Nitidine chloride-induced CYP1 enzyme inhibition and alteration of estradiol metabolism

Article abstract of DOI:10.1124/dmd.119.086892

The cytochrome P450 (P450) 1 family is an important phase I enzyme involved in carcinogen activation. Nitidine chloride (NC) is a pharmacologically active alkaloid with polyaromatic hydrocarbon found in the roots of Zanthoxylum nitidum (Roxb.) DC, a traditional medicinal herb widely used in China. We examined the inhibitory effects of NC on CYP1A1, 1B1, and 1A2. NC significantly inhibited CYP1A1- and 1B1-catalyzed ethoxyresorufin O-deethylation activity (IC50 5 0.28±0.06 and 0.32±0.02 mM, respectively) in a concentration- dependent manner, but only showed slight inhibition of CYP1A2 activity (IC50 > 50 mM). Kinetic analysis revealed that NC competitively inhibited CYP1B1 with a Ki value of 0.47±0.05 mM, whereas NC caused a mixed type of inhibition on CYP1A1 with Ki and KI values of 0.14±0.04 and 0.19±0.09 mM, respectively. The observed enzyme inhibition neither required NADPH nor revealed time dependency. Molecular docking manifested the generation of strong hydrogen-bonding interactions of Ser116 in CYP1A1 and Ser127 in CYP1B1 with methoxy moiety of NC. Additionally, NC-induced alteration of estradiol (E2) metabolism was also investigated in the present study. Hydroxyestradiols, including 2-hydroxyestradiol [(2-OHE2) nontoxic] and 4-hydroxyestradiol [(4-OHE2) genotoxic] generated in recombinant enzyme incubation systems and cultured MCF-7 cells were analyzed, and NC was found to preferentially inhibit the nontoxic 2-hydroxylation activity of E2 mediated by CYP1A1. In conclusion, NCwas a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. The remarkable inhibition on E2 2-hydroxylation might increase the risk of 4- OHE2-induced genotoxicity.

Full text of DOI:10.1124/dmd.119.086892

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TITLE PAGE
Nitidine Chloride-Induced CYP1 Enzyme Inhibition and
Alteration of Estradiol Metabolism
Xu Mao, Jian Wang, Qian Wang, Lan Yang, Yilin Li, Hao Lin,
Ying Peng*, and Jiang Zheng*
Wuya College of Innovation (X.M., Q.W., L.Y., Y.L., H.L., Y.P., J.Z.), Shenyang
Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China School of
Pharmacy. State Key Laboratory of Functions and Applications of Medicinal Plants
(J.Z.), Key Laboratory of Pharmaceutics of Guizhou Province and Guizhou Medical
University, Guiyang, Guizhou 550004, P. R. China. School of Pharmaceutical
Engineering (J.W.), Shenyang Pharmaceutical University, Shenyang, Liaoning, P.R.
China
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RUNNING TITLE PAGE
Running Title: Inhibition of CYP1s by nitidine chloride
Corresponding Authors:
Jiang Zheng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, P. R.
China; State Key Laboratory of Functions and Applications of Medicinal Plants, Key
Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University,
Guiyang, Guizhou, 550004, P. R. China
E-mail: zhengneu@yahoo.com
Tel: +86-24-23984215
Fax: +86-24-23986510
The two corresponding units contributed equally to this work.
Ying Peng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang,
Liaoning, 110016, P. R. China
E-mail: yingpeng1999@163.com
Tel: +86-24-23986361
Fax: +86-24-23986510
The number of text pages: 45
The number of figures: 9
The number of references: 35
The number of words in the Abstract: 242
The number of words in the Introduction: 456
The number of words in the Discussion: 1043
Abbreviations: NC, nitidine chloride; ERF, ethoxyresorufin; α-NF, α-naphthoflavone;
E2, estradiol; 2-OHE2, 2-hydroxyestradiol; 4-OHE2, 4-hydroxyestradiol; MDP,
methylenedioxyphenyl; NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate
reduced tetrasodium salt; CYPs, Cytochrome P450 enzymes; LC-MS/MS, liquid
chromatography coupled to tandem mass spectrometry; MRM, multiple reaction
monitoring; EPI, enhanced product ion; IS, internal standard; EROD, ethoxyresorufin
O-deethylation.
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Abstract
Cytochrome P450 (CYPs) 1 family is an important phase Ⅰ enzyme involved in
carcinogen activation. Nitidine chloride (NC) is a pharmacologically active alkaloid
with polyaromatic hydrocarbon found in the roots of Zanthoxylum nitidum (Roxb.) DC,
a traditional medicinal herb widely used in China. We examined the inhibitory effects
of NC on CYPs1A1, 1B1, and 1A2. NC significantly inhibited CYPs1A1 and 1B1-
catalyzed ethoxyresorufin O-deethylation (EROD) activity (IC50 = 0.28 ± 0.06 and 0.32
± 0.02 μM, respectively) in a concentration-dependent manner, but only showed slight
inhibition of CYP1A2 activity (IC50＞50 μM). Kinetic analysis revealed that NC
competitively inhibited CYP1B1 with Ki of 0.47 ± 0.05 μM, whereas NC caused a
mixed type of inhibition on CYP1A1 with Ki and KI of 0.14 ± 0.04 and 0.19 ± 0.09 μM,
respectively. The observed enzyme inhibition neither required NADPH nor revealed
time dependency. Molecular docking manifested the generation of strong hydrogen-
bonding interactions of Ser116 of CYP1A1 and Ser127 of CYP1B1 with methoxy
moiety of NC. Additionally, NC-induced alteration of estradiol (E2) metabolism was
also investigated in the present study.
Hydroxyestradiols, including 2-
hydroxyestradiol (nontoxic) and 4-hydroxyestradiol (genotoxic) generated in
recombinant enzyme incubation systems and cultured MCF-7 cells were analyzed, and
NC was found to preferentially inhibit the nontoxic 2-hydroxylation activity of E2
mediated by CYP1A1. In conclusion, NC was a mixed type inhibitor of CYP1A1 and
a competitive inhibitor of CYP1B1. The remarkable inhibition on E2 2-hydroxylation
might increase the risk of 4-OHE2-induced genotoxicity.
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Significance Statement
CYP1 enzymes catalyze oxidative metabolism of a variety of compounds and are
known to play a crucial role in the development of cancer. CYP1A1 and CYP1A2 are
responsible for hydroxylation of estradiol (E2) at C-2 position, resulting in the
formation of 2-hydroxyestradiol (2-OHE2), which is proposed to be a detoxification
pathway. However, CYP1B1-mediated hydroxylation of E2 at C-4 position has been
suggested to be a tumor initiator. The present study found that nitidine chloride (NC)
is a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. NC
preferentially inhibited the nontoxic E2 2-hydroxylation pathway mediated by
CYP1A1, which might increase the risk of 4-OHE2-induced genotoxicity and cause
severe drug-drug interactions.
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Introduction
Zanthoxylum nitidum (Roxb.) DC as a traditional herbal medicine is mainly
distributed in Asian nations (Li et al., 2017; Liao et al., 2013). The roots of the plant
have been used extensively in China for treatment of various diseases, including
neuralgia, toothache, rheumatism, and swelling of throat (Liu et al., 2017).
Meanwhile, it also usually serves as a natural ingredient of commodities such as
toothpaste. An abundance of alkaloids has been isolated and identified from herbal
roots, and nitidine chloride (NC, Scheme 1) is the major one of them (Arthur et al.,
1959).
The reported pharmacological activities of NC included anti-human
immunodeficiency virus (Tan et al., 1991), anti-inflammatory (Hu et al., 2006; Wang et
al., 2012), anti-malarial (Gakunju et al., 1995; Bouquet et al., 2012), and anti-tumor
agent (Ou et al., 2015; Iwasaki et al., 2010; Pan et al., 2011; Chen et al., 2011; Fang et
al., 2014).
Cytochromes P450 (CYPs) are heme-containing enzymes primarily responsible
for oxidation or reduction of the majority of pharmaceutical agents currently in use
(Foti and Dalvie, 2016). Comprised of vast number of isoforms, this gene superfamily
of proteins mostly belongs to intrahepatic enzymes (Vrba et al., 2004). CYP1 family
plays a crucial role in phase I metabolism of endogenous and exogenous compounds
such as estrogen and polyaromatic hydrocarbon (PAH) and is known to be involved in
generation of carcinogens (Lee et al., 2016; Lo et al., 2013). Furthermore, CYP1
members exhibit differences in tissue distribution. Constitutive expression and
production of CYP1A2 occur in human liver, whereas CYPs1A1 and 1B1 are mainly
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localized in extrahepatic tissues (Lo et al., 2013).
Estradiol (E2, Scheme 1) is a natural estrogen with many physiological effects
(Lozan et al., 2017). Oxidation of E2 to 2-hydroxyestradiol (2-OHE2) and 4-
hydroxyestradiol (4-OHE2) mediated by CYPs1A1, 1B1, and 1A2 has been suggested
to be associated with detoxification or tumorigenesis, respectively (Lakhan et al., 2003;
Lee et al., 2003). Many CYP1 inhibitors have demonstrated to share several structural
motifs which are planar with two or more hydrophobic aromatic rings (Lee et al., 2016;
Zanger and Schwab, 2013). For example, sanguinarine is identified to be a
noncompetitive inhibitor of CYP1A1 and a competitive inhibitor of CYP1A2 (Vrba et
al., 2004). Berberine potently inhibits CYP1B1 activity, whereas CYP1A2 exhibits
resistance to the inhibition (Lo et al., 2015). This led us to speculate that NC may
induce CYP1 enzyme inhibition.
With the rapidly growing global interest in the use of natural products as dietary
supplements and medical remedies, rigorous interdisciplinary studies on the efficacy,
potential toxicity, and health benefits of these botanicals have become increasingly and
considerably significant. In the present study, we investigated the interactions of NC
with human recombinant CYP1 enzymes and explored the alteration of E2 metabolism
mediated by NC.
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Materials and Methods
Chemicals and materials. Nitidine chloride (NC) was used in the study with purity >
98% and acquired from Chengdu Push Bio-Technology Co., Ltd. (Chengdu, China).
Human recombinant CYP1A2 and CYP1B1 were obtained from BD Gentest (Woburn,
MA). Human recombinant CYP1A1 was purchased from Cypex (Dundee, UK).
Ethoxyresorufin (ERF), resorufin, estradiol (E2), ethinyloestradiol (EE2), dansyl
chloride, α-naphthoflavone (α-NF), and propranolol were supplied by Aladdin
Industrial Corporation (Shanghai, China). NADPH was acquired from Sigma-Aldrich
(St. Louis, MO). 2-Hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2)
were purchased from Toronto Research Chemicals Inc. (Toronto, Canada).
Determination of IC50 values of NC-mediated inhibition of CYPs1A1, 1B1, and
1A2. Ethoxyresorufin O-deethylation (EROD) assays (Scheme 1) were performed with
CYP1s to measure enzyme inhibition, according to a published method (Henderson et
al., 2000). Briefly, CYP1A1, CYP1B1, or CYP1A2 (5.0 nM) was incubated with ERF
(2.0 μM) in 200 μL of 100 mM potassium phosphate buffer (pH 7.4) containing NC (0-
100 μM) and MgCl2 (3.2 mM). The incubations were started by adding NADPH (1.0
mM). In addition, α-NF, a known selective inhibitor of CYP1s (Shimada, 2017), was
incubated with ERF as described above to ensure the involvement of CYP1s in EROD
metabolism. After 10 min of incubation at 37 °C, the reaction mixtures were
quenched with 200 μL ice-cold acetonitrile consisting of 50 ng/mL propranolol (IS) and
centrifuged at 19,000 g for 10 min to remove protein. The supernatants were analyzed
by the LC-MS/MS system as described below. The formation of de-ethylation
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metabolite was linear for the first 10 min, and the concentrations of NC and α-NF
required for 50% inhibition of catalytic activity (IC50 values) were calculated by curve
fitting.
Determination of kinetic values for NC-induced inhibition of CYP1A1 and
CYP1B1. Kinetic values for NC-induced inhibition of CYP1 enzymes were determined
by EROD assays. ERF (0.05-5 μM) was mixed with CYP1A1 or CYP1B1 (5.0 nM)
in the presence of NC at concentrations of 0, 0.1, 0.5, or 2.5 μM. The same procedure
was carried out for reaction initiation, reaction termination, and sample preparation as
described above. EROD activity was measured by LC-MS/MS-based monitoring of
resorufin formation as follows. Enzyme activities were assessed by an established
resorufin standard curve. Specifically, calibration standards were obtained by serially
diluting resorufin stock solutions with PBS to the final concentrations of 0.5, 1.0, 2.0,
5.0, 25, 100, and 500 nM, followed by adding equal volume ice-cold acetonitrile with
50 ng/mL propranolol (IS). Seven-point calibration curves were estimated by the plot
of the response ratios of analytes to the internal standard (resorufin/IS) against the
resorufin concentrations, and the linearity was verified by applying 1/x-weighted linear
regression method.
Determination of time- and NADPH-dependent inhibitory effects of NC on
CYP1A1 and CYP1B1. Recombinant CYP1A1 or CYP1B1 (50 nM), NC (0 and 100
μM), and MgCl2 (3.2 mM) were mixed in 120 μLPBS, followed by addition of NADPH
(1.0 mM) for the initiation of the reaction. After incubation at 37 °C for 0, 5, 15, and
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30 min, aliquots (20 μL) were withdrawn and transferred to the secondary incubation
mixtures containing ERF (2.0 μM), MgCl2 (2.0 mM), and NADPH (0.5 mM). The
resulting mixture was incubated 37 °C for 10 min. In a separate study, similar
microsomal incubation without NADPH was carried out to determine the effect of
NADPH on the enzyme inhibition. The reactions were terminated by adding equal
volume ice-cold acetonitrile which contained propranolol as the IS.
After
deproteination by centrifuging, the resulting supernatants were submitted to LC-
MS/MS analysis for the assessment of the residual catalytic activity.
Determination of IC50 values of NC-induced inhibition of 2- and 4-hydroxylation
of E2 mediated by CYP1s. The incubation mixtures were composed of E2 (20 μM),
NC (0-100 μM), and MgCl2 (3.2 mM) fortified with CYP1A1, CYP1B1, or CYP1A2
(10 nM) in 100 μL of potassium phosphate buffer (100 mM, pH 7.4) containing 0.1%
ascorbic acid. The enzymatic reactions were initiated by addition of NADPH (1.0
mM), followed by incubation at 37 °C for 10 min. Reactions were stopped by mixing
with 500 μL of ethyl acetate with EE2 (IS, 25 nM). After vortexing for 30 s, the
organic layer was collected and concentrated under a stream of nitrogen gas, and the
residues were dissolved in 100 μL of sodium bicarbonate buffer (0.1 M, pH 9.0) and
100 μL dansyl chloride (1.0 mg/mL in acetone). The solution was incubated at 60 °C
for 20 min. The resulting samples were cooled on ice and centrifuged at 19,000 g for
10 min. The supernatants were injected into the LC-MS/MS system for analysis as
subsequently described.
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Determination of kinetic parameters for NC-induced inhibition of E2
hydroxylation mediated by CYP1A1. The incubations containing CYP1A1 (10 nM)
and E2 (0.5-80 μM) were performed with NC at concentrations of 0, 0.05, 0.1, 0.2, or
0.5 μM and NADPH (1.0 mM) at 37 °C for 10 min. The resulting incubation mixtures
and metabolite standards (2-OHE2 and 4-OHE2) were extracted and derivatized
following the same procedure as above. After derivatization, all samples were
analyzed by LC-MS/MS.
Cell culture. MCF-7 cells were generously provided by Dr. F.J. Zhang (Shenyang
Pharmaceutical University, China). Cells were cultured in Dulbecco's Modified Eagle
Medium (DMEM, HyClone, GE Healthcare Life Sciences, China) fortified with 10%
fetal bovine serum (FBS, Gibco, Australia) and 1% penicillin-streptomycin solution
(HyClone, GE Healthcare Life Sciences, China) at 37°C in a 5% CO2 environment.
Evaluation of NC-induced inhibition of 2- and 4-hydroxylation of E2 mediated by
CYP1s in MCF-7 cells. MCF-7 cells were seeded on 24-well plates at a density of 1.2
× 105 cells per well. Following 24 h of stabilization, cells were treated with DMEM
containing NC (5.0, 10, and 20 μM). Dimethyl sulfoxide (DMSO, 0.4% v/v) was used
as a negative control. After incubating for 1 h, culture media were removed. The
cells were washed once with PBS, followed by addition of E2 (1.0 μM) and 24 h
incubation. Cell media (1.0 mL) were then collected and extracted with ethyl acetate
(2 × 2 mL) containing EE2 (IS, 5.0 nM) and 0.1% ascorbic acid. The organic layers
were combined and dried under a gentle flow of nitrogen. The residues were
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derivatized as described above, and the resultant samples were submitted to LC-MS/MS
for analysis.
Simulation of binding of NC with CYP1 enzymes by computer modeling and
docking. LibDock module with LibDockScore scoring function of Discovery Studio
v3.0 (DS) was employed to perform the computer modeling of NC. The docking of
NC to putative active sites of three CYP1 enzymes was generated using
crystallographic structures of CYP1A2 (PDB ID: 2HI4), CYP1A1 (PDB ID: 4I8V) and
CYP1B1 (PDB ID: 3PM0). During the docking process, top 10 conformations were
saved for each ligand based on dock score value after the energy minimization using
the smart minimize method through LibDock program. Finally, docking models of
NC with CYP1 members were displayed by PyMOL software (http://www.pymol.org).
LC-MS/MS analysis method. NC-mediated inhibition of EROD and E2 hydroxylation
activity catalyzed by CYP1 enzymes was assessed by monitoring the generation of de-
ethylation and hydroxylation metabolites, respectively. The products were analyzed
on a 5500 triple quadrupole mass spectrometer from AB Sciex (Applied Biosystems,
Foster City, CA) interfaced online with an Agilent 1260 Series HPLC (Agilent
Technologies, Santa Clara, CA). Chromatographic separation was achieved on an
analytical column Promosil C18 (100 × 4.6 mm, 5.0 μm, Agela Technologies, Inc.,
Tianjin, China) at the temperature of 25 °C. Gradient elution was performed for
resorufin with a flow rate of 0.8 mL/min, including mobile phase A (acetonitrile with
0.1% formic acid) and mobile phase B (water with 0.1% formic acid). HPLC gradient
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was set as follows: 10% A at 0-2.0 min; 10-40% A at 2.0-2.5 min; 40-95% A at 2.5-6.0
min; 95% A at 6.0-9.0 min; 95-10% A at 9.0-9.5 min; and 10% A at 9.5-11.0 min.
Isocratic elution was conducted for derivatized 2-OHE2 and 4-OHE2 with 88% (A) at
1.0 mL/min flow rate. The injection volume was 5.0 μL. Quantification was
conducted with an electrospray ionization source (ESI) source in positive mode.
Multiple-reaction monitoring (MRM) scanning was used for targeted analysis with ion
transitions (corresponding to collision energy and declustering potential) m/z
214.0→186.0 for metabolite resorufin (35, 70), m/z 755.0→521.0 for derivatized
metabolites 2-OHE2 and 4-OHE2 (44, 50), m/z 260.3→116.3 for IS propranolol (44,
77), and m/z 530.0→171.0 for derivatized IS EE2 (35, 30), respectively. All
operations and data analysis were processed by Analyst software (version 1.6.3,
Applied Biosystems).
Data analysis and statistics. To evaluate NC-induced inhibition of CYPs1A1 and 1B1
and alteration of E2 metabolism, kinetic analyses were conducted following Michaelis-
Menten kinetic property. Velocity-substrate concentration (v-S) curves were fitted by
no-weighting nonlinear least-squares regression, according to the Michaelis-Menten
equation of GraphPad Prism 5 software (GraphPad Co. Ltd., San Diego, CA). The
data were expressed as the mean ± SD of three independent experiments. For different
types of inhibition, kinetic values (KI and Ki) were calculated as follows.
푉
∙푆
푚푎푥
Competitive inhibition: 푣 = 푆+퐾
[
(
)]
1+ 퐼/퐾
푖
푚
푉
∙푆
푚푎푥
Noncompetitive inhibition: 푣 = [
][ )]
푆+퐾 1+ 퐼/퐾
푖
(
푚
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푉
∙푆
푚푎푥
Mixed type of inhibition: 푣 = 푆 1+ 퐼/퐾
[
(
)]
[
(
)]
+퐾 1+ 퐼/퐾
푖
퐼
푚
I and Vmax are NC concentration and maximal velocity, respectively. Km is the
substrate concentration at half Vmax of the reaction. Ki and KI are the inhibition
constants for the binding of an inhibitor to enzyme and enzyme-substrate complex,
respectively.
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Results
NC-induced inhibition of CYP1s. EROD assays with recombinant enzymes were
conducted to investigate NC-induced inhibitory effects on CYP1s. Among the three
isozymes, NC was found to cause a potent inhibition of CYP1A1 and CYP1B1
activities in a concentration-dependent manner with IC50 values of 0.28 ± 0.06 and 0.32
± 0.02 μM, respectively (Table 1). However, the inhibitory efficiency of NC on
CYP1A2 (IC50＞50 μM) was considerably lower than that with CYP1A1 or CYP1B1
(Figure 1). IC50 values obtained from inhibition tests by α-NF are also listed in Table
1. These data indicate that NC selectively inhibited CYP1A1 and CYP1B1 activities,
especially CYP1A1 activity.
Time- and NADPH-dependent inhibition of NC on CYP1A1 and CYP1B1. We
initially probed different preincubation times on the inhibitory effects of CYP1A1 and
CYP1B1 activities mediated by NC. As shown in Figures 2A and B, NC showed no
time-dependent inhibition on CYPs1A1 and 1B1. We also tested whether the presence
of NADPH is required for enzymatic inactivation induced by NC. No significant
difference of CYP1A1 and CYP1B1 activities was found after 30 min microsomal
incubation with or without NADPH. These results suggest that NC is reversible
inhibitors of CYP1A1 and CYP1B1.
Kinetic analysis of NC-induced inhibition on CYP1A1 and CYP1B1. According to
the IC50 results above, CYP1A2-mediated EROD activity was hardly inhibited by NC.
Thus, kinetic studies were carried out to further evaluate NC-induced inhibition on
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CYP1A1 and CYP1B1. Nonlinear regression analysis for competitive or mixed
inhibition of GraphPad Prism 5 software was utilized to determine the apparent Ki
values and the mode of inhibition. The correlation coefficients (r) were 0.968-0.995.
CYP1A1 catalyzed EROD activity with Vmax and Km values of 21.0 ± 2.3
nmol/min/nmol P450 and 1.30 ± 0.36 μM, respectively. CYP1B1 catalyzed EROD
activity with Vmax and Km values of 5.71 ± 0.39 nmol/min/nmol P450 and 0.31 ± 0.03
μM, respectively. NC competitively inhibited CYP1B1 activity with Ki of 0.47 ± 0.05
μM. However, NC produced mixed type of inhibition of CYP1A1 with Ki and KI of
0.14 ± 0.04 and 0.19 ± 0.09 μM, respectively (Figure 3 and Table 2). Kinetic values
(Ki) of CYP1B1 inhibition by NC was higher than that of CYP1A1 (Ki and KI), which
is in accordance with the IC50 results obtained.
Molecular docking of NC with CYPs1A1, 1A2 and 1B1. To assist the explanation for
the observed inhibition of CYPs1A1 and 1B1, computer modeling of the binding of NC
to CYP1 enzymes was performed to compare NC affinity with the three CYP1s and to
define the interactions with crucial amino acid residue(s). LibDock was used to
perform the interplay of NC with CYP1 enzymes (Figure 4). The dockings of NC to
the active sites of CYP1s showed LibDockScore values in the order of CYP1A1
(153.191) ＞ CYP1B1 (151.352) ＞ CYP1A2 (120.382). The binding of NC to
CYP1A1 displayed a higher score with a lower IC50 value in activity inhibition (Table
1). The methylenedioxy ring of NC lay closely to the heme moiety of the three CYP1
members. Ser127 of CYP1B1 and Ser116 of CYP1A1 were found to interact with
one of the methoxy moieties of NC via hydrogen bonds, respectively. However, no
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hydrogen bond formation was found between the methoxy moieties of NC and
CYP1A2. Instead, two unfavorable interaction bonds were observed in CYP1A2.
Besides, other chemical interactions, including π-π, π-alkyl, etc., have been presented
in Figure 4. There was no significant difference between CYP1 isozymes in forming
hydrophobic interactions with NC. The docking results suggest that Ser127 of
CYP1B1 and Ser116 of CYP1A1 might be of significance for NC-induced enzyme
inhibition.
NC-induced inhibition of 2- and 4-hydroxylation of E2 mediated by CYP1s.
Coincubation studies were performed to determine inhibitory effects of NC on CYP1s-
mediated E2 hydroxylation. NC showed a prominent inhibition on CYP1A1-
catalyzed 2- and 4-hydroxylation of E2 with IC50 values of 0.16 ± 0.004 μM and 0.25
± 0.04 μM, respectively, significantly lower than those of CYP1A2 (IC50 = 13.2 ± 5.56
and 5.35 ± 0.62 μM) and CYP1B1 (IC50 = 35.0 ± 6.44 and 8.27 ± 2.40 μM) (Figure 5
and Table 3). Furthermore, the IC50 for the inhibition of 2-hydroxylation activity of
CYP1A1 was about 60% of that of 4-hydroxylation, revealing that NC preferentially
inhibited CYP1A1-mediated E2 2-hydroxylation activity.
Kinetic parameters for NC-induced inhibition of E2 hydroxylation mediated by
CYP1A1. Due to the strong inhibition of CYP1A1-mediated 2- and 4-hydroxylation of
E2 by NC, kinetic analyses were conducted with various concentrations of NC in the
CYP1A1 system to characterize the type of reversible inhibition. The plots of the
velocity vs. substrate concentrations in CYP1A1-catalyzed 2- and 4-hydroxylation of
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E2 showed a hyperbolicity (Figures 6A and C). The Km and Vmax values for 2-
hydroxylation of E2 by CYPs1A1, 1B1, and 1A2 were 3.38 ± 0.69, 2.26 ± 0.30, and
23.7 ± 4.06 μM, and 6.91 ± 0.33, 0.45 ± 0.04, and 50.6 ± 4.93 nmol/min/nmol P450,
respectively. The Km and Vmax values for 4-hydroxylation of E2 by CYPs1A1, 1B1,
and 1A2 were 28.9 ± 11.5, 0.87 ± 0.16, and 24.8 ± 4.78 μM, and 0.22 ± 0.04, 1.21 ±
0.12, and 0.97 ± 0.14 nmol/min/nmol P450, respectively (Table 3). The catalytic
efficiency (Vmax/Km) of CYP1s-mediated 2- and 4-hydroxylation of E2 were 2.04 and
0.008 ml/min/nmol P450 (CYP1A1), 0.20 and 1.39 ml/min/nmol P450 (CYP1B1), and
2.14 and 0.039 ml/min/nmol P450 (CYP1A2), respectively (Table 3). The calculated
activity ratio (2-/4-hydroxylation) of CYPs1A1, 1B1, and 1A2 were 255, 0.14, and 55,
respectively, indicating that CYPs1A1 and 1A2 primarily catalyzed the formation of 2-
OHE2, and CYP1B1 primarily catalyzed the formation of 4-OHE2 (Scheme 2 and
Table 3). Lineweaver-Burk plots showed that NC-induced inhibition of E2 2-
hydroxylation activity mediated by CYP1A1 was best fit to a mixed-type way with Ki
and KI values were 0.052 ± 0.007 and 0.210 ± 0.016 μM (Figure 6B and Table 4). E2
4-hydroxylation activity inhibition by NC was fit well to a competitive inhibition model
with Ki of 0.26 ± 0.03 μM (Figure 6D and Table 4).
Effects of NC on the formation of E2-hydroxylation metabolites mediated by
CYP1s in MCF-7 cells. As further investigation, the potential inhibitory effects of NC
on CYPs1A1 and 1B1 were evaluated in MCF-7 cells. As shown in Figure 7, both 2-
and 4-OHE2 were detected in cell media after E2 (1.0 μM) treatment with or without
NC. The relative amounts of 2- and 4-OHE2 generated in vehicle- and NC-treated
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groups were estimated. As expected, NC elicited inhibitory effects on 2- and 4-
hydroxylation of E2 in a concentration-dependent manner. Additionally, significantly
decreased levels of 2-OHE2 was found after cells were treated with NC at
concentrations of 10 (P＜0.05) or 20 μM (P＜0.01). The results suggest that NC had
a stronger inhibition on the formation of nontoxic 2-OHE2 in MCF-7 cells.
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Discussion
Botanical dietary supplements consisting of a variety of chemopreventive
constituents with many pharmacological activities are popularly used as natural
alternatives to medicines. However, drug-drug interactions are frequently caused by
some natural product-mediated CYP enzyme inhibition, possibly resulting in severe
adverse effects (Danton et al., 2013). CYP inhibition can be classified into reversible
and irreversible inhibition (VandenBrink, 2010). CYPs are important phase I enzymes
responsible for various biotransformation of endogenous substrates and xenobiotics,
not only existing in intrahepatic tissues but also extensively distributing across
extrahepatic tissues (Liu et al., 2017; Vrba et al., 2004). Thus, safe consumption of
such natural products is becoming a public concern. NC is the major component in
Zanthoxylum nitidum (Roxb.) DC widely used in traditional Chinese medicine. Our
previous research demonstrated that NC shows no remarkable time-dependent
inhibition on other major CYP enzymes in liver except for CYP2D6 (Mao et al., 2018).
Carbene intermediate produced in the metabolic pathway of methylenedioxyphenyl
group of NC might lead to the quasi-irreversible inactivation of CYP2D6. In the
present study, we examined the inhibitory effects of NC on three CYP1 isozymes which
are considered to be linked with the generation of carcinogens. The majority of CYP1
inhibitors contain two or more fused aromatic, exhibiting molecular planarity.
Heterocyclic rings and nitrogen frequently occur in such inhibitors as part of the ring
system and/or in a substituent group (Lee et al., 2016; Zanger and Schwab, 2013). NC
is such a polyaromatic hydrocarbon (PAH) compound containing nitrogen heterocycle.
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Our findings revealed that NC efficiently inhibited EROD activities mediated by
CYP1A1 and CYP1B1 in a concentration-dependent manner with IC50 values of 0.28
and 0.32 μM, respectively, but CYP1A2 was resistant to the inhibition by NC (Figure1
and Table 1). However, time- and NADPH-dependent inhibition of CYP1s by NC
was not observed under the present conditions (Figure 2), indicating that NC is a
reversible inhibitor of CYPs1A1 and 1B1.
Further kinetic studies clearly
demonstrated that NC exhibited competitive inhibition of CYP1B1 with Ki of 0.47 μM,
suggesting that NC bound to the same pocket of CYP1B1 enzyme as that for ERF
(Figure 3 and Table 2). NC showed the mixed-type inhibition of CYP1A1 with lower
Ki and KI of 0.14 and 0.19 μM, respectively, which suggests that NC can not only share
the identical binding pocket with probe substrate (ERF) but also bound to the different
pocket of CYP1A1 enzyme. The observation indicates that NC demonstrated more
significant inhibitory effects on CYP1A1 than that of CYP1B1. In addition,
molecular modeling of NC with CYP1s was conducted to help clarification of NC-
induced difference in inhibition of the three enzymes. Hydrogen-bonding and π-π
interactions were proposed to play a critical role in the binding of alkoxy derivatives of
heterocyclic compounds to CYP1 enzymes (Lo et al., 2013). NC was found to interact
with amino acid residue Ser127 of CYP1B1 or Ser116 of CYP1A1 via hydrogen-
bonding interactions, respectively, but no such hydrogen bond was generated in
CYP1A2 (Figure 4). LibDockScore results also showed that the affinities of NC with
CYPs1A1 and 1B1 higher than that of CYP1A2. Based on the docking score and
molecular interactions with active site residues, the formed hydrogen-bonding of
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CYP1A1 and CYP1B1 with NC might increase the inhibitory susceptibility of the two
enzymes.
E2 is one of the major estrogens in women and has been the standard treatment
option for postmenopausal symptom relief for decades (Wang et al., 2016). However,
it is well known that prolonged exposure of estrogens plays a role in breast cancer
etiology, especially in post-menopausal women (Takemura et al., 2010). As in most
other countries, breast cancer is now the most common cancer in Chinese women; cases
in China account for 9.6% of all deaths from breast cancer worldwide (Fan et al., 2014).
CYPs1A1, 1B1, and 1A2 were demonstrated to mainly catalyze the oxidation of E2 to
2-OHE2 and 4-OHE2 (Lee et al., 2003). 2-Hydroxylation pathway negatively
correlates with breast cancer risk, whereas 4-hydroxylation pathway is likely an
important initiator and promotor in breast cancer (Dunlap et al., 2017). 4-OHE2 is
biotransformed to 3,4-E2-quinone, a known electrophilic species, causing the formation
of DNAadducts and tumorigenesis (Wang et al., 2016; Yager, 2015). Reactive oxygen
species (ROS) can be also produced in this metabolic pathway, contributing to oxidative
DNA damage. 2-Methoxyestradiol (2-MeOE2), generated from the metabolism of
catechol by catechol-O-methyltransferase (COMT), blocks the further oxidation of 2-
OHE2.
Meanwhile, 2-MeOE2, an inhibitor of CYP1B1 responsible for 4-
hydroxylation pathway, has been recently considered to be a promising agent to fight
metastatic breast cancer (Schwarz et al., 2011). Thus, 2-OHE2 and 4-OHE2 may be
regarded as a detoxification and genotoxic biomarker, respectively (Scheme 2). Our
results showed that the formation of 2-OHE2 was primarily mediated by CYP1A1 and
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CYP1A2, and the formation of 4-OHE2 was primarily mediated by CYP1B1 (Scheme
2), which is consistent with the previous report (Itoh et al., 2010). The catalytic
efficiency of E2 2-hydroxylation and 4-hydroxylation by CYP1A1 is approximately
255-fold higher than those of CYP1A2 (55-fold) and CYP1B1 (0.14-fold) (Table 3).
Nevertheless, the IC50 value for NC-induced inhibition of E2 2-hydroxylation of
CYP1A1 was about 60% of the value of 4-hydroxylation, indicating that NC is more
potent to inhibit the generation of 2-OHE2 (Table 3). Kinetic analysis displayed that
NC is a mixed type inhibitor of E2 2-hydroxylation with Ki and KI of 0.052 and 0.21
μM, and a competitive inhibitor of E2 4-hydroxylation with Ki of 0.26 μM (Figure 6
and Table 4). No significant inhibition of E2 metabolism induced by NC was
observed in CYP1B1 or CYP1A2 incubation systems. Additionally, MCF-7 cell
model study showed that NC significantly decreased the formation of 2-OHE2 (Figure
7). Given together, the effect of NC on E2 chemical carcinogenesis is proposed as
shown in Scheme 2. These data suggest that NC-induced inhibitory effect on E2
oxidative metabolism in vitro was mainly contributed from CYP1A1-mediated 2-
hydroxylation pathway. The reduced production of 2-MeOE2 may relatively result in
the accumulation of 4-OHE2. Thus, potential drug-drug interaction should be
considered in the dosage regimen of NC for chemoprotection against the toxicity of 4-
OHE2.
In conclusion, NC is a mixed type inhibitor of CYP1A1 and a competitive inhibitor
of CYP1B1. The observed preferential inhibition of NC on CYP1A1-mediated E2 2-
hydroxylation suggests that 4-OHE2-mediated genotoxicity might be increased by NC.
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Authorship Contributions
Participated in research design: Mao, Zheng, and Peng.
Conducted experiments: Mao, Q. Wang, Yang, Li, and Lin.
Performed data analysis: Mao and J. Wang.
Wrote or contributed to the writing of the manuscript: Mao and Zheng.
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References
Arthur HR, Hui WH, and Ng YL (1959) An examination of the rutaceae of Hong Kong.
Part II. The alkaloids, nitidine and oxynitidine, from Zanthoxylum nitidum. J Chem
Soc O: 1840-1845.
Bouquet J, Rivaud M, Chevalley S, Deharo E, Jullian V, and Valentin A (2012)
Biological activities of nitidine, a potential antimalarial lead compound. Malar J 11:
67.
Chen J, Wang J, Lin L, He L, Wu Y, Zhang L, Yi Z, Chen Y, Pang X, and Liu M (2011)
Inhibition of STAT3 signaling pathway by nitidine chloride suppressed the
angiogenesis and growth of human gastric cancer. Mol Cancer Ther 11(2): 277-287.
Danton AC, Montastruc F, Sommet A, Durrieu G, Bagheri H, Bondon-Guitton E,
Lapeyre-Mestre M, and Montastruc JL (2013) Importance of cytochrome P450
(CYP450) in adverse drug reactions due to drug-drug interactions: a
pharmacovigilance study in France. Eur J Clin Pharmacol 69(4): 885-888.
Dunlap TL, Howell CE, Mukand N, Chen SN, Pauli GF, Dietz BM, and Bolton JL(2017)
Red clover aryl hydrocarbon receptor (AhR) and estrogen receptor (ER) agonists
enhance genotoxic estrogen metabolism. Chem Res Toxicol 30(11): 2084-2092.
Fan L, Strasser-Weippl K, Li JJ, St Louis J, Finkelstein DM, Yu KD, Chen WQ, Shao
ZM, and Goss PE (2014) Breast cancer in China. Lancet Oncol 15(7): e279-289.
Fang Z, Tang Y, Jiao W, Xing Z, Guo Z, Wang W, Xu Z, and Liu Z (2014) Nitidine
24

DMD Fast Forward. Published on May 30, 2019 as DOI: 10.1124/dmd.119.086892
This article has not been copyedited and formatted. The final version may differ from this version.
DMD/2019/086892
chloride induces apoptosis and inhibits tumor cell proliferation via suppressing ERK
signaling pathway in renal cancer. Food Chem Toxicol 66: 210-216.
Foti RS and Dalvie DK (2016) Cytochrome P450 and non-cytochrome P450 oxidative
metabolism: contributions to the pharmacokinetics, safety and efficacy of xenobiotics.
Drug Metab Dispos 44(8): 1229-1245.
Gakunju DM, Mberu EK, Dossaji SF, Gray AI, Waigh RD, Waterman PG, and Watkins
WM (1995) Potent antimalarial activity of the alkaloid nitidine, isolated from a
Kenyan herbal remedy. Antimicrob Agents Chemother 39(12): 2606-2609.
Henderson MC, Miranda CL, Stevens JF, Deinzer ML, and Buhler DR (2000) In vitro
inhibition of human P450 enzymes by prenylated flavonoids from hops, Humulus
lupulus. Xenobiotica 30(3): 235-251.
Hu J, Zhang WD, Liu RH, Zhang C, Shen YH, Li HL, Liang MJ, and Xu XK (2006)
Benzophenanthridine alkaloids from Zanthoxylum nitidum (Roxb.) DC, and their
analgesic and anti-inflammatory activities. Chem Biodivers 3(9): 990-995.
Itoh T, Takemura H, Shimoi K, and Yamamoto K (2010) A 3D model of CYP1B1
explains the dominant 4-hydroxylation of estradiol. J Chem Inf Model 50(6): 1173-
1178.
Iwasaki H, Okabe T, Takara K, Toda T, Shimatani M, and Oku H (2010) Tumor-
selective cytotoxicity of benzo[c]phenanthridine derivatives from Toddalia asiatica
Lam. Cancer Chemother Pharmacol 65(4): 719-726.
25

DMD Fast Forward. Published on May 30, 2019 as DOI: 10.1124/dmd.119.086892
This article has not been copyedited and formatted. The final version may differ from this version.
DMD/2019/086892
Lakhani NJ, Sarkar MA, Venitz J, and Figg WD (2003) 2-Methoxyestradiol, a
promising anticancer agent. Pharmacotherapy 23(2): 165-172.
Lee AJ, Cai MX, Thomas PE, Conney AH, and Zhu BT (2003) Characterization of the
oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively
expressed human cytochrome P450 isoforms. Endocrinology 144(8): 3382-3398.
Lee D, Perez P, Jackson W, Chin T, Galbreath M, Fronczek FR, Isovitsch R, and Iimoto
DS (2016) Aryl morpholino triazenes inhibit cytochrome P450 1A1 and 1B1. Bioorg
Med Chem Lett 26(14): 3243-3247.
Liao J, Xu T, Zheng JX, Lin JM, Cai QY, Yu DB, and Peng J (2013) Nitidine chloride
inhibits hepatocellular carcinoma cell growth in vivo through the suppression of the
JAK1/STAT3 signaling pathway. Int J Mol Med 32(1): 79-84.
Li W, Yin H, Bardelang D, Xiao J, Zheng Y, and Wang R (2017) Supramolecular
formulation of nitidine chloride can alleviate its hepatotoxicity and improve its
anticancer activity. Food Chem Toxicol 109: 923-929.
Liu L, Sun S, Rui H, and Li X (2017) In vitro inhibitory effects of dihydromyricetin on
human liver cytochrome P450 enzymes. Pharm Biol 55(1): 1868-1874.
Lo SN, Chang YP, Tsai KC, Chang CY, Wu TS, and Ueng YF (2013) Inhibition of
CYP1 by berberine, palmatine, and jatrorrhizine: selectivity, kinetic characterization,
and molecular modeling. Toxicol Appl Pharmacol 272(3): 671-680.
Lo SN, Shen CC, Chang CY, Tsai KC, Huang CC, Wu TS, and Ueng YF (2015) The
26

DMD Fast Forward. Published on May 30, 2019 as DOI: 10.1124/dmd.119.086892
This article has not been copyedited and formatted. The final version may differ from this version.
DMD/2019/086892
effect of oxidation on berberine-mediated CYP1 Inhibition: oxidation behavior and
metabolite-mediated inhibition. Drug Metab Dispos 43(7): 1100-1107.
Lozan E, Shinkaruk S, Al Abed SA, Lamothe V, Potier M, Marighetto A, Schmitter JM,
Bennetau-Pelissero C, and Buré C (2017) Derivatization-free LC-MS/MS method for
estrogen quantification in mouse brain highlights a local metabolic regulation after
oral versus subcutaneous administration. Anal Bioanal Chem 409(22): 5279-5289.
Mao X, Hu Z, Wang Q, Zhang N, Zhou S, Peng Y, and Zheng J (2018) Nitidine chloride
is a mechanism-based inactivator of CYP2D6. Drug Metab Dispos 46(8): 1137-1145.
Ou X, Lu Y, Liao L, Li D, Liu L, Liu H, and Xu H (2015) Nitidine chloride induces
apoptosis in human hepatocellular carcinoma cells through a pathway involving p53,
p21, Bax and Bcl-2. Oncol Rep 33(3): 1264-1274.
Pan X, Han H, Wang L, Yang L, Li R, Li Z, Liu J, Zhao Q, Qian M, Liu M, and Du B
(2011) Nitidine chloride inhibits breast cancer cells migration and invasion by
suppressing c-Src/FAK associated signaling pathway. Cancer Lett 313(2): 181-191.
Schwarz D, Kisselev P, Schunck WH, and Roots I (2011) Inhibition of 17β-estradiol
activation by CYP1A1: genotype- and regioselective inhibition by St. John's Wort and
several natural polyphenols. Biochim Biophys Acta 1814(1): 168-174.
Shimada T (2017) Inhibition of carcinogen-activating cytochrome P450 enzymes by
xenobiotic chemicals in relation to antimutagenicity and anticarcinogenicity. Toxicol
Res 33(2): 79-96.
27

DMD Fast Forward. Published on May 30, 2019 as DOI: 10.1124/dmd.119.086892
This article has not been copyedited and formatted. The final version may differ from this version.
DMD/2019/086892
Takemura H, Uchiyama H, Ohura T, Sakakibara H, Kuruto R, Amagai T, and Shimoi K
(2010) A methoxyflavonoid, chrysoeriol, selectively inhibits the formation of a
carcinogenic estrogen metabolite in MCF-7 breast cancer cells. J Steroid Biochem Mol
Biol 118(1-2): 70-76.
Tan GT, Pezzuto JM, Kinghorn AD, and Hughes SH (1991) Evaluation of natural
products as inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse
transcriptase. J Nat Prod 54: 143-154.
VandenBrink BM and Isoherranen N (2010) The role of metabolites in predicting drug-
drug interactions: focus on irreversible cytochrome P450 inhibition. Curr Opin Drug
Discov Devel 13(1): 66-77.
Vrba J, Kosina P, Ulrichová J, and Modrianský M (2004) Involvement of cytochrome
P450 1A in sanguinarine detoxication. Toxicol Lett 151(2): 375-387.
Wang S, Dunlap TL, Howell CE, Mbachu OC, Rue EA, Phansalkar R, Chen SN, Pauli
GF, Dietz BM, and Bolton JL (2016) Hop (Humulus lupulus L.) extract and 6-
prenylnaringenin induce P450 1A1 catalyzed estrogen 2-hydroxylation. Chem Res
Toxicol 29(7): 1142-1150.
Wang Z, Jiang W, Zhang Z, Qian M, and Du B (2012) Nitidine chloride inhibits LPS-
induced inflammatory cytokines production via MAPK and NF-kappaB pathway in
RAW 264.7 cells. J Ethnopharmacol 144(1): 145-150.
Yager JD (2015) Mechanisms of estrogen carcinogenesis: The role of E2/E1-quinone
metabolites suggests new approaches to preventive intervention--A review. Steroids
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This article has not been copyedited and formatted. The final version may differ from this version.
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99(Pt A): 56-60.
Zanger UM and Schwab M (2013) Cytochrome P450 enzymes in drug metabolism:
regulation of gene expression, enzyme activities, and impact of genetic variation.
Pharmacol Ther 138(1): 103-141.
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Footnotes. This work was supported in part by the National Natural Science
Foundation of China [Grant 81830104, 81430086, 81773813, U1812403].
Conflict of interest. The authors declare that there are no conflicts of interest.
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