues published the identification of
a novel benzoimidazole-derived drug ethyl 3-hydroxy-3-[2-(2-
phenylethyl)benzoimidazol-4-yl]propanoate (M50367) (Figure
1), capable of significantly reducing disease scores of experi-
mentally induced asthma and airway hyperresponsiveness in
mice (12). This compound was shown to inhibit important signal
transduction pathways, leading to airway hyperresponsiveness
and asthma, by blocking IL-4 (e.g., important for IgE class
switching) and IL-5 production (e.g., important for the homing
of eosinophils at inflammation sites), respectively (12). M50367
was also capable of enhancing production of the TH1 cytokine
INF-γ in bronchoalveolar lavage fluid and cultured spleenocytes
Introduction
Atopy is an inherited tendency to develop chronic allergic
responses that manifest themselves in the form of asthma,
eczema, and anaphylaxis, among other predominantly T lym-
phocyte helper type 2 (TH2)1-driven disorders. The etiologies
of these allergic conditions have not been fully established,
although polymorphisms in genetic loci coding for proteins
involved in immune system function are often reported (1–4).
Biological parameters such as increased IgE production, eosin-
ophilia, GATA-3 expression, increases in IL-4, IL-5, IL-10, and
tumor necrosis factor-R cytokine production are among a
complex array of markers for TH2-driven immune responses.
Accordingly, immune system disorders are often classified into
T lymphocyte helper type 1 (TH1)- and TH2-biased responses
* To whom correspondence should be addressed. Tel: 814-865-0400.
Fax: 814-863-1696. E-mail: ghp2@psu.edu.
† Graduate Program in Biochemistry, Microbiology, and Molecular
Biology.
‡ Department of Pharmacology, College of Medicine.
§ Center for Molecular Toxicology and Carcinogenesis and the Depart-
ment of Veterinary and Biomedical Sciences.
1 Abbreviations: AhR, aryl hydrocarbon receptor; TCDD, 2,3,7,8-
tetrachlorodibenzo-p-dioxin; B[a]P, benzo[a]pyrene; ꢀ-NF, ꢀ-naphthofla-
vone; R-NF, R-naphthoflavone; 3-MC, 3-methylcholanthrene; M50354, 3-[2-
(2-phenylethyl)benzoimidazole-4-yl]-3-hydroxypropanoic acid; M50367,
ethyl 3-hydroxy-3-[2-(2-phenylethyl)benzoimidazol-4-yl]propanoate; XAP2,
hepatitis B virus X-associated protein 2 also known as ARA9 and AIP;
ARNT, AhR nuclear translocator; hsp90, 90 kDa heat shock protein; TSDS-
PAGE, tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
HOP, hsp90/hsp70 organizing protein; PBS, phosphate-buffered saline;
CYP1A1, cytochrome P450 family I subfamily A isoform 1; TH1 or TH2,
T lymphocyte helper type 1 and type 2, respectively; NF-κB, nuclear factor-κ
B; DRE, dioxin responsive element.
10.1021/tx700350v CCC: $40.75
2008 American Chemical Society
Published on Web 01/08/2008

Two Antiallergic Drugs Are Full AhR Agonists
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 473
3-carbinol (29), bilirubin (30), curcumin (31), quercetin (32),
resveratrol (33), galangin (34), the synthetic compound R-naph-
thoflavone (R-NF) (35), as well as suspected arachidonic acid
metabolites (36).
One of the key observations linking the AhR with normal
immune system function came from the generation of AhR
knockout mice (AhR-/-). The spleen and lymph nodes of
AhR-/- mice display a reduced presence, or perhaps recruit-
ment, of T lymphocytes during the first 10 weeks after birth
(37). Consequently, some of these mice succumb early to
opportunistic infections (37). Others have challenged this
evidence as being limited to the particular strain of mouse
utilized since other AhR-/- mice display normal antigen-
mediated immune responses (38). However, a recent study on
AhR-/- mice revealed that these mice exhibit abnormally
elevated expression of both TH1 and TH2 cytokines in OVA-
sensitized AhR-null mice (14). Therefore, although the mech-
anisms have not been fully established, evidence suggests that
the AhR could still play a balancing role in the immune system.
When M50367 and M50354 were identified as AhR ligands
(14), it was stated that these drugs could only partially induce
CYP1A1 activity when compared to other established AhR
ligands. In addition, these compounds appeared to exhibit unique
AhR-dependent gene expression profiles. These results were
rather surprising and captured our interest due to a lack of
planarity in these molecules, a characteristic that is typical of
most high-affinity AhR ligands (e.g., TCDD). Analogous to the
effects seen for selective modulators of the steroid receptors
and their role in the modulation of immunological responses
(discussed in ref 39), the M50367 and M50354 compounds were
suggested to be selective AhR modulators, driving receptor-
dependent immunological changes without significant induction
of CYP1A1 or other dioxin responsive element (DRE)-driven
genes (14). In this study, the synthesis and further characteriza-
tion of M50367 and M50354 within the AhR signal transduction
pathway are reported. In contrast to published reports, we
demonstrate that both M50367 and M50354 behave as transient
but full AhR agonists, at previously indicated therapeutically
relevant doses (12, 13). The transient but significant nature of
M50354 and M50367 AhR-driven gene expression may have
accounted for the underestimation of this response, due to the
methods and time frame chosen for analyses by previous authors
(14). The implications of these findings, on the potential use of
these drugs in the treatment of atopic allergic diseases, are
further discussed.
Figure 1. Structures of M50354 and M50367.
and had no effect on IL-2 expression (e.g., important for TH1-
driven responses). Consequently, and in contrast to prednisone,
M50367 does not suppress the innate immune system but it is
active in ameliorating both OVA and DNP ascaris-induced
asthma and airway hyperresponsiveness in mice (12). Further-
more, no weight loss in mice or toxicity to cultured spleenocytes
could be observed in vitro, which is in contrast to some of the
side effects exerted by prednisone. Later studies suggested that,
following an oral administration of M50367, the compound is
rapidly metabolized to yield 3-[2-(2-phenylethyl)benzoimida-
zole-4-yl]-3-hydroxypropanoic acid (M50354), which was coined
the “active metabolite” of M50367 (13). The target cells for
M50354 and M50367 were subsequently suggested to be
differentiating naïve TH cells, since none of the drugs affected
mature TH1 or TH2 cell functions (13). The mechanisms by
which M50367 and M50354 elicited these immunomodulatory
activities remained partially unknown until they were identified
as ligands for the aryl hydrocarbon receptor (AhR), a receptor
whose protein expression levels are also modulated during the
differentiation program of naïve TH cells (14).
The AhR is a basic helix–loop–helix and PAS domain
transcription factor and an orphan receptor that mediates most
of the toxic effects elicited by several halogenated polycyclic
aromatic compounds (HPAHs) and nonhalogenated polycyclic
aromatic compounds (PAHs) (15). In its unliganded state, the
AhR is found in the cytoplasmic compartment primarily as a
tetrameric complex composed of two molecules of 90 kDa heat
shock protein (hsp90) and the immunophilin-like protein,
hepatitis B virus X-associated protein 2 (XAP2) (16–18). After
the association of a ligand with the AhR, the tetrameric complex
translocates into the nucleus where the AhR heterodimerizes
with AhR nuclear translocator (ARNT) and dissociates from
the hsp90 dimer and XAP2. The transformed AhR-ARNT
heterodimer then binds xenobiotic responsive elements with the
consensus sequence 5′-TNGCGTGA-3′ and upregulates target
gene expression (19). Activation of the AhR leads primarily to
the upregulation of phase I, II, and III metabolism gene products,
which are important for the clearance and/or activation of
various endogenous and exogenous substances. However,
chronic exposure to high-affinity AhR ligands such as 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) may lead to toxic end
points such as immune system suppression, hydronephrosis, and
porphyric disorders (20–23). The mechanism by which the AhR
is responsible for these conditions remains largely unknown,
but it has been associated with the expression of some of its
target genes such as cytochrome P450 family I subfamily A
isoform 1 (CYP1A1) (24). Moreover, repression of the nuclear
factor-κ B (NF-κB) and AP1 by the AhR signaling pathway
has also been documented (25–28). Even though suspected to
exist, no endogenous high-affinity ligand has been identified
for the AhR that would make clear its role in normal physiologic
homeostasis or a potential role in the immune system. Yet, the
AhR can bind numerous synthetic and naturally occurring
substances that serve as agonists or antagonists such as indole-
Materials and Methods
Chemistry. Synthesis of M50354 and M50367. All solvents
and reagents were used as purchased, without further purification,
unless noted otherwise. Anhydrous tetrahydrofuran (THF) was
prepared by distillation from sodium and benzophenone. Melting
points were recorded on a Fisher-Johns melting point apparatus
1
and are uncorrected. H NMR spectra were recorded on a Bruker
500 MHz spectrometer. Chemical shifts are expressed in ppm with
respect to TMS (tetramethylsilane) as the internal standard. Column
chromatography was performed on a silica gel 60 Å 70–230 mesh
or flash on 230–320 mesh. For thin-layer chromatography, alumi-
num plates precoated with silica gel 60 F254 (0.2 mm) were used.
2-(3-Phenylethyl)benzimidazole-4-carboxylic Acid. To a solu-
tion of 2,3-diaminobenzoic acid (7.8 g, 51.3 mmol) in 120 mL of
dimethylacetamide (11 g, 82.08 mmol, 1.6 equiv), 3-phenylpropi-
onaldehyde was added slowly with stirring followed by potassium
ferricyanide (33 g, 0.1 mol, 2 equiv). The mixture was heated and
stirred at 60 °C under N2 for 12 h. The deep blue solid was filtered
out and washed with ethyl acetate, washings were combined with

474 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Scheme 1. Synthesis of M50354 and M50367
Morales et al.
1
filtrate, and the solvents were removed in vacuo. The residue was
purified by silica gel flash column chromatography using CHCl3
with an increasing amount of EtOH (2–20%) to give 11.5 g (40%)
of acid intermediate 3 (see Scheme 1) as an off-white solid; mp
220 °C (sample was recrystallized from CHCl3:MeOH). 1H NMR
(acetone-d6): δ 3.21–3.25 (m, 2H), 3.32–3.36 (m, 2H), 7.15–7.19
(m, 1H), 7.27–7.35 (m, 5H), 7.86 (d, J ) 8.0 Hz, 1H), 7.90 (d, J
) 8.0 Hz, 1H).
1 as a white solid; mp 123–124 °C. H NMR (CDCl3): δ 1.32 (t,
J ) 7.0 Hz, 3H), 2.87 (d, J ) 6.5 Hz, 2H), 3.17–3.21 (m, 2H),
3.23–3.27 (m, 2H), 4.24 (q, J ) 7.0 Hz, 2H), 5.50 (t, J ) 6.5 Hz,
1H), 7.02 (d, J ) 7.5 Hz, 1H), 7.19 (dd, J ) 8.0 and 7.5 Hz, 1H),
7.24–7.34 (m, 6H), 7.56 (br d, J ) 7.0 Hz, 1H).
The enantiomers of 1 were separated by HPLC using Pirkle
Covalent (S,S) Whelk-O1 column 250 mm × 4.6 mm #786101
(Regis Technologies Inc.). Hexanes/ethanol (92:8) + 8 mM
ammonium acetate were used as a mobile phase in isocratic mode
(flow rate, 1.5 mL/min.). Baseline separation was achieved, and
fractions corresponding to the peaks at 22 and 24 min, respectively,
were collected. Each of them contained single enantiomers as yet
chiroptically unassigned.
3-Hydroxy-3-[2-(ꢀ-phenylethyl)-1H-benzimidazole-4-yl]pro-
pionic Acid. To a solution of 1 (0.2 g, 0.591 mmol) in MeOH (3
mL) was added 0.2 N LiOH in 3:1 MeOH/H2O (6 mL). The
reaction progress was monitored by TLC (4:1 ethyl acetate/MeOH).
After 40 min of stirring at room temperature, 50 mL of water was
added and the pH was brought to 5–5.5 with 25% AcOH. The
mixture was extracted with ethyl acetate, the extract was dried
(MgSO4), and the solvent was removed to give 0.12 g (66%) of 2
Ethyl 3-Oxo-3-[2-(ꢀ-phenylethyl)-1H-benzimidazole-4-yl]pro-
pionate. A mixture of acid 3 (4 g, 15.8 mmol) and thionyl chloride
(80 mL) was refluxed for 3 h. Benzene (250 mL) was then added,
and the solid material was filtered off. It was washed twice with
both benzene and Et2O and dried in vacuo to yield 4.1 g of acid
chloride 4 as a white powder. To a solution of monoethyl malonate
(2.6 g, 20 mmol, 2.5 equiv) in anhydrous THF (45 mL) under N2
was added a few milligrams of 2,2′-bipyridyl as an indicator. The
solution was cooled to -70 °C, and n-BuLi (2.5 M in hexanes, 16
mL, 40 mmol) was added dropwise allowing the temperature to
rise to -5 °C at the end of the addition. The mixture was maintained
at this temperature for 10 min observing the persistence of pink
indicator color. It was then recooled to -65 °C, and the acid
chloride 4 suspension (2 g, 8.0 mmol) in THF (20 mL) was added
dropwise over 5 min. After 1.5 h at -65 °C, the mixture was diluted
with Et2O (90 mL) and acidified to pH 2–3 with ∼60 mL of 1 N
HCl, and the phases were separated. The water phase was alkalized
to pH 8 with 10 N NaOH and extracted either, and the combined
extracts were dried over MgSO4 and evaporated. The crude product
was purified by flash column chromatography (gradient: hexanes/
ethyl acetate from 5 to 50% v/v) to give 1.4 g of pure ꢀ-ketoester
5 as a white solid. The workup of organic (Et2O) phase yielded an
additional 0.4 g of 5; combined yield, 1.8 g (72%); mp 100–101
1
as a white solid; mp 110–112 °C. H NMR (acetone-d6): δ 2.78
(dd, J ) 15.6 and 9.2 Hz, 1H, -CH2-), 2.94 (dd, J ) 15.6 and
3.3 Hz, 1H, -CH2-), 3.17–3.21 (m, 2H), 3.23–3.26 (m, 2H), 5.58
(dd, J ) 9.0 and 3,5 Hz), 7.11–7.21 (m, 3H), 7.25–7.30 (m, 4H),
7.39 (d, J ) 8.0 Hz, 1H).
Biology. Antibodies. The primary antibodies utilized included
mouse anti-AhR monoclonal IgG1 (RPT1) (Affinity BioReagents),
rabbit anti-XAP2 IgG (40), rabbit anti-CYP1A1 IgG (H-70) (Santa
Cruz), and mouse anti-HOP (hsp90/hsp70 organizing protein) IgG
(F5) (David Toft, Department of Biochemistry and Molecular
Biology, Mayo Clinic, Rochester, MN), which were used to detect
the AhR, XAP2, CYP1A1, and HOP proteins, respectively.
Cell Culture. Cells were maintained in modified Eagle’s
R-minimum essential medium (Sigma, St. Louis, MO), supple-
mented with 1000 units/mL penicillin, 100 µg/mL streptomycin
(Sigma), and 7% fetal bovine serum (HyClone, Logan, UT) and
kept in a humidified incubator at 37 °C and 5% CO2. The growth
medium was replaced every 2 days.
Cell Culture Treatments To Determine AhR Transcriptional
Activity. A thousand-fold working stocks of TCDD (10 µM, 0.5
µM, etc.), benzo[a]pyrene (B[a]P) (10 mM), M50354 (10 mM),
and M50367 (10 mM) were prepared in DMSO (Sigma). Unless
otherwise noted in figure legends, cells were dosed by adding each
compound directly into the cell culture plates, mixed, and incubated
for the stated period in the figures and legends. The amount of
DMSO never exceeded 0.1%. Notice that TCDD and B[a]P are
very toxic substances (41). In addition, toxicological information
for M50354 and M50367 has yet to be established. Therefore, these
1
°C. H NMR (CDCl3): δ 1.27 (t, J ) 7.0 Hz, 3H), 3.22–3.25 (m,
2H), 3.28–3.32 (m, 2H), 4.11 (s, 2H), 4.26 (q, J ) 7.0 Hz, 2H),
7.24–7.28 (m, 3H), 7.32–7.36 (m, 3H), 7.74 (d, J ) 7.5 Hz, 1H),
8.01 (d, J ) 8.0 Hz, 1H).
Ethyl 3-Hydroxy-3-[2-(ꢀ-phenylethyl)-1H-benzimidazole-4-
yl]propionate. To a solution of ketoester 5 (0.95 g, 2.83 mmol) in
20 mL of 95% EtOH was added dropwise a solution of sodium
borohydride (44 mg, 1.16 mmol) in EtOH (5 mL) at room
temperature. The progress of the reaction was monitored by TLC
(hexanes/ethyl acetate 1:1). After 1.5 h, the next portion of NaBH4
(10 mg, 0.264) in EtOH (1.5 mL) was added and a half of this
amount was added again after 1.5 h. The reaction was continued
for another 1 h, after which time no starting material was detected
(TLC). The solution was poured into 70 mL of water, acidified to
pH 5–5.5 with 25% AcOH, extracted with ethyl acetate, and dried
(MgSO4). The extract was stripped of solvent to leave a glassy
solid, which, after flash chromatography (hexanes/ethyl acetate,
gradient from 10 to 30% of ethyl acetate), gave 0.82 (86%) of pure

Two Antiallergic Drugs Are Full AhR Agonists
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 475
compounds, including the solvents and reactants utilized to
synthesize them, should be treated as potentially harmful and must
be disposed according to toxicological waste disposal guidelines
established by your institution.
Results
Chemistry. Synthesis of M50367 and M50354. During the
course of this study, the antiallergic agent M50367 [ethyl
3-hydroxy-3-[2-(ꢀ-phenylethyl)-1H-benzimidazole-4-yl]propi-
onate (1)] and its active metabolite M50354 [3-hydroxy-3-[2-
(ꢀ-phenylethyl)-1H-benzimidazole-4-yl]propionate (2)] (Figure
1) were synthesized. The synthetic strategy is outlined in Scheme
1. In the initial step, suitably substituted 2,4-benzimidazole
derivative was synthesized, adopting a published method with
modification (44). Oxidative cyclization of 2,3-diaminobenzoic
acid and 3-phenylpropionaldehyde utilizing potassium ferricya-
nide as an oxidant gave 2-(3-phenylethyl)benzimidazole-4-
carboxylic acid (3) with moderate yield. Acid chloride 4
prepared by standard procedure using thionyl chloride was
without further purification converted to ꢀ-ketoester 5 (45) by
condensation with monethyl malonate. Finally, reduction of 5
with NaBH4 in ethanol furnished the desired ester 1, which upon
hydrolysis (46) gave metabolite 2. In an attempt to obtain pure
enantiomers, 1 was derivatized with auxiliary chiral reagent
(N,N-dimethyl-L-phenylalanine), thus creating diastereomeric
mixture of amino esters. The diastereomers were separated on
flash silica column and characterized by 1H NMR spectroscopy.
However, efforts to restore original secondary hydroxyl func-
tionality from amino ester protection were unsuccessful. Sec-
ondary reactions might have occurred leading to other products,
the nature of which was not investigated. Required separation
and purification of unmodified enantiomers of 1 were finally
achieved by enantioselective HPLC on Whelk-O1 chiral station-
ary phase column.
Biology. M50367 and M50354 Exhibit Significant AhR
Agonistic Activity. The recently published work of Negishi and
colleagues suggested that two potentially useful antiasthmatic
drugs, namely, M50354 and M50367 (Figure 1), had immuno-
modulatory activities that were dependent on the functional
expression of the AhR (14). These substances were identified
as AhR ligands in competition ligand-binding experiments,
although their ability to induce classic AhR-dependent CYP1A1
gene expression was considerably lower when compared to
relatively potent AhR ligands [e.g., ꢀ-naphthoflavone (ꢀ-NF)
and 3-methylcholanthrene (3-MC)]. To further assess the ability
of M50354 and M50367 to activate the AhR, the Hepa-1c1c7-
derived mouse cell line H1L1.1c2 that stably expresses a DRE-
driven luciferase reporter gene was utilized to determine the
extent of AhR activation by M50354 and M50367 vs the
prototypical high-affinity AhR ligand TCDD. In contrast to
previous observations (14), this assay revealed that both M50354
and M50367 could induce significant activity (Figure 2A). In
addition, consistent with the higher in vivo effects of M50367
(12, 13), the M50367 compound was more active than M50354
in this gene reporter test.
Western Blots. Proteins were resolved by tricine sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (TSDS-PAGE) and
transferred to PVDF membranes (Millipore, Billerica, MA) using
standard procedures. Membranes were preincubated with blocking
buffer [10 mM monobasic sodium phosphate mono hydrate, 150
mM sodium chloride, 0.5% (v/v) Tween 20 (Sigma), and 3% (m/
v) bovine serum albumin fraction V (EMD Chemicals Inc., San
Diego, CA)] for 1 h. Primary antibodies were diluted in wash buffer
[10 mM monobasic sodium phosphate monohydrate, 150 mM
sodium chloride, 0.5% (v/v) Tween 20 (Sigma), and 0.1% (m/v)
bovine serum albumin fraction V (EMD Chemicals Inc.)] and
incubated for an additional 1 h at room temperature. Blots were
washed three times in a 30 min period. A 1 h incubation with biotin-
conjugated secondary antibodies was followed by a 15 min
incubation with [125I]streptavidin. Blots were washed, and proteins
were visualized by autoradiography. The relative protein levels were
determined by phosphor image analysis.
Real-Time PCR Analysis. Total mRNA was isolated using
Sigma’s TRI Reagent mRNA isolation reagent (Sigma) and
amplified using the High Capacity cDNA Archive Kit from Applied
Biosystems (Foster City, CA). The levels of CYP1A1, CYP1B1,
and UGT1A2 mRNA were assessed by real-time qPCR using the
MyIQ single-color PCR detection system (BioRad, Hercules, CA)
and the iQ SYBR Green supermix (BioRad).
Luciferase Reporter Gene Experiment. The previously estab-
lished DRE-driven reporter cell lines HG40/6 (42) and H1L1.1c2
(43), derived from the Hep-G2 and Hepa-1c1c7 cell lines, respec-
tively, were utilized to monitor AhR transcriptional activity. Two
days before treatments with ligands, cells were fed with complete
medium containing no antibiotics. Ligands were added at the
specified concentrations for the given period as stated in the figure
legends. After treatments, the luciferase activity was measured using
Promega’s (Madison, WI) Luciferase Assay System following
manufacturer’s instructions.
Determination of the CYP1A1 and CYP1B1 Activity
Directly in Cells. The combined CYP1A1 and CYP1B1 activity
in Hepa-1c1c7 cells was measured using Promega’s P450-Glo
microsomal assay with luciferin-CEE, a substrate primarily me-
tabolized by CYP1A1, CYP1B1, and CYP3A7 and, to a much lesser
extent, by CYP1A2 (<7% CYP1A1) and even lower by other
cytochrome P450s. Briefly, cells grown on 12 well plates were
dosed with the appropriate CYP1A1-inducing agent (e.g., TCDD)
for 6 and/or 24 h. The growth medium was then discarded, and
500 µL of fresh serum-free DMEM containing 8 µL of luciferin-
CEE was added to each well and incubated for an additional 3 h.
The metabolism of luciferin-CEE was then determined according
to the manufacturer’s instructions using a Turner TD-20e lumi-
nometer (Turner BioSystems, Sunnyvale, CA).
Competition Binding Experiments. Hepa-1c1c7 cytosolic
extracts were prepared in MENGM buffer [16.2 mM 3-(N-
morpholino)propanesulfonic acid sodium salt, 10 mM free acid
3-(N-morpholino)propanesulfonic acid, 0.02% (m/v) sodium azide,
10% (m/v) glycerol, 4 mM EDTA, and 20 mM molybdate]. Briefly,
a 150 µL of cytosol at 2 mg/mL was transferred to 12 mm × 75
mm borosilicate glass tubes. Samples were preincubated with cold
competitor for 10 min at room temperature prior to the addition of
radioligand. A total of 0.13 pmol of 2-azido-3-[125I]iodo-7,8-
dibromodibenzo-p-dioxin (photoaffinity ligand) was added per
sample and incubated at room temperature for 30 min in the
presence or absence of competitor. The samples were finally
exposed to 15 W UV lamps (>302 nm) at 8 cm for 4 min. Sixty
microliters of each supernatant (120 µg of total protein) were then
combined with 5× Laemli buffer containing ꢀ-mercaptoethanol,
heated at 95 °C for 5 min, and analyzed by TSDS-PAGE. Resolved
proteins were then transferred to PVDF membranes, and autora-
diographs were generated. The protein bands were quantified via
phosphor image analysis.
However, because reporter gene data alone may not com-
pletely recapitulate the induction of an endogenous AhR target
gene, Promega’s P450-Glo Luciferin-CEE assay was utilized.
Although this assay detects the combined activity of CYP1A1,
CYP1B1, and CYP3A7, the CYP3A7 gene is not a direct target
of the activated AhR. Therefore, in our assay, the ligand-treated
samples predominantly represent the combined activity of
CYP1A1 and CYP1B1. Accordingly, both M50367 and M50354
were capable of inducing a significant level of CYP1A1 and
CYP1B1 catalytic activity, especially when compared to a
saturating dose of TCDD (Figure 2B). Consistent with the
reporter data, M50367 could induce microsomal enzymatic
activity to a higher degree than M50354.

476 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Morales et al.
obtained utilizing the racemic mixture of M50367 (Figure 2),
each M50367 enantiomer at 10 µM could individually induce
similar reporter activity in H1L1.1c2 cells in a time-course
reporter experiment (Figure 3A). However, the extent of
induction was less than a saturating dose of TCDD. Regardless,
as predicted from the H1L1.1c2 reporter cell line results (Figure
2) and Negishi’s report (14), the induction of reporter activity
approached near basal expression by 24 h, while TCDD-induced
reporter activity remained relatively high (Figure 3A). Both
enantiomers were also tested in the human cell line HG40/6
that stably expresses a DRE-driven luciferase reporter gene.
Interestingly, in the HG40/6 reporter line, there was a modest
increase of reporter activity displayed by the P2 enantiomer
(Figure 3B). This higher reporter activity seen for P2 in HG40/6
cells was also consistent with higher CYP1A1 mRNA levels in
the same cell line (Figure 3C). Given the absence of this effect
in the H1L1.1c2 cell line, it was important to test whether such
differences could be recapitulated on an endogenous AhR target
gene and in other cells. Hence, CYP1A1 mRNA expression was
also analyzed via real-time PCR in the wild-type murine Hepa-
1c1c7 and the human keratinocyte-derived cell line HaCaT. Just
as in the reporter cell line H1L1.1c2 (Figure 3A), these
experiments revealed no differences in CYP1A1 mRNA induc-
tion by the M50367 enantiomers (Figure 3D,E). Furthermore,
while neither M50354 nor M50367 could activate the reporter
gene comparably to a saturating dose of TCDD in HG40/6 and
H1L1.1c2 cells (Figure 3A,B), both M50367 enantiomers could
readily induce CYP1A1 mRNA expression to levels comparable
to a saturating dose of TCDD in Hepa-1 and HaCaT cells at
4 h post-treatment (Figure 3D,E). Notably, when either of the
M50367 enantiomers was administered at 1 µM, they ap-
proached the activity of a saturating dose of TCDD in Hepa-
1c1c7 cells and to a lesser degree in HaCaT cells. At 10 µM,
both P1 and P2 induced CYP1A1 mRNA levels to the same
level as a saturating dose of TCDD at 4 h.
Having established that both M50367 enantiomers could
readily activate CYP1A1 gene expression, we examined their
ability to modulate expression of some known AhR target genes
such as CYP1B1, UGT1A2, and the recently identified AhR
regulated gene epiregulin2 (47) in Hepa-1c1c7 and/or HaCaT
cells. Consistent with M50367’s AhR full agonistic activity on
CYP1A1 expression, at 10 µM, both enantiomers could readily
activate other AhR target genes to levels comparable of a
saturating dose of TCDD (Figure 4) in a 4 h dose. Interestingly,
the concentration of M50367 enantiomers required to elicit
maximal induction of these genes varied between cell lines and
in a gene-specific manner.
Figure 2. M50354- and M50367-mediated AhR transcriptional activity.
(A) H1L1.1c2 cells were treated with vehicle (DMSO), TCDD (500
pM), M50354 (10 µM), or M50367 (10 µM) for 6 h. The vehicle
concentration never exceeded 0.1% in all treatments. Cells were
harvested, and the luciferase enzymatic activity was measured. (B)
Hepa-1c1c7 cells were treated with TCDD (10 nM), M50354 (10 µM),
or M50367 (10 µM) for 6 or 24 h. The CYP1A1 enzymatic activity
was measured using Promega’s P450-Glo Assay with Luciferin-CEE
as a CYP1A1 and CYP1B1 substrate, according to the manufacturer’s
instructions. For both A and B, all samples were run in triplicate, and
the results are representative of three independent experiments.
M50367 and M50354 Can Compete with 2-Azido-3-
iodo-7,8-dibromodibenzo-p-dioxin for Binding to the AhR
in a Dose-Dependent Manner. The ability of M50354 and
M50367 compounds to displace [3H]M50354 were initially
provided in Negishi’s report (14), although the experiment was
limited to one saturating concentration and provided no infor-
mation on the relative affinity of M50354 and M50367 for the
AhR. Competition-binding experiments were carried with the
AhR photoaffinity radioligand 2-azido-3-[125I]-iodo-7,8-dibro-
modibenzo-p-dioxin to determine the relative affinity of the
M50354 and M50367 compounds for the AhR and to clearly
demonstrate that these compounds are direct AhR ligands.
Therefore, cytosolic extracts from Hepa-1c1c7 cells were utilized
for competition ligand-binding experiments with the photo-
affinity ligand. These results indicated that the M50354 was
Characterization of M50367 Enantiomers. The approach
utilized for the synthesis of both M50354 and M50367
compounds results in the formation of a racemic mixture of
stereoisomers. M50367 and M50354 enantiomers could be
readily detected via the appropriate HPLC analysis migrating
as two equimolar peaks. For simplicity, we will refer to each
enantiomer of M50367 as the 22 min peak 1 (P1) and 24 min
peak 2 (P2). A method was designed for the separation of
M50367 enantiomers, given that it was important to determine
whether there was a stereospecific requirement for each M50367
enantiomer to function as an AhR agonist or even possibly as
an antagonist. Because M50367 displayed higher activity than
M50354 in our luciferin-CEE-based CYP1A1 assays and the
indication that M50367 has a higher therapeutic effect in vivo
(e.g., mice) (14), we pursued the characterization of both
M50367 enantiomers individually. Consistent with the results
2 Unpublished authors’ observations.

Two Antiallergic Drugs Are Full AhR Agonists
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 477
Figure 3. Characterization of M50367 enantiomers. (A) DRE-driven luciferase reporter gene experiments were performed as in Figure 2A, except
that cells were treated at the concentrations stated in the figure and samples were analyzed at the 0, 4, 8, 16, and 24 h time points. The culture
medium was replaced with fresh medium containing each compound at the stated concentrations. (B) Performed as in panel A, except samples were
collected at 0, 8, and 24 h. (C-E) Following the treatment cells with each compound at the indicated concentrations and a 4 h incubation period,
mRNA was isolated using the Trireagent (Sigma) RNA isolation method and analyzed by RT-PCR analysis. The quantities of mRNA were normalized
to GAPDH mRNA values. All experimental samples were run in triplicate, and the results are representative of three isolated experiments.
capable of displacing the radioligand to a higher degree than
M50367 at 46 vs 24.1% at 10 µM and 90.4 vs 74.5% at 100
µM, respectively (Figure 5). As expected, ꢀ-NF and R-NF could
readily displace the radioligand. As a negative control, an equal
dose with the estrogen receptor ligand 17ꢀ-estradiol was unable
to significantly displace the radioligand at 100 µM. Finally,
M50367 and M50354 could compete specifically with the
radioligand and thus are bonafide AhR ligands.
M50354 and M50367-Promoted AhR Protein Turnover
and Their Effect on the Temporal Expression of CYP1A1
Protein. A key aspect of AhR activation by high affinity ligands
is the rapid loss of AhR protein to proteasome-mediated
degradation (48, 49). To look for potential differences between
these two drugs vs known AhR ligands, their ability to induce
AhR protein degradation was examined. Comparably to TCDD
and B[a]P, M50367 was capable of causing a high degree of
AhR protein turnover after a 6 h incubation period in Hepa-
1c1c7 cells (Figure 6). The M50354 compound was also capable
of inducing AhR degradation, although to a lesser extent than
M50367. In summary, the consistent ranking order for each
agent in causing AhR protein degradation within a 6 h period
was TCDD > M50367 > B[a]P > M50354. However, the level
of CYP1A1 protein expression was comparable in the TCDD,
M50367, and B[a]P experimental samples at 6 h, with lower
but significant levels induced by M50354.
Having established that M50367 and M50354 can cause a
high degree of AhR protein turnover, we wanted to determine
whether CYP1A1 protein upregulation was transient in nature.
Therefore, Hepa-1c1c7 cells were pulsed with each compound
for a total of 6 and 36 h. However, the 6 h treatment group
(group A), just like the 36 h (group B), was also harvested at
the 36 h time point and protein levels were analyzed. As
expected, the level of CYP1A1 expression seen with TCDD at
36 h for group A was comparable to group B, perhaps
accounting for its nonmetabolizable nature and high affinity for
the AhR. For group A, measurable differences in CYP1A1
protein expression could only be observed for TCDD, B[a]P,
and M50367. Thus, even after the M50354-dependent induction
of CYP1A1 seen in our 6 h experiment (Figure 6), the levels
of CYP1A1 expression had returned to basal levels by 36 h in
group A (Figure 7). Surprisingly, the level of CYP1A1
expression remained relatively high for M50367 for group B
and unexpectedly even above B[a]P. Finally, an approximately
2-fold CYP1A1 protein expression was seen for M50354 in
group B.

478 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Morales et al.
Figure 4. M50367 activates other AhR-regulated genes. For panels A-C, the mRNA levels were determined as in Figure 3C-E. All experimental
samples were run in triplicate, and results are representative of two isolated experiments.
Figure 5. M50354 and M50367 are direct ligands for the AhR. Hepa-
1c1c7 cytosol was preincubated with each competing ligand at the
indicated concentrations for 10 min at 25 °C followed by a 30 min
incubation with the photoaffinity radioligand also at 25 °C. Samples
were then exposed to 15 W UV lamps (>302 nm) at a distance of 8
cm for 4 min. Sixty microliters of each supernatant (120 µg of protein)
were then combined with 5× SDS sample buffer supplemented with
ꢀ-mercaptoethanol and heated at 95 °C for 5 min. Proteins were
resolved by TSDS-PAGE and transferred to PVDF membranes, and
Figure 6. M50354 and M50367 induce AhR protein turnover. Following
a 6 h treatment with TCDD (10 nM), B[a]P (10 µM), M50354 (10 µM),
or M50367 (10 µM), whole cell extracts were prepared in RIPA buffer,
and proteins were resolved by TSDS-PAGE and blotted into PVDF
membranes. Primary antibodies mouse monoclonal IgG1 RPT1 (Affinity
BioReagents), rabbit IgG H-70 (Santa Cruz), and mouse IgG F5 (kind
gift from David Toft) were used to identify the AhR, CYP1A1, and HOP
proteins, respectively. To quantitatively detect proteins, these were labeled
with biotin-conjugated secondary antibodies and [125I]streptavidin. The
relative protein levels were determined by phosphor image analysis and
normalized to HOP protein levels, which were not affected by treatments.
Proteins were visualized by autoradiography.
autoradiographs were generated. The protein bands were quantified via
phosphor image analysis and visualized by autoradiography. *Notice
the 1000-fold lower concentration for ꢀ-NF.
Discussion
The antiasthmatic properties of the drug M50367 and its
active metabolite M50354 were revealed in a study designed
to identify novel substances capable of reducing allergic disease
scores in a mouse model of atopic asthma and airway hyper-
responsiveness (12, 13). It was established that these compounds
were capable of promoting a shift in T-helper cell balance
toward a TH1 phenotype, while promoting a reduction in IgE
production and eosinophil infiltration at sites of inflammation.
The AhR became a prime suspect in this M50354/M50367-
elicited TH1 shift, given the ability of both compounds to induce

Two Antiallergic Drugs Are Full AhR Agonists
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 479
Figure 7. CYP1A1-induced activity after a short and long exposure to M50354 and M50367. Hepa-1c1c7 cells were pulsed with TCDD (10 nM),
B[a]P (10 µM), M50354 (10 µM), or M50367 (10 µM) for a total of 6 or 36 h. The 6 h samples (group A) were washed with PBS three times, and
fresh medium was administered and continued to incubate for the remaining 30 h period. The quantification of CYP1A1 and XAP2 protein expression
was determined after 36 h, following the same protocol as in Figure 6, except that CYP1A1 values were normalized to XAP2 protein levels, which
were also not affected by the treatments.
CYP1A1, a gene directly regulated by the AhR (50), and the
absence of a therapeutic effect in AhR-null mice (14). Paradoxi-
cally, these drugs were coined as mild CYP1A1 inducers (14).
However, as shown in Figure 2, these two substances were
potent inducers of CYP1A1 activity in Hepa-1c1c7 cells, well
beyond what was appreciable through reporter assays. Neverthe-
less, these discrepancies are reconciled from our DRE-driven
reporter gene model results (Figure 3A,B), in which a
time-course experiment in HG40/6 and H1L1.1c2 cells with
M50367 enantiomers revealed that reporter gene expression had
returned to near basal levels by 24 h, while induction by TCDD
remained highly elevated (Figure 3A,B). If this time point in a
reporter gene experimental approach was considered alone, as
in a previous report (14), the ability of M50367/M50354 to
activate the AhR would be clearly underestimated. Furthermore,
our survey of other AhR target genes such as CYP1B1,
UGT1A2 (Figure 4), and epiregulin,2 further illustrated that all
were readily activated by either of the M50367 enantiomers.
These results are all consistent with the notion of M50367 as a
full AhR agonist at the dose and time frames examined, while
in comparison, M50354 exhibited similar activity (Figure 2B).
Given that M50354 was coined the active metabolite (13), these
minor differences are likely the result of M50367 having higher
bioavailability in cells as stated previously (12, 13). An
interesting aspect of these results is the lower reporter gene
expression (Figure 4) and higher CYP1A1 activity (Figure 2B)
observed by 24 h. However, these discrepancies could be related
to differences in the half-life of the firefly luciferase and
CYP1A1 proteins. As a result, induced metabolic enzymes (e.g.,
CYP1A1) would remain in the cell well beyond the initial rise
and fall of AhR transcriptional activity as seen in reporter
experiments, which is also consistent with the combined results
shown in Figures 6 and 7.
Interestingly, while no difference could be established
between the M50367 enantiomers in their ability to induce
CYP1A1 and CYP1B1 mRNA expression in Hepa-1c1c7 and
HaCaT cells (Figures 3 and 4), the evaluation of the Hep-G2-
derived HG40/6 reporter cell line revealed that P2 was modestly
more active than P1 in both reporter and real-time PCR
experiments (Figure 3B,C). While the biological significance
or importance of this result remains to be further established, it
indicates that variations in the cell-specific magnitude of
responses are theoretically possible for each enantiomer. This
is consistent with the absence of this effect in HaCaT cells,
which are also of human origin (Figure 3E). The possibility
that the P1 enantiomer could be metabolized more readily than
the P2 in a cell-specific manner is a feasible scenario. Future
studies may further assess whether such differences can be
observed in a tissue-specific manner in vivo.
Competition-binding experiments established that M50354
could displace the photoaffinity ligand more readily than
M50367 (Figure 5). This result is consistent with previous work
suggesting that M50354 was the active metabolite of M50367
(14). Negishi’s conclusion was based on the observation that
shortly after an oral dose with M50367, the compound M50367
could not be detected in plasma but only as its metabolite
M50354 (13). It is important to note that while M50354
displaced the photoaffinity radioligand more readily than
M50367 (Figure 5), this is not sufficient evidence to suggest
that M50354 is the “only” active metabolite. Clearly, R-NF, a
partial AhR antagonist and agonist, can displace the radioligand
more readily than either M50354 or M50367; yet, both M50354

480 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Morales et al.
and M50367 were more active at inducing CYP1A1 and reporter
activity than R-NF at 10 µM in 6 h treatments.2 In addition,
M50367 had more profound and long-lasting effects in cells
than M50354 (Figure 7). Perhaps differences in the ability of
these compounds to transverse the plasma membrane and
activate the AhR may account for the reduced activity of
M50354 in our experiments when compared to M50367 (Figure
2). However, it is also probable that M50367 is simply not
hydrolyzed into M50354 as readily in cells as it is in vivo (13).
The slow hydrolysis of M50367 into M50354 in cell culture
would then account for extended activation of the AhR, as seen
with the induction of CYP1A1 in Figure 7. These issues can
certainly be addressed in future metabolic studies. It was also
evident that at the concentrations of 1 and 10 µM, the level of
radioligand displaced was not as high as expected, especially
since, at these concentrations, a high level of AhR-driven gene
expression could be detected in cell culture experiments.
However, the explanation for such discrepancies remains unclear
and will be addressed in future studies.
antibody production (14). Therefore, our report does not
necessarily negate the potential use of these drugs or derivatives
for the treatment of atopic allergic diseases. Because these drugs
are able to fully induce AhR-mediated microsomal activity in
our tests (Figures 2B, 6, and 7), their structures may still allow
the AhR to achieve an unusual conformation that selectively
modulates specific signaling cascade pathways of the immune
system. Alternatively, the metabolism of these drugs by AhR-
induced metabolic enzymes may activate them into modulators
of the immune system. It is currently unclear whether M50354
and M50367 could also have antagonistic effects on the
enzymatic activity of metabolic enzymes as seen with imidazole-
derived antimycotic drugs, which could also play a role in its
therapeutic effect (57–59). Estrogen signaling has also been
implicated in the regulation of the TH cell balance (60). Because
the activated AhR has been shown to interact with the estrogen
receptor (ER) and attenuate its transcriptional output (61–64),
perhaps the immunomodulatory mechanisms of M50354 or
M50367 could also be associated with AhR-dependent modula-
tion of the ER pathway. Finally, GATA-3 gene downregulation
was among the effects exerted by M50354 and M50367 and
was the presumed explanation for favoring TH1 cytokine
production (14). Future studies can address whether the AhR
can tether to the promoter of GATA-3 and transrepress its
expression, although the repression mechanism could still be
mediated through an alteration in gene expression of other
factors involved in GATA-3 regulation.
We have established that both M50354 and M50367 are
potent but relatively transient inducers of direct AhR target
genes, which is in contrast to a previous publication (14). Given
the suitability of M50354 for a diverse series of chemical
modifications, this compound may be an invaluable tool for
quantitative structure–activity relationship (QSAR) studies. With
specific measurable immunological parameters already estab-
lished (12–14, 65) (e.g., specific cytokines, transcription factor
activation, and so on), perhaps more insights into AhR cellular
functions could be obtained with these compounds serving as
molecular backbones in future QSAR studies.
Another aspect that we examined was whether these sub-
stances can cause AhR protein turnover. This was important
since the reported actions of M50354 and M50367 could have
AhR-dependent immunomodulatory functions without affecting
AhR protein levels. However, consistent with the ability of high-
affinity AhR ligands to induce degradation of the AhR (49, 51),
treatment with either M50367 or M50354 could induce AhR
turnover (Figure 6). Interestingly, the higher turnover observed
between each ligand tested did not correlate with the level of
CYP1A1 inducibility. For example, whereas B[a]P could induce
AhR degradation to a lesser degree than M50367 in the time
frame examined, the level of CYP1A1 protein induction was
still comparable. This result suggests that AhR degradation and
transcriptional activity are differentially regulated.
As seen in Figure 7, the analysis of CYP1A1 expression after
a 6 h pulse or 36 h pulse with each compound revealed that
M50367 had greater potency and lasting effects than M50354
on the expression of CYP1A1 protein, when analyzed at the
36 h time point. While M50354 and M50367 could both induce
CYP1A1 activity to a similar degree in a 6 h dose analysis
(Figure 2B), noticeably after 36 h, M50367 had a profound
effect on CYP1A1 protein expression (Figure 7). Given that
chronic expression of CYP1A1 and other AhR target genes has
been implicated in carcinogenesis (52), perhaps our results
would indicate that M50354 may be a more favorable candidate
to use in vivo. However, the therapeutic index for these
substances has yet to be determined.
Acknowledgment. We thank David Toft for providing us
with the F5 anti-HOP mouse monoclonal antibody, Dr. Graeme
Bolger for the anti-XAP2 antibody, and Steve Safe for TCDD.
We thank Dr. Michael Denison for H1L1.1c2 cells. We also
thank Marcia Perdew for editorial assistance. Rushang Patel,
M.D., is acknowledged for supplying the epiregulin and
UGT1A2 real-time PCR primers. This work was supported by
NIEHS, National Institutes of Health Grants ES04869 and
ES011834.
A potential caveat for the use of these drugs (M50354 and
M50367) in the treatment of human atopic allergic diseases is
warranted by the data presented in this report. The chronic
activation of CYP1A1 and other AhR target genes by TCDD
and several polycyclic aromatic hydrocarbons have often been
associated with several toxic end-points (53, 54). Accordingly,
the upregulation of AhR target genes by M50354 or M50367
would be of concern given the chronic nature of atopic allergic
diseases and the potential need for continuous administration
of these drugs. However, it is possible that the transient nature
of AhR transcriptional activity elicited by M50354 and M50367,
possibly due to their rapid metabolism, may not result in a toxic
end-point as seen with persistent AhR activation by nonme-
tabolizable ligands like TCDD (there is an excellent discussion
about this issue in ref 55). Furthermore, although high-affinity
AhR ligands exert pleiotropic immunosuppressive effects on
the production of various antibody isotypes and cytokines (56),
M50367 preferentially affected TH2-specific cytokine and
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