Metabolism of 7-ethoxycoumarin, safrole, flavanone and hydroxyflavanone by cytochrome P450 2A6 variants

Article abstract of DOI:10.1002/bdd.1825

CYP 2A6 is a human enzyme that metabolizes many xenobiotics including coumarin, indole, nicotine and carcinogenic nitrosamines. The gene for CYP2A6 is polymorphic. There are few data available to clarify the relationship between P450 genetic variants and the metabolism of materials in food. The CYP 2A6 wild-type protein and 13 mutants (CYP2A6.1, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16, CYP2A6.17, CYP2A6.18, CYP2A6.21, CYP2A6.23 and CYP2A6.25) were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activities toward 7-ethoxycoumarin, coumarin, safrole, flavanone and hydroxyflavanone were examined. Ten types of CYP2A6 variants except for CYP2A6.2, CYP2A6.5 and CYP2A6.6 showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra and had 7-ethoxycoumarin O-deethylase activities. CYP2A6.15 and CYP2A6.18 showed higher activities for safrole 1′-hydroxylation than CYP2A6.1. CYP2A6.25 and CYP2A6.7 had lower safrole 1′-hydroxylase activities. CYP2A6.7 had lower flavanone 6- and 2′-hydroxylase activities, whereas CYP2A6.25 had higher 6-hydroxylase activity and lower 2′-hydroxylase activity. Hydroxyflavanone was metabolized by CYP2A6.25, but was not metabolized by wild-type CYP2A6.1. These results indicate that CYP2A6.25 possessed new substrate specificity toward flavonoids. Copyright 2012 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/bdd.1825

BIOPHARMACEUTICS & DRUG DISPOSITION
Biopharm. Drug Dispos. 34: 87–97 (2013)
Published online 4 December 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/bdd.1825
Metabolism of 7-ethoxycoumarin, safrole, flavanone and
hydroxyflavanone by cytochrome P450 2A6 variants
a,
a
a
b
a
c
Tomohide Uno *, Yuichiro Obe , Chika Ogura , Tatsushi Goto , Kohei Yamamoto , Masahiko Nakamura ,
a
a
b
Kengo Kanamaru , Hiroshi Yamagata , and Hiromasa Imaishi
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo
a
6
57-8501, Japan
b
Functional Analysis of Environmental Genes, Research Center for Environmental Genomics, Kobe University, Nada-ku, Kobe, Hyogo
6
57-8501, Japan
c
Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Kyoto Gakuen University, 1-1 Nanjo, Sogabe, Kameoka,
Kyoto 621-8555, Japan
ABSTRACT: CYP 2A6 is a human enzyme that metabolizes many xenobiotics including coumarin,
indole, nicotine and carcinogenic nitrosamines. The gene for CYP2A6 is polymorphic. There are
few data available to clarify the relationship between P450 genetic variants and the metabolism of
materials in food. The CYP 2A6 wild-type protein and 13 mutants (CYP2A6.1, CYP2A6.2, CYP2A6.5,
CYP2A6.6, CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16, CYP2A6.17, CYP2A6.18,
CYP2A6.21, CYP2A6.23 and CYP2A6.25) were co-expressed with NADPH-cytochrome P450 reductase
in E. coli. The hydroxylase activities toward 7-ethoxycoumarin, coumarin, safrole, flavanone and
hydroxyflavanone were examined. Ten types of CYP2A6 variants except for CYP2A6.2, CYP2A6.5
and CYP2A6.6 showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra
and had 7-ethoxycoumarin O-deethylase activities. CYP2A6.15 and CYP2A6.18 showed higher
0
activities for safrole 1 -hydroxylation than CYP2A6.1. CYP2A6.25 and CYP2A6.7 had lower safrole
0
0
1
-hydroxylase activities. CYP2A6.7 had lower flavanone 6- and 2 -hydroxylase activities, whereas
0
CYP2A6.25 had higher 6-hydroxylase activity and lower 2 -hydroxylase activity. Hydroxyflavanone
was metabolized by CYP2A6.25, but was not metabolized by wild-type CYP2A6.1. These results
indicate that CYP2A6.25 possessed new substrate specificity toward flavonoids. Copyright ©
2
012 John Wiley & Sons, Ltd.
Key words: HPLC; monooxygenase; polymorphism; flavanone; P450
Introduction
vertebrates and insects [1–3]. Besides the degra-
dation of chemicals native to the environment,
these enzymes have also been shown to metabolize
highly complex procarcinogenic atmospheric
pollutants such as PAHs (polycyclic aromatic
hydrocarbons) – organic compounds containing
two or more fused aromatic or heterocyclic rings [4].
Human CYP 2A6 is a major hepatic isoform
specifically involved in the oxidative metabolism
of nicotine [5], a major constituent in tobacco
smoke [6]. CYP2A6 also metabolizes pharma-
ceutical agents such as methoxyflurane, halothane,
The cytochrome P450 monooxygenases are capable
of the metabolism of a number of substrates.
Enzymes from the CYP family metabolize toxic nat-
ural products present in the diets of many terrestrial
*
Correspondence to: Laboratory of Biological Chemistry,
Department of Biofunctional Chemistry, Faculty of Agriculture,
Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan.
E-mail: unotom@kobe-u.ac.jp
Received 22 June 2012
Revised 16 October 2012
Accepted 23 October 2012
Copyright © 2012 John Wiley & Sons, Ltd.

88
T. UNO ET AL.
losigamone, letrozole, valproic acid, disulfiram and
fadrozole and activates some procarcinogens such
as 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone
and N-nitrosodiethylamine [7].
There are genetic polymorphisms present in the
CYP2A6 genes. Some variants have been reported
to decrease or abolish enzymatic activity. Alleles
possessing a single nucleotide polymorphism
Its hepatocarcinogenicity was reported to correlate
closely with its DNA adduct formation in the
liver [24]. The cytochrome P450s (CYP1A2, 2A6,
2C9, 2C19, 2D6 and 2E1) mediate the bioactiva-
tion of safrole to its proximate carcinogenic
0
metabolite, 1 -hydroxysafrole [25]. Polymorphism
in P450s, leading to poor metabolizer phenotypes,
may reduce the relative risk of the harmful effects
of safrole.
(
(
(
SNP), such as CYP2A6*2(L160H) [8], CYP2A6*6
R128Q) [9], CYP2A6*7(I471T) [10], CYP2A6*11
S224P) [11] and CYP2A6*17(V365M) [12], have been
Flavonoids are a diverse group of natural
products consisting of more than 500 compounds.
Many beneficial pharmacological properties have
been attributed to flavonoids including antioxidant,
anti-inflammatory, anticarcinogenic and chemopre-
ventive activities [26,27]. Many flavonoids are metab-
olized by mammalian hepatic microsomes [28–31].
Flavanone, a flavonoid, has important antioxidant
activities [32–35]. Flavanone is the precursor to all
flavonoid structures. Other flavonoids may have
mutagenic and/or prooxidant effects [31,36] and
depending on the P450 polymorphism, a beneficial
effect may be shifted to an antioxidant or mutagenic
effect. CYP1A1, CYP1A2 and CYP2B6 convert
flavanone to flavone and 2,3-trans-flavanonol [37].
In order to reveal the effect of SNPs within human
CYP2A6 on the metabolism of 7-ethoxycoumarin,
safrole, flavanone and hydroxyflavanone, this
study used recombinant CYP2A6 enzyme variants
overexpressed in Escherichia coli.
reported. These gene products showed decreased
enzymatic activities in vitro and in vivo. CYP2A6.15,
CYP2A6.16 and CYP2A6.21 exhibited coumarin
7-hydroxylation activities [13]. CYP2A6.25 showed
a decrease of nicotine C-oxidation [14]. CYP2A6.5
expressed in yeast was a very unstable enzyme [15].
CYP2A6.18 showed coumarin 7-hydroxylation
and 5-fluorouracil formation [16]. CYP2A6*8 is
unlikely to affect catalytic activity in vivo [17].
CYP2A6*23 allele impairs enzyme function in vitro
and in vivo and trends toward an association with
a lower risk of smoking [18].
The functional consequences of these mutations
on the enzymatic catalytic activity were mainly
examined for coumarin 7-hydroxylase activities
and nicotine C-oxidation [12–14,19]. There are few
reports that clarify the relationship between the
other possible substrates and CYP2A6 variants.
At present, there is an increasing market for food
products with perceived and real health benefits.
This development, combined with the consumers’
perception that ‘natural equals safe’, results in a
tendency for the increased use of botanical products
as bioactive ingredients in functional foods, as food
supplements and as herbal teas and food flavors.
However, in spite of a long history of use, botanical
or herb-based preparations may contain individual
ingredients known to be toxic and even genotoxic
and carcinogenic. The present article focuses on
safrole and flavonoids in food.
Materials and Methods
Materials
Isopropyl-thio-b-D-thiogalactopyranoside, methanol,
flavanone, safrole, 7-ethoxycoumarin, coumarin,
7-hydroxycoumarin and ethyl acetate were obtained
from Wako Pure Chemical Industries (Osaka, Japan).
All chemicals were of the purest grade commercially
available. The QuickChange site-directed muta-
genesis system was from Stratagene (LaJolla, CA).
Safrole (1-allyl-3,4-methylenedioxybenzene) is a
natural constituent of a number of spices such as
nutmeg, mace cinnamon, anise, black pepper and
sweet basil. Safrole is also present in cola drinks.
Safrole is the main component of Piper betel
inflorescence [20] that is frequently included in
the Taiwanese betel quid for its aromatic flavor.
Safrole is an alkenylbenzene that is recognized to be
a genotoxic carcinogenic agent in rodents [21–23].
0
6-Hydroxyflavanone and 2 -hydroxyflavanone were
from Extrasynthese (Genay Cedex, France) and
0
4 -hydroxyflavanone and 7-hydroxyflavanone
were from Tokyo Chemical Industry Co., Ltd
0
(Tokyo, Japan). 1 -Hydroxysafrole was a gift
from Dr Kunio Issiki (Bioresource Laboratories,
Mercian Corporation, Japan).
Copyright © 2012 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 87–97 (2013)
DOI: 10.1002/bdd

CHARACTERIZATION OF CYP 2A6 ALLELIC VARIANTS
89
Construction of CYP2A6 gene expression vectors
reductase cDNA. To verify that the correct inserts
were ligated into the plasmids, the inserts were
excised and electrophoresed to confirm that they
were the correct size.
Full-length cDNA fragments of the CYP2A6 gene
were amplified by polymerase chain reaction
methods from a human liver cDNA library by
using gene-specific primers. To generate protein in
an E. coli system, the N-terminal transmembrane
sequences were deleted and several amino acid
P450 production and culture conditions
Escherichia coli DH5a cells were transformed with
the pCW vector containing a P450 insert. The trans-
formed cells were preincubated in Luria broth (LB)
24
24
residue substitutions ( WQQRKSK to AKKTSSK)
were made in the fragments. The amplified frag-
ment was inserted into the NdeI and SalI sites of
the pT7 blue vector, designated as the pT7-CYP2A6
vector (Takara Bio Inc., Otsu, Japan).
ꢀ
containing 100 mg/ml ampicillin at 37 C for 1 day,
and 3 ml of this culture was then added to 300ml
of LB containing 100 mg/ml ampicillin and incu-
ꢀ
Site-directed mutagenesis on CYP2A6 cDNAwas
performed using the QuickChange II site-directed
mutagenesis system (Stratagene, La Jolla, CA)
according to the manufacturer’s instructions. The
DNA template was a pT7-CYP2A6 vector. Muta-
genesis primers that were designed for generating
mutant alleles of CYP2A6*2(L160H), CYP2A6*5
bated at 37 C to an OD6 of 0.2–0.3. After the
00
addition of isopropyl-1-thio-b-D-galactopyranoside
(0.1 mM) and 5-aminolevulinic acid (0.5 mM), cultures
ꢀ
were grown at 25 C for 40–48 h. The cells were centri-
fuged at 8000 ꢁ g for 10 min, suspended in 4 ml of
100 mM potassium phosphate buffer (pH 7.25) con-
ꢀ
taining 20% glycerol, and stored at –80 C. Membrane
(
G479V), CYP2A6*6(R128Q), CYP2A6*7(I471T),
CYP2A6*8(R485L), CYP2A6*11(S224P), CYP2A6*15
K194E), CYP2A6*16(R203S), CYP2A6*17(V365M),
CYP2A6*18(V392F), CYP2A6*21(K476R), CYP2A6*23
fractions were prepared as described previously [38].
The P450 content of the cells was determined
by CO-difference spectroscopy as described previ-
ously [39] using a dual-beam spectrophotometer
(U-3000 T, Hitachi, Tokyo, Japan). Duplicate
matched cuvettes of 2 ml of E. coli cell suspensions
in 100 mM potassium phosphate buffer (pH 7.25)
containing 20% glycerol were used to record a
baseline spectrum at 400–500 nm. After adding a
few grains of sodium dithionite crystals to two
cuvettes, carbon monoxide was bubbled through
(
(R203C) and CYP2A6*25(F118L) are listed in Table 1.
The substitutions were confirmed by DNA
sequence analysis.
All plasmids including CYP2A6 variants were
cut with NdeI and SalI, and the fragment was
subcloned into the NdeI and SalI sites of the
pCW vector containing the human NADPH-P450
Table 1. Specifically designed oligonucleotide primers that have been used in the generation of CYP2A6*2, CYP2A6*5, CYP2A6*6,
CYP2A6*7, CYP2A6*8, CYP2A6*11, CYP2A6*15, CYP2A6*16, CYP2A6*17, CYP2A6*18, CYP2A6*21, CYP2A6*23 and CYP2A6*25
mutant cDNAs
Primer
Primer sequence
0
0
CYP2A6*2
CYP2A6*5
CYP2A6*6
CYP2A6*7
CYP2A6*8
CYP2A6*11
CYP2A6*15
CYP2A6*16
CYP2A6*17
CYP2A6*18
CYP2A6*21
CYP2A6*23
CYP2A6*25
5 CATCGACGCCCACCGGGGCACTG-3
0
0
5 -CCCCAAACACGTGGTCTTTGCCACGATCC-3
0
0
5 -GCCAAGCAGCTCCAGCGCTTCTCCATC-3
0
0
5 -CCTCCCAGTCACCTAAGGACACTGACGTGTCCC-3
0
0
5 -CTTTGCCACGATCCCACTAAACTACACCATGAGCT-3
0
0
5 -CAGCTCTATGAGATGTTCCCTTCGGTGATGAAACACC-3
0
0
5 -TCTTTGGGGACCGCTTTGACTATGAGGACAAAGAGT-3
0
0
5 -AGTTCCTGTCACTGTTGAGCATGATGCTAGGAATC-3
0
0
5 -ATCCAAAGATTTGGAGACATGATCCCCATGAGTTTGG-3
0
0
5 -TAAGGGCACCGAAGTGTTCCCTATGCTGGG-3
0
0
5 -ATTGACGTGTCCCCCAGACACGTGGGCTTTG-3
0
0
5 -AGTTCCTGTCACTGTTGTGCATGATGCTAGGAATC-3
0
0
5 -TCAAAGGCTATGGCGTGGTACTCAGCAACGGGG-3
Sequences shown are those of the forward primer, the reverse primers contained sequences opposite to those of the forward primer and annealed to
the same sequences on opposite strands of the plasmid template. Nucleotides that were changed to make the desired mutation are shown in bold and
underlined.
Copyright © 2012 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 87–97 (2013)
DOI: 10.1002/bdd

90
T. UNO ET AL.
one cuvette. The resulting trace gave the spectral
difference between the dithionite-reduced CO-
bound P450 and dithionite-reduced P450. The
P450 content was expressed using an extinction
coefficient of 91 mM cm . NADPH-cytochrome
c reduction activity was determined as described
previously [40].
volume, 0.5 ml). The reaction was started by the
addition of hydroxyflavanone, safrole or flava-
none (final concentration, 335 mM). The incubation
ꢀ
mixture was shaken at 37 C for 30min in the dark.
-1
-1
Stock solutions of substrates were prepared in
methanol. Reactions were stopped by the addition
of 1 ml of ethyl acetate, vortexed for 5 min and
centrifuged at 1000ꢁ g for 8 min. The upper phase
was extracted with 1 ml of ethyl acetate three
times, and the resulting solution was air-dried.
The residual material was reconstituted in 50 ml
of 50% methanol and 10 ml aliquots were injected
into a high-performance liquid chromatography
Assays for 7-ethoxycoumarin O-deethylase and
coumarin 7-hydroxylase activity
A total of 50 pmol of P450 was added to 100 mM
potassium phosphate buffer (pH 7.25) containing
20% glycerol (total reaction volume, 0.5 ml); the
(
HPLC) system (LaChrom, Hitachi, Tokyo, Japan)
reaction was started by the addition of 7-ethoxy-
equipped with an L-7100 pump, an L-7300
injector and an L-7400 UV absorbance detector.
coumarin or coumarin (final concentration,
ꢀ
3
35 mM). The solution was then shaken at 37 C
ꢀ
The metabolites were separated at 40 C on a
for 30 min in the dark. Reactions were stopped
by the addition of 1 ml of ethyl acetate, vortexed
for 5 min and centrifuged at 1000 ꢁ g for 8 min.
The upper phase was extracted with 1 ml of ethyl
acetate three times, and the resulting solution was
air-dried in the dark. Then, 50 ml of methanol or
dimethyl sulfoxide was added to reconstitute the
pellet, followed by the addition of 2.5 ml of 0.01M
NaOH/0.1 M NaCl. The amounts of de-ethylated
products were quantified by using standard
solutions of 7-hydroxycoumarin. The concentrations
of 7-hydroxycoumarin were determined fluorome-
trically (excitation maximum at 368 and emission
at 530 nm) with a spectrofluorometer (F-2500,
TSK-gel Super ODS column (4 mm, 4.6 ꢁ 100 mm,
Tosoh, Tokyo, Japan) using 40% methanol at a
flow rate of 1.2 ml/min. Metabolite peaks were
monitored at 210 nm. Each value is the mean of
triplicate measurements. Testosterone (100 mM)
was added as an internal control after the reaction
was stopped by adding ethyl acetate. Statistical
analysis was performed by Tukey-Kramer method
and Scheffe’s F-test.
Results
ꢀ
Expression of CYP2A6 variants in E. coli
Hitachi, Tokyo, Japan) at 25 C as described
previously [41,42]. Since 7-hydroxycoumarin is
photosensitive, the working substrate and standard
solutions were prepared daily from stock solutions.
Each sample was measured in triplicate. Hydroxyl
coumarin production of CYP2A6.1 (wild type)
was linear with time for 60min (data not shown).
The Km value of CYP2A6.1 for 7-ethoxycoumarin
deethylase activity was 33.5 mM (data not shown).
So a concentration of ten times of Km was used.
The assay was done at 335 mM 7-ethoxycoumarin
for 30 min.
Fourteen expression vectors were constructed
encoding CYP2A6*1 (wild type), CYP2A6*2(L160H),
CYP2A6*5(G479V), CYP2A6*6(R128Q), CYP2A6*7
(
I471T), CYP2A6*8(R485L), CYP2A6*11(S224P),
CYP2A6*15 (K194E), CYP2A6*16(R203S), CYP2A6*17
V365M), CYP2A6*18(V392F), CYP2A6*21(K476R),
(
CYP2A6*23(R203C) and CYP2A6*25(F118L).
Escherichia coli DH5a cells were transformed with
the vector constructs, cultured, collected, and the
amount of P450 was measured with a spectropho-
tometer. Whereas cells transformed with empty
pCW vector lacked a peak at 450 nm, the reduced
CO-difference spectra of the 10 allelic variants
showed Soret peaks (450 nm) typical of P450s.
CYP2A6.2, CYP2A6.5 and CYP2A6.6 lacked a peak
at 450 nm (data not shown). Membrane fractions
were isolated and the NADPH-P450 reductase
activities were measured. NADPH-P450 reductase
Assays for safrole, flavanone, and hydroxyflavanone
hydroxylase activities, and HPLC analysis
A total of 50 pmol of P450 was added to 100 mM
potassium phosphate buffer (pH 7.25) containing
20% glycerol and 1 mM NADPH (total reaction
Copyright © 2012 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 87–97 (2013)
DOI: 10.1002/bdd

CHARACTERIZATION OF CYP 2A6 ALLELIC VARIANTS
91
activities of CYP2A6.1 and CYP2A6.25 were 5.14
and 5.41 nmol/min/mg protein, respectively. The
expression levels of CYP2A6.1 and CYP2A6.25 were
CYP2A6.8, CYP2A6.11, CYP2A6.16, CYP2A6.17,
CYP2A6.21 and CYP2A6.23 had similar activities to
CYP2A6.1. Thus, four CYP2A6 variants (CYP2A6.7,
CYP2A6.15, CYP2A6.18 and CYP2A6.25) were
used to test for coumarin 7-hydroxylation.
135 and 124 pmol/mg total proteins, respectively.
Coumarin 7-hydroxylase activities, which are
widely used activity to CYP2A6, were examined
using four CYP2A6 variants and compared with
wild type. Coumarin 7-hydroxylase activities are
shown in Figure 2. The hydroxylase activities of
CYP2A6.1, CYP2A6.7, CYP2A6.15, CYP2A6.18 and
CYP2A6.25 were 0.39ꢂ 0.02, 0.12ꢂ 0.03, 0.43ꢂ 0.01,
0.34ꢂ 0.01 and 0.16ꢂ 0.01 nmol/min/nmol P450
(meanꢂ SD), respectively. Data are meanꢂ SD of
three independent experiments. These activities were
low, compared with the values reported previously
[12,16]. CYP2A6.15 had 1.11-fold higher activities
than CYP2A6.1, respectively, whereas CYP2A6.7,
CYP2A6.18 and CYP2A6.25 were 0.31- 0.89- and
0.41-fold less active than CYP2A6.1, respectively.
The differences in the activities of the mutants
(CYP2A6.7 and CYP2A6.25) and wild type were
statistically significant (p < 0.01). The trends in
coumarin 7-hydroxylase activities of the CYP2A6
variants showed similar activity patterns to those
observed for 7-ethoxycoumarin O-deethylase.
Deethylation of 7-ethoxycoumarin and coumarin
-hydroxylation
7
The study first tested if each CYP2A6 variant
could O-deethylate the drug 7-ethoxycoumarin.
7
-Ethoxycoumarin O-deethylase activities are shown
in Figure 1. Activities of CYP2A6.1, CYP2A6.7,
CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16,
CYP2A6.17, CYP2A6.18, CYP2A6.21, CYP2A6.23
and CYP2A6.25 were 0.34ꢂ 0.01, 0.25 ꢂ 0.00,
0
0
0
.30ꢂ 0.01, 0.35ꢂ 0.02, 0.45ꢂ 0.02, 0.37ꢂ 0.01,
.35ꢂ 0.03, 0.44ꢂ 0.02, 0.29ꢂ 0.01, 0.31ꢂ 0.01 and
.24ꢂ 0.01 nmol/min/nmol of P450 (meanꢂ SD),
respectively. Data are mean ꢂ SD of three indepen-
dent experiments. CYP2A6.15 and CYP2A6.18
were 1.3 fold more active than CYP2A6.1. The
differences were statistically significant (p < 0.05).
CYP2A6.7 and CYP2A6.25 had 0.72- and 0.69-fold
lower activities than CYP2A6.1, respectively. The
differences were statistically significant (p < 0.01).
Figure 1. 7-Ethoxycoumarin O-deethylase activities of wild
type and mutants of CYP2A6. Control, cells were transformed
with empty pCW vector. A total of 50 pmol of P450 was added
to 100 mM potassium phosphate buffer (pH 7.25) containing
Figure 2. Coumarin 7-hydroxylase activities of wild type and
mutants of CYP2A6. Control, cells were transformed with
empty pCW vector. A total of 50 pmol of P450 was added to
100 mM potassium phosphate buffer (pH 7.25) containing 20%
glycerol; the reaction was started by the addition of coumarin
2
7
0% glycerol; the reaction was started by the addition of
ꢀ
-ethoxycoumarin (final concentration, 335mM). The solution
(final concentration, 335mM). The solution was shaken at 37 C
ꢀ
was shaken at 37 C for 30min in the dark. Reactions were
stopped by the addition of ethyl acetate. The extracted solution
was air-dried in the dark. The amounts of de-ethylated products
were quantified by using standard solutions of 7-hydroxycou-
marin. SD is represented by vertical bar. **p < 0.01,*p < 0.05
for 30min in the dark. Reactions were stopped by the addition
of ethyl acetate. The extracted solution was air-dried in the dark.
The amounts of de-ethylated products were quantified by using
standard solutions of 7-hydroxycoumarin. SD is represented by
a vertical bar. **p < 0.01
Copyright © 2012 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 87–97 (2013)
DOI: 10.1002/bdd

92
T. UNO ET AL.
Safrole hydroxylation
differences in the activities of the mutants
(
CYP2A6.7 and CYP2A6.15) and wild type were
Safrole hydroxylase activities were examined by
HPLC using four CYP2A6 variants and compared
with wild type. One product was obtained with a
retention time of 3.4 min. The hydroxylase activities
are shown in Figure 3. To identify the hydroxylated
safrole, metabolite standards of safrole were used.
statistically significant (p< 0.01, p < 0.05). The
trends in safrole hydroxylase activities of the
CYP2A6 variants showed similar activity patterns
to those observed for coumarin 7-hydroxylase.
0
The main hydroxylated product was 1 -hydroxy
Flavanone hydroxylation
safrole (data not shown). The hydroxylase activities
of CYP2A6.1, CYP2A6.7, CYP2A6.15, CYP2A6.18
and CYP2A6.25 were 1.02 ꢂ 0.08, 0.49 ꢂ 0.01,
Flavanone was recently reported to be metabo-
lized by mammalian hepatic P450s [43]. The study
sought to determine whether CYP2A6 variant
enzymes would hydroxylate flavanone. CYP2A6
variants were incubated separately with flava-
none and two products were obtained with reten-
tion times of 12.3 and 17.7 min (Figure 4). These
peaks were identified by comparing with the
1
.79ꢂ 0.01, 1.31ꢂ 0.18, and 0.73ꢂ 0.04 nmol/min/
nmol P450 (mean ꢂ SD), respectively. Data are mean
ꢂ SD of three independent experiments. CYP2A6.15
and CYP2A6.18 had 1.75- and 1.28-fold higher
activities than CYP2A6.1, respectively, whereas
CYP2A6.7 and CYP2A6.25 were 0.48- and 0.72-
fold less active than CYP2A6.1, respectively. The
authentic standards as 6-hydroxyflavanone and
0
2
-hydroxyflavanone at 12.3 min and 17.7 min,
respectively (Figure 4E). These peaks were not
observed in the control (Figure 4A and 4B). These
results suggest that flavanone is metabolized by
CYP2A6 variants. As shown in Figure 5, the
6
-hydroxylase activities of CYP2A6.1, CYP2A6.7,
CYP2A6.15, CYP2A6.18 and CYP2A6.25 were
0
1
.25ꢂ 0.00, 0.15ꢂ 0.01, 0.31ꢂ 0.02, 0.53ꢂ 0.04 and
.56 ꢂ 0.05 nmol/min/nmol P450 (mean ꢂ SD),
respectively. Data are mean ꢂ SD of three inde-
pendent experiments, respectively. CYP2A6.15,
CYP2A6.18 and CYP2A6.25 had 1.24-, 2.12- and
6
.24-fold higher activities than CYP2A6.1, respec-
tively, whereas CYP2A6.7 was 0.6-fold less active
than CYP2A6.1. The differences in the activities of
the mutants (CYP2A6.18 and CYP2A6.25) and
wild type were statistically significant (p < 0.01).
0
The 2 -hydroxylation activities of CYP2A6.1,
CYP2A6.7, CYP2A6.15, CYP2A6.18 and CYP2A6.25
were 0.32ꢂ 0.02, 0.10 ꢂ 0.01, 0.64ꢂ 0.04, 0.54ꢂ 0.03
and 0.14ꢂ 0.01 nmol/min/nmol P450 (meanꢂ SD),
respectively. Data are meanꢂ SD of three indepen-
dent experiments. CYP2A6.15 and CYP2A6.18
were 2.0- and 1.69-fold more active than CYP2A6.1,
respectively, whereas CYP2A6.7 and CYP2A6.25
were 0.33- and 0.43-fold less active than CYP2A6.1,
respectively. The differences in the activities of the
mutants and wild type were statistically significant
Figure 3. Safrole hydroxylase activities of wild type and
mutants of CYP2A6. A total of 50 pmol of P450 was added to
1
00 mM potassium phosphate buffer (pH 7.25) containing 20%
glycerol; the reaction was started by the addition of substrate
final concentration, 335 mM). The solution was shaken at 37
(
ꢀ
C for 30 min in the dark. Reactions were stopped by the addi-
tion of ethyl acetate. The extracted solution was air-dried. The
residual material was reconstituted in 50 ml of 50% methanol
and 10 ml aliquots were injected into an HPLC system. The
(
p < 0.01).
ꢀ
metabolites were separated at 40 C on a TSK-gel Super ODS
0
The 2 -hydroxylation activities of the two
column using 40% aqueous methanol at a flow rate of 1.2 ml/
min. Metabolite peaks were monitored at 210 nm. SD is repre-
sented by a vertical bar. **p < 0.01,*p < 0.05
variants were lower than CYP2A6.1, similar to the
other substrates, whereas the 6-hydroxylation
Copyright © 2012 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 87–97 (2013)
DOI: 10.1002/bdd

CHARACTERIZATION OF CYP 2A6 ALLELIC VARIANTS
93
Figure 5. Flavanone hydroxylase activities of wild type and
mutants of CYP2A6 (A) 6-hydroxylation (B) 2’-hydroxylation.
SD is represented by a vertical bar. **p < 0.01
the effect of an amino acid substitution on flava-
none hydroxylation.
Figure 4. HPLC analysis of flavanone metabolites produced by
the action of P450s. Open arrows, substrates; filled arrows,
products. (A) Inoculate lacking P450, (B) Inoculate lacking
flavanone, (C) Inoculate containing both flavanone and wild
type CYP2A6*1. (D) Inoculate containing both flavanone and
Hydroxylation of hydroxyflavanones
0
Next, the metabolism of 4 -hydroxyflavanone by
CYP2A6.1 and CYP2A6.25 was investigated. HPLC
analysis indicated that the metabolite produced
0
CYP2A6*25. (E) 6-hydroxyflavanone and 2 -hydroxyflavanone.
0
0
The retention times for 6-hydroxyflavanone and 2 -hydroxyfla-
from 4 -hydroxyflavanone by CYP2A6.1 was
vanone are at 12.3 min and 17.7 min, respectively. Fifty pmol of
P450 was added to 100 mM potassium phosphate buffer
eluted at 12.4 min, whereas the two metabolites
produced by CYP2A6.25 were detected at
(pH 7.25) containing 20% glycerol; the reaction was started
7
.5 and 12.4 min (Figure 6). Attempts were
by the addition of substrate. The incubate was shaken at
ꢀ
made to clarify the structure of the metabolites
formed using MS/MS, but they were not suc-
cessful. No product was observed in the recon-
3
7
C for 30 min in the dark. Reactions were stopped by the
addition of ethyl acetate. The extracted solution was air-dried.
The residual material was reconstituted in 50ml of 50% methanol
and 10 ml aliquots were injected into an HPLC system. The
0
stituted system containing 2 -hydroxyflavanone,
ꢀ
metabolites were separated at 40 C on a TSK-gel Super ODS
6
-hydroxyflavanone or 7-hydroxyflavanone as
column using 40% aqueous methanol at a flow rate of
a substrate. These results suggest that hydroxy-
flavanone is metabolized by CYP2A6.1 and
CYP2A6.25 demonstrated a new substrate specific-
ity to 4 -hydroxyflavanone. This is the first report to
clarify the effect of an amino acid substitution on 4 -
1
.2 ml/min. Metabolite peaks were monitored at 210 nm
0
activity of CYP2A6.25 was higher than CYP2A6.1.
CYP2A6.25 demonstrated a new substrate specificity
toward flavanone. This is the first report to clarify
0
hydroxyflavanone hydroxylation.
Copyright © 2012 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 87–97 (2013)
DOI: 10.1002/bdd

94
T. UNO ET AL.
Discussion
CYP 2A6 plays a crucial role in the metabolism of
many therapeutic drugs as well as metabolic activa-
tion of procarcinogens [44–46]. Genetic polymor-
phism of CYP2A6 is believed to the major cause of
individual variation in enzymatic activities for
various CYP2A6 substrates and, thus, it is critical
to characterize the enzymatic properties caused by
the allelic polymorphism [9].
CYP2A6.2 expressed in HepG2 cells showed
no activity toward coumarin 7-hydroxylation and
7-ethoxycoumarin O-deethylation [8]. CYP2A6.2
was not expressed as the active form in E. coli.
CYP2A6.2 expressed in E. coli may be unstable.
CYP2A6*5 possesses SNPs that cause the amino
acid change G479V. When expressed in yeast [15],
this substitution resulted in a very unstable
enzyme. The CYP2A6*6 variant contained the
R128Q substitution. When expressed in insect
cells, the reduced carbon oxide-bound Soret peak
was lost [9]. These two substitutions may disor-
der the holoprotein structure. The structure of
CYP2A6 bound with substrate and inhibitor
was determined by x-ray crystallography. The
CYP2A6 structure shows an enzyme that is
well adapted for the oxidation of small planar sub-
strates, such as coumarin, that fit within the narrow,
hydrophobic active site cavity of the enzyme [47].
CYP2A6*7 possesses SNPs that cause I471 to be
changed to T. The amino acid change of I471T is
known to decrease the enzymatic activities (coumarin
0
Figure 6. HPLC analysis of the formed metabolites from 4 -
hydroxyflavanone by CYP2A6. Open arrows, substrates; filled
arrows, products. (A) Inoculate lacking P450, (B) Inoculate
lacking hydroxyflavanone, (C) Inoculate containing both hy-
droxy flavanone and CYP2A6.1. (D) Inoculate containing both
hydroxyflavanone and CYP2A6.25. 50 pmol of P450 was
added to 100 mM potassium phosphate buffer (pH 7.25) con-
taining 20% glycerol; the reaction was started by the addition
7-hydroxylation and nicotine C-oxidation) in vitro
and in vivo [10,17]. In this study, CYP2A6.7
decreased 7-ethoxycoumarin O-deethylation and
flavanone and safrole hydroxylation (Figures 2, 3
and 5). The isoleucine at residue 471 is located
within the b4(1) strand and near the SRS (substrate
recognition site) 6. The amino acid change may
affect the folding of SRS 6.
CYP2A6.15 and CYP2A6.18 showed higher
activities of 7-ethoxycoumarin deethylation and
safrole and flavanone hydroxylation than CYP2A6.1
ꢀ
of substrate. The incubate was shaken at 37 C for 30 min in
the dark. Reactions were stopped by the addition of ethyl ace-
tate. The extracted solution was air-dried. The residual material
was reconstituted in 50 ml of 50% methanol and 10 ml aliquots
were injected into an HPLC system. The metabolites were sep-
arated at 40 C on a TSK-gel Super ODS column using 40%
aqueous methanol at a flow rate of 1.2 ml/min. Metabolite
peaks were monitored at 210 nm. The metabolite produced
from 4 -hydroxyflavanone by CYP2A6.1 was eluted at
2.4 min (C) whereas the two metabolites produced by
ꢀ
0
1
(Figures 2, 3 and 5). For coumarin 7-hydroxylation,
CYP2A6.25 were detected at 7.5 and 12.4 min (D)
CYP2A6.15 exhibited a higher Vmax than the wild
type [13]. The single point mutation that causes the
K194E substitution in variant CYP2A6*15 is located
not within any particular SRS site but adjacent to
helix F, which partially embraces SRS-2. Thus, the
K194E change may affect the substrate recognition
indirectly. Although the tyrosine at residue 392 is
located within the b1(3) strand (48) and not in
the SRS site, the amino acid change may affect
substrate recognition, affinity toward substrate,
or turn-over number.
Copyright © 2012 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 87–97 (2013)
DOI: 10.1002/bdd

CHARACTERIZATION OF CYP 2A6 ALLELIC VARIANTS
95
CYP2A6*25 exhibits a single amino acid muta-
tion, F118L, which is located in helix B within
the highly conserved SRS-1 region of the CYP2A
family (13) and close to the coumarin binding site
in the x-ray structure [47]. Mwenifumbo et al.
indicated that CYP2A6.25 showed a lower
nicotine C-oxidase activity than CYP2A6.1 [14].
In this study, CYP2A6.25 had lower safrole
hydroxylase activity (Figure 3). CYP2A6.25
Acknowledgements
This work was supported by the Program for Pro-
motion of Basic Research Activities for Innovative
Bioscience.
Conflict of Interest
0
had lower flavanone 2 -hydroxylation activity,
The authors report no conflict of interest.
but had higher 6-hydroxylase activity (Figures 4
0
and 5). Furthermore, a new metabolite of 4 -
hydroxyflavanone was detected after incubation
with CYP2A6.25 (Figure 6). Obviously, the F118L
mutation affects substrate recognition, especially
for flavonoids.
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