Functional characterization of 34 CYP2A6 allelic variants by assessment of nicotine C-oxidation and coumarin 7-hydroxylation activities

Article abstract of DOI:10.1124/dmd.116.073494

CYP2A6, amember of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activitieswere assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.

Full text of DOI:10.1124/dmd.116.073494

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Functional characterization of 34 CYP2A6 allelic variants by
assessment of nicotine C-oxidation and coumarin 7-hydroxylation
activities
Hiroki HOSONO, Masaki KUMONDAI, Masamitsu MAEKAWA, Hiroaki
YAMAGUCHI, Nariyasu MANO, Akifumi ODA, Noriyasu HIRASAWA, Masahiro
HIRATSUKA
Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of
Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan (H.H., M.K.,
N.H., M.H.)
Department of Pharmacy, Tohoku University Hospital, Sendai 980-8574, Japan (M.M.,
H.Y., N.M.)
Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan (A.O.)

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Running title: Functional characterization of 34 CYP2A6 variants
Corresponding author:
Dr. Masahiro Hiratsuka, Ph.D.
Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of
Pharmaceutical Sciences, Tohoku University, Sendai, Japan, 6-3, Aoba, Aramaki,
Aoba-ku, Sendai 980-8578, Japan
Tel & Fax: +81-22-717-7049
E-mail: mhira@m.tohoku.ac.jp
Number of text pages: 29
Number of tables: 1
Number of figures: 5
Number of references: 38
Number of words in Abstract: 143
Number of words in Introduction: 648
Number of words in Discussion: 1059

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Abbreviations: CLint, intrinsic clearance; CYP2A6, cytochrome P450 2A6; NADP+,
oxide -nicotinamide-adenine dinucleotide phosphate; G-6-P, glucose-6-phosphate;
G-6-PDH, glucose-6-phosphate dehydrogenase; HPLC, high-performance liquid
chromatography; Km, Michaelis constant; Vmax, maximum velocity.

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Abstract
Cytochrome P450 2A6 (CYP2A6) is one of the enzymes responsible for the
metabolism of therapeutic drugs and tobacco components, such as nicotine,
4-methylnitrosoamino-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic
polymorphisms in CYP2A6 are associated with individual variation in smoking
behavior, drug toxicities and the risk of developing several cancers. In this study, we
conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and
coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were
heterologously expressed in 293FT cells, and their enzymatic activities were assessed
based on nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34
CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26,
CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, while 14 other variants
exhibited markedly reduced activity toward both nicotine and coumarin. These
comprehensive in vitro findings may provide useful insight into individual differences
in smoking behavior, drug efficacy, and cancer susceptibility.

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Introduction
Cytochrome P450 (CYP) 2A6 is the primary enzyme involved in the oxidative
metabolism of nicotine and clinically useful drugs such as metronidazole,
methoxyflurane, tegafur, valproic acid, and fadrozole (Kharasch et al., 1995;
Nakajima et al., 1996; Sadeque et al., 1997; Komatsu et al., 2000; Pelkonen et al., 2000;
Pearce et al., 2013). This enzyme also plays an important role in the metabolic
activation
of
4-methylnitrosoamino-1-(3-pyridyl)-1-butanone
(NNK)
and
N-nitrosodiethylamine (NNN), major constituents in tobacco smoke (Crespi et al., 1991).
Nicotine, the main driver in the development and maintenance of tobacco dependence,
is primarily metabolized to cotinine (70–80%) by CYP2A6 (Fig. 1) (Benowitz et al.,
1995; Fagerstrom and Balfour, 2006). A phenotype study using nicotine as the CYP2A6
probe substrate discovered significant individual variability in CYP2A6 activity
(Nakajima et al., 2006). A meta-analysis suggested that reduced metabolizers (patients
with low CYP2A6 activity) smoke fewer cigarettes per day than normal metabolizers
(Pan et al., 2015). The presence of several CYP2A6 variants explained as to why large
deviation fractions where observed in estimated nicotine biomarker metabolism rates,
identified in a genome-wide association study (Loukola et al., 2015). Furthermore,
CYP2A6 activity was significantly correlated with not only increased nicotine uptake

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but also with the effect that causes smokers to smoke more extensively (Patel et al.,
2016). Tegafur, a prodrug of 5-fluoroulacil (5-FU), is another clinically important
CYP2A6 substrate in human liver microsomes (Yamamiya et al., 2014). CYP2A6
enzymatic activity variation would affect the therapeutic effect through 5-FU
biokinetics.
Investigating the enzymatic functions of individual CYP2A6 allelic variants in
vivo is difficult being that the number of patients carrying low-frequency alleles is
limited (Soriano et al., 2011). Therefore, in vitro characterization of CYP2A6 variants
activity, using a recombinant enzyme system is essential for a deeper understanding of
the individual variability in drug response and toxicity. Various heterologous expression
systems, including bacteria, yeast, insect cells, and mammalian cells, have been used in
previous studies (Hiratsuka, 2012). Through the use of these systems, several CYP2A6
variants have been found to exhibit reduced catalytic activities toward various CYP2A6
substrates compared with that of the wild type, CYP2A6.1 (Kitagawa et al., 2001;
Daigo et al., 2002; Han et al., 2012; Yamamiya et al., 2014). However, these studies
assessed different kinetic parameters (Km, Vmax, and CLint). In addition, posttranslational
processing of proteins expressed in bacteria, yeast, and baculoviruses differs from that
in mammalian cells (Oldham, 1998; Hirathuka, 2012). Such differences in expression

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systems may result in discrepancies in findings and conclusions.
A large number of genetic polymorphisms in the CYP2A6 gene have been
identified in several ethnic groups and shown to contribute to altered enzyme activation
and expression (Fernandez-Salguero et al., 1995; Oscarson et al., 1999; Kiyotani et al.,
2002; Haberl et al., 2005; Al Koudsi et al., 2009; Piliguian et al., 2014). These
polymorphisms are thought to produce individual variations in pharmacokinetics, drug
efficacy, adverse drug reactions, and drug toxicities (Fujieda et al., 2004; Wang et al.,
2011; Yamamiya et al., 2014; Hosono et al., 2015; Kumondai et al., 2016). To date, 45
CYP2A6 allelic variants have been identified (http://www.cypalleles.ki.se/cyp2a6.htm)
[Accessed September 5th 2016]. Although most of these alleles carry amino acid
substitutions, CYP2A6*3, CYP2A6*4, CYP2A6*12, CYP2A6*20, CYP2A6*27, and
CYP2A6*34 are known to be inactive as a result of frame-shift mutations, gene
conversion with the pseudogene CYP2A7 (another member of the human CYP2A
subfamily), or whole-gene deletion. CYP2A6*9 is reported to exhibit reduced
transcriptional activity due to a point mutation in the TATA box, located in the 5
flanking region. Information regarding CYP2A6*29, CYP2A6*30, CYP2A6*32, and
CYP2A6*33 is currently unavailable. After removal of the above mentioned alleles, 34
CYP2A6 variants remained for characterization. To characterize these 34 CYP2A6
variants, we assessed their functional activities over a nicotine substrate following
protein expression in 293FT cells. Additionally, coumarin was used as another substrate
for CYP2A6 to determine whether the observed functional changes were
substrate-dependent (Miles et al., 1990).

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Materials and Methods
Chemicals
(-)-Nicotine, (-)-cotinine, and (±)-cotinine-(methyl-d3) derivatives, as well as
coumarin, 7-hydroxycoumarin, and 4-methyl-7-hydroxycoumarin were purchased from
Sigma-Aldrich (Tokyo, Japan). CYP2A6 baculosomes and Ultrapool 150 Human Liver
Cytosol were purchased from Corning Incorporated (Corning, NY, USA). Oxidized
-nicotinamide-adenine dinucleotide phosphate (NADP+), glucose-6-phosphate (G-6-P),
and glucose-6-phosphate dehydrogenase (G-6-PDH) were purchased from Oriental
Yeast (Tokyo, Japan). The polyclonal anti-human CYP2A6 antibody was purchased
from Nosan Corporation (Kanagawa, Japan). The polyclonal anti-calnexin antibody was
purchased from Enzo Life Sciences (Farmingdale, NY, USA). Horseradish
peroxidase-conjugated goat anti-rabbit IgG was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA).
CYP2A6 cDNA cloning and construction of expression vectors
CYP2A6 cDNA fragments, obtained from a human liver complementary DNA
(cDNA) library, (Takara, Shiga, Japan) were amplified by PCR with a forward primer
(5
-CACCATGCTGGCCTCAGGGATGCTTC-3
) and reverse primer (5

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-TCAGCGGGGCAGGAAGCTCATGGTGTAG-3 ) using PfuUltra High-Fidelity
DNA Polymerase (Agilent Technologies, Santa Clara, CA, USA). The underlined
sequences in the forward primer were introduced for directional TOPO cloning. The
amplified fragments were subcloned into the pENTR/D-TOPO vector (Thermo Fisher
Scientific, Waltham, MA, USA). Plasmids carrying CYP2A6*1 cDNA (wild-type) were
used as
CYP2A6*5–CYP2A6*8,
CYP2A6*21–CYP2A6*23, CYP2A6*25, CYP2A6*28, CYP2A6*31, CYP2A6*35,
CYP2A6*38–CYP2A6*43, and CYP2A6*45) using QuikChange Lightning
a
template to generate various CYP2A6 constructs (CYP2A6*2,
CYP2A6*11, CYP2A6*13–CYP2A6*18,
a
Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s
instructions. Other CYP2A6 constructs were generated from plasmids carrying other
cDNA templates: CYP2A6*10, CYP2A6*19, and CYP2A6*36 from CYP2A6*7 cDNA;
CYP2A6*24 from CYP2A6*35 cDNA; CYP2A6*26 from CYP2A6*25 cDNA;
CYP2A6*37 from CYP2A6*10 cDNA; and CYP2A6*44 from CYP2A6*28 cDNA. All
prepared constructs were confirmed by direct sequencing. The Wild-type and variant
CYP2A6 cDNA sequences were subsequently subcloned into the mammalian expression
vector pcDNA3.4 (Thermo Fisher Scientific).

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Expression of CYP2A6 variant proteins in 293FT cells
We cultured 293FT cells in Dulbecco’s modified Eagle’s medium (Nacalai
Tesque, Kyoto, Japan) containing 10% fetal bovine serum at 37ºC in the presence of 5%
CO2. Cells were transfected with plasmid (5 g) carrying CYP2A6 cDNA using
TransFectin lipid reagent (Bio-Rad Laboratories, Hercules, CA, USA), according to the
manufacturer’s instructions. After incubation for 24 h at 37ºC, cells were scraped off,
centrifuged at 1500 g for 5 min, and re-suspended in homogenization buffer [10 mM
Tris-HCl (pH 7.4), 1 mM ethylenediaminetetraacetic acid (EDTA), and 10% glycerol].
Microsomal fractions were prepared by differential centrifugation at 9000g for 20 min,
followed by centrifugation of the resulting supernatant at 105,000g for 60 min. The
microsomal pellet was re-suspended in 50 mM Tris-HCl (pH 7.4) containing 1 mM
EDTA, 20% glycerol, 150 mM KCl, and Protease Inhibitor Cocktail for Use with
Mammalian Cell and Tissue Extract (Nacalai Tesque) and stored at -80ºC. The protein
concentration was determined using a BCA Protein Assay Kit (Thermo Fisher
Scientific).
Determination of protein expression levels by immunoblotting
We separated 293FT microsomal fractions (4 g of microsomal protein) by

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electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels. Western blotting
was performed in triplicate for each CYP2A6 variant protein in accordance with
standard procedures. Recombinant CYP2A6 supersome was used as the standard
(0.06–0.50 pmol) in each gel to quantify the CYP2A6 protein level. CYP2A6 protein
was detected using a polyclonal anti-human CYP2A6 antibody (diluted at 1:10,000),
and calnexin, used as a loading control, was detected using a polyclonal anti-calnexin
antibody (diluted at 1:5000). Secondary detection was carried out with horseradish
peroxidase-conjugated goat anti-rabbit IgG (diluted at 1:10,000). Immunoblots were
visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher
Scientific). Chemiluminescence was quantified using a ChemiDoc XRS+ with the help
of Image Lab Software (Bio-Rad Laboratories).
Nicotine C-oxidation assay
Nicotine C-oxidation by CYP2A6 was measured as reported previously
(Nakajima et al., 1996) with several modifications. The incubation mixture consisted of
the microsomal fraction (20 g), Ultrapool 150 Human Liver Cytosol as a source of
aldehyde oxidase (10 g), nicotine (10, 25, 50, 100, 250, 500, or 1000 M), and 50 mM
potassium phosphate buffer (pH 7.4) in a total volume of 100 L. Following

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pre-incubation at 37ºC for 3 min, reactions were initiated by addition of
NADPH-generating medium, consisting of 0.5 mM NADP+, 5 mM G-6-P, 5 mM MgCl,
and 1 U/mL G-6-PDH. The mixture was incubated at 37ºC for 20 min. Reactions were
terminated by adding 100 L of acetonitrile containing 1 M of cotinine-(methyl-d3)
(internal standard). Measurement of nicotine C-oxidation using 100 M of nicotine and
20 g of microsomal fraction containing the wild-type and variant CYP2A6 showed that
cotinine formation was linear for incubations of up to 20 min. Moreover, when the
reaction containing 100 M of nicotine was incubated for 20 min, cotinine formation
was linear in the presence of up to 20 g of microsomal protein (data not shown).
After protein removal by centrifugation at 15,400 g for 10 min), the
supernatant was diluted twice with water, and 10 L of diluted solution was injected
into a liquid chromatography tandem mass spectrometry (LC-MS/MS) system. Cotinine
was measured using the LC-MS/MS system in the positive ion detection mode at the
electrospray ionization interface (TSQ Vantage Triple Stage Quadrupole Mass
Spectrometer; Thermo Fisher Scientific). Separation by ultra-high-performance liquid
chromatography (HPLC) was conducted using the Nexera Ultra High Performance
Liquid Chromatography system (SHIMADZU, Kyoto, Japan). Chromatographic
separation was performed using a CAPCELLPAK ADME S2 (2.1 × 50 mm, 2.0- m

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particle size; Shiseido, Tokyo, Japan) maintained at 35ºC. Cotinine was eluted
isocratically with a mobile phase consisting of 0.05% (v/v) formic acid in water and
0.05% (v/v) formic acid in acetonitrile (90:10, v/v) at a flow rate of 200 L/min.
Quantitative MS/MS analyses were performed in the selective reaction
monitoring mode. The each area under the peak of m/z 177
80 (collision energy, 24
80 (collision
V; S-Lens RF amplitude voltage, 85 V) for cotinine and of m/z 180
energy, 29 V; S-Lens RF amplitude voltage, 55 V) for cotinine-(methyl-d3) was
measured. The optimized parameters for MS were as follows: spray voltage, 4.5 kV;
sheath gas pressure, 60 psi; vaporizer temperature, 200ºC; capillary temperature, 205ºC;
and collision pressure, 1.7 mTorr. The sheath gas was nitrogen, and the collision gas
was argon. The LC-MS/MS system was controlled by Xcalibur software (Thermo
Fisher Scientific), which was also used to analyze the data. The lower limit of cotinine
quantification was 10 nM. Standard curves for cotinine were constructed in the 0.01–30
M range using authentic metabolite standards. The enzymatic activity was normalized
to the corresponding CYP2A6 expression level.
Coumarin 7-hydroxylation assay
The coumarin 7-hydroxylation activity of CYP2A6 was determined using

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previously reported methods (Ho et al., 2008) with minor modifications. The reaction
mixture contained the microsomal fraction (30 g), coumarin (0.1, 0.25, 0.5, 1.0, 2.5,
5.0, 10, or 25 M), and 50 mM potassium phosphate buffer (pH 7.4) in a total volume
of 100 L. Following pre-incubation at 37ºC for 3 min, reactions were initiated by the
addition of NADPH-generating medium (see above). The mixture was incubated for an
additional 10 min at 37ºC. Reactions were terminated by adding 100 L of acetonitrile
containing 250 nM of 4-methyl-7-hydroxycoumarin (internal standard). Determination
of coumarin 7-hydroxylation activity in the microsomal fraction containing CYP2A6.1
(30
g of microsomal protein) and 2.5
M of coumarin revealed that
7-hydroxycoumarin formation was linear for incubations of up to 10 min. When the
reaction containing 2.5 M of coumarin was incubated for 10 min, 7-hydroxycoumarin
formation was linear in the presence of up to 30 g of microsomal protein (data not
shown).
After protein removal by centrifugation at 15,400 g for 10 min, the supernatant
was diluted twice with 50 mM potassium phosphate buffer (pH 7.4), and 50 L of the
diluted solution was subjected to HPLC. The HPLC system consisted of a Waters 2695
Separations Module, a Waters 2475 multi fluorescence detector (Waters, Milford, MA,
USA), and a SunFire C18 column (4.6 × 150 mm, 5- m particle size; Waters)

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maintained at 40ºC. We eluted 7-hydroxycoumarin isocratically with 20 mM potassium
phosphate buffer (pH 7.4) and acetonitrile (70:30, v/v) at a flow rate of 0.5 mL/min. The
quantity of 7-hydroxycoumarin was measured at an excitation wavelength of 376 nm
and emission wavelength of 454 nm. The lower limit of 7-hydroxycoumarin
quantification was 25 nM. Standard curves for 7-hydroxycoumarin were constructed in
the 25–2000 nM range using authentic metabolite The enzymatic activity was
normalized to the CYP2A6 expression level.
Data analysis
The kinetic data were analyzed using the Enzyme Kinetics Module of
SigmaPlot 12.5 (Systat Software, Inc., Chicago, IL, USA), a curve-fitting program
based on nonlinear regression analysis. Km, maximum velocity (Vmax), and intrinsic
clearance (CLint = Vmax/Km) values were determined by using this software. Vmax was
calculated using the average CYP2A6 expression level values in triplicate. All values
are expressed as the mean ± standard deviation (SD) of experiments performed in
triplicate. Statistical analyses of enzymatic activity and kinetic parameters were
performed by analysis of variance using Dunnett’s T3 test or the Kruskal-Wallis method
(IBM SPSS Statistics Ver. 22, International Business Machines, Armonk, NY, USA).

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The correlation between the CLint ratios of nicotine C-oxidation and coumarin
7-hydroxylation among CYP2A6 variants was analyzed using weighted linear
regression analysis (IBM SPSS Statistics). Differences or correlations with P-values of
less than 0.05 were defined as significant.
Three-dimensional structural modeling of CYP2A6
Three-dimensional (3D) structural modeling of CYP2A6 and nicotine binding
was based on the DeVore and Scott (2012) CYP2A6 X-ray structure (Protein Data Bank
code 4EJJ), while 3D structural modeling of CYP2A6 and coumarin docking was based
on the Yano et al. (2005) CYP2A6 X-ray structure (Protein Data Bank code 1Z10).
Maestro 9.7.012 (Schrodinger, LLC, and New York, USA) was used for 3D imaging of
CYP2A6.

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Results
Protein levels of CYP2A6 variants expressed in 293FT cells were measured by
immunoblotting. Polyclonal CYP2A6 antibodies recognized all CYP2A6 variant
proteins (Fig. 2). Only CYP2A6.13 exhibited a double band, the bands at both
molecular weights were quantified. Expression levels for calnexin, an endoplasmic
reticulum-resident protein, were virtually constant in the microsomes of transfected
cells. CYP2A6 protein was not detected in cells transfected with empty vector (mock
transfection).
The kinetic parameters of nicotine C-oxidation were determined for the wild
type (CYP2A6.1) and 26 CYP2A6 variants (Table 1). Km, Vmax, and CLint values for
nicotine C-oxidation by CYP2A6.1 were 33.3 M, 5.91 pmol min-1 pmol-1 CYP2A6,
and 176 nL min-1 pmol-1 CYP2A6, respectively. The kinetic parameters of four variants
(CYP2A6.2, CYP2A6.5, CYP2A6.6, and CYP2A6.26) could not be determined because
no nicotine C-oxidation activity was detected. The kinetic parameters of three variants
(CYP2A6.10, CYP2A6.36, and CYP2A6.37) could not be determined because the
amount of product was at or below the lower quantification limit at low substrate
concentrations. The Km value of CYP2A6.38 (108 M) was significantly higher than
that of the wild type. Vmax values of CYP2A6 allelic variants were not significantly
lower than those of the wild-type CYP2A6. The CLint value for CYP2A6.15 was 1.2

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times higher than that of CYP2A6.1.
The kinetic parameters of coumarin 7-hydroxylation were determined for 25
CYP2A6 variants, as shown in Table 1. The Km, Vmax, and CLint values for coumarin
7-hydroxylation by the wild-type enzyme were 1.94 M, 5.89 pmol min-1 pmol-1
CYP2A6, and 3.04 L min-1 pmol-1 CYP2A6, respectively. The kinetic parameters of
CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.26, and CYP2A6.37 could not be
determined because no coumarin 7-hydroxylation activity was detected. The kinetic
parameters of four variants (CYP2A6.10, CYP2A6.25, CYP2A6.36, and CYP2A6.44)
could not be determined because the amount of 7-hydroxycoumarin product produced
by these variants was below the quantification limit at low substrate concentrations.
Compared to the Km value for CYP2A6.1, that for CYP2A6.19 was significantly
elevated by 2.4-fold. In contrast, the Km values of CYP2A6.13, CYP2A6.15, and
CYP2A6.40 were significantly reduced. CYP2A6.15 exhibited a Vmax value that was
1.5-fold higher than that of the wild-type CYP2A6. The Vmax values of eleven variants
(CYP2A6.8, CYP2A6.11, CYP2A6.17, CYP2A6.19, CYP2A6.38, CYP2A6.39,
CYP2A6.40, CYP2A6.41, CYP2A6.42, CYP2A6.43, and CYP2A6.45) were
significantly lower than that of CYP2A6.1. The CLint values for CYP2A6.14 and
CYP2A6.15 were significantly higher than that of the wild type, and those of 13

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variants (CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.17, CYP2A6.19, CYP2A6.22,
CYP2A6.24, CYP2A6.35, CYP2A6.40, CYP2A6.41, CYP2A6.42, CYP2A6.43, and
CYP2A6.45) were significantly lower than that of the wild type.
After calculating the CLint values for nicotine C-oxidation and coumarin
7-hydroxylation, a significant correlation was found between these values among the
different variants (R2 = 0.970, P < 0.001), as shown in Fig. 3. CYP2A6.2, CYP2A6.5,
CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 were catalytically
inactive toward both nicotine and coumarin.

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Discussion
CYP2A6 is an important enzyme that metabolizes several tobacco components,
including nicotine, NNK, and NNN. CYP2A6 genetic polymorphisms are believed to
affect individual variation in smoking behavior and tobacco-related lung cancer risk. In
the present study, the effects of 34 CYP2A6 variant alleles on enzyme activity were
characterized by overexpressing the variant proteins in 293FT cells. Among the
CYP2A6 variants, the kinetic parameters for nicotine C-oxidation and coumarin
7-hydroxylation were determined for 27 and 25 variants, respectively. Thus, it is likely
that the measured enzymatic properties were affected by the allelic polymorphisms
present in each variant.
Our results revealed that there was a significant correlation between the CLint
values for nicotine C-oxidation and coumarin 7-hydroxylation among CYP2A6 variants.
In particular, metabolic activity on both nicotine and coumarin substrates was virtually
abolished in CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26,
CYP2A6.36, and CYP2A6.37. Similarly, the CLint of CYP2A6.10 expressed in Sf9 cells
was shown to reduce the metabolism of tegafur to 0.6% of the wild-type CYP2A6
(Yamamiya et al., 2014). Thus, these inactive variant enzymes are predicted to exhibit
markedly reduced catalytic metabolism in the presence of other clinical drugs and

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activation of procarcinogens similar to nicotine and coumarin. Such variants may
influence individual differences in drug efficacies, adverse effects, and cancer risks.
The nicotine C-oxidation and coumarin 7-hydroxylation activities of
CYP2A6.17, CYP2A6.19, CYP2A6.25, CYP2A6.26, CYP2A6.35, CYP2A6.36,
CYP2A6.37, and CYP2A6.43 were reduced compared to that of CYP2A6.1. The amino
acid substitutions in these variant alleles exists in conserved regions among CYP
isoforms and are critical to enzymatic function. CYP2A6*17, carrying the Val365Met
amino acid substitution, was found in African-American subjects with a frequency of
9.4% (Fukami et al., 2004).
The substituted residue is located in the substrate recognition site (SRS)-5, some of
these amino acid chains form the heme pocket. CYP2A6*25 and CYP2A6*26 were
identified in an African population and include the Phe118Leu substitution
(Mwenifumbo et al., 2008). The nicotine metabolic activities for these variants were
reduced to 90% when expressed in Escherichia coli cells (CYP2A6*25) and to 0%
(CYP2A6*26) for the wild-type. Phe118 is situated in SRS-1 and is predicted to be
involved in substrate stability. Moreover, CYP2A6*26 contains the Arg128Leu
substitution, which is thought to be related to holoprotein formation. CYP2A6*35,
CYP2A6*36, and CYP2A6*37 were also discovered in an African population

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(Mwenifumbo et al., 2008; Al Koudsi et al., 2009). These variant alleles include
Asn438Tyr, caused by the 6458A>T mutation. Asn438 is located in a heme binding site,
suggesting the possibility that the amino acid substitution alters the binding stability of
heme. CYP2A6*43 (4406C>T, Thr303Ile) was identified in African Americans, and its
catalytic efficiency was reported to be reduced compared to that of CYP2A6.1
(Piliguian et al., 2014). Thr303 is positioned in SRS-4, and its substitution may
negatively impact function and stability. Thus, these mutations in highly conserved CYP
regions may affect enzyme activity by altering the stability of the enzyme and/or its
affinity to substrates and heme. However, the quantitative protein expression levels
differ between CYP2A6 variants due to alterations in protein stability caused by amino
acid replacement variations (Fig 4). However, it is the limitation of this study that the
transfection efficiency for expressing CYP2A6 protein was not evaluated.
The CYP2A6*7 allele contains a 6558T>C nucleotide substitution causing the
Ile471Thr substitution in the 4(1) strand near SRS-6; this substitution is known to
decrease the catalytic activity of the enzyme (Ariyoshi et al., 2001; Uno et al., 2013).
This amino acid may be involved in determining the conformation of SRS-6. In our
study, CYP2A6.8, including the Arg485Leu substitution, exhibited reduced enzymatic
activity. This substitution also occurs near SRS-6 and may alter the folding of SRS-6

DMD Fast Forward. Published on December 14, 2016 as DOI: 10.1124/dmd.116.073494
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(Ariyoshi et al., 2001). Both of the above amino acid changes are also included in
several other variant alleles (CYP2A6*10, CYP2A6*19, CYP2A6*36, and CYP2A6*37)
and are likely to affect the enzymatic activities for these alleles as well. For CYP2A6*13,
two protein bands were detected by immunoblotting. The Gly5Arg amino acid
substitution in CYP2A6.13, occurring in the membrane anchor region, may alter the
protein’s digestion by proteases explaining the additional band. Further studies are
needed to confirm these findings.
The 1703G>A single point mutation in CYP2A6*6 leads to the Arg128Gln
substitution, which is present in the Japanese population with a frequency of 0.4%
(Kitagawa et al., 2001). The Arg128 residue is located in a highly conserved region of
the CYP superfamily and results in a reduced CO difference spectrum for
hemin-fortified CYP2A6.6 compared with that of the wild-type enzyme. In the 3D
structure model of the human CYP2A6, this residue forms hydrogen bonds with Asn438,
which is located next to the heme-binding Cys439 (Fig. 5). By eliminating this
hydrogen bond, the Arg128Gln substitution may affect the binding of heme and disorder
the holoprotein structure. The CYP2A6*11 allele (3391T>C, Ser224Pro) was identified
in a Japanese patient exhibiting a high plasma tegafur concentration-time curve (Daigo
et al., 2002). According to our analysis, the coumarin 7-hydroxylation activity of

DMD Fast Forward. Published on December 14, 2016 as DOI: 10.1124/dmd.116.073494
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CYP2A6.11 was significantly reduced by 26% compared to that of CYP2A6.1. While
Ser224 is not located in a highly conserved region, this amino acid forms a hydrogen
bond with Tyr220 and contributes to the folding of an -helix. The Ser224Pro
substitution may abrogate the hydrogen bond and alter the 3D structure of the CYP2A6
enzyme. The single-nucleotide polymorphism in CYP2A6*15 results in the Lys194Glu
substitution and significantly higher metabolic activity with both substrates. While
Lys194 is not located within a highly conserved region, the substitution is considered to
be indirectly adjacent to the F-helix and SRS-2 (Uno et al., 2013). CYP2A6.44 was also
found to exhibit markedly reduced enzymatic activity in the presence of both substrates.
CYP2A6*44 was identified in an African American and contains the Glu390Lys amino
acid substitution (Piliguian et al., 2014). Glu390 is positioned in a -sheet and forms
hydrogen bonds with Glu103 in SRS-1 and Arg76. CYP2A6*44 alleles may affect the
protein conformation of SRS-1, resulting in reduced affinity to CYP2A6 substrates.
In conclusion, we expressed 34 CYP2A6 protein variants in 293FT cell and
determined their metabolic activities in vitro. Our results demonstrated the presence of
functional variability in enzyme activities due to amino acid substitutions caused by
genetic polymorphisms. CYP2A6 is an important CYP isoform involved in the
metabolism of nicotine, many clinical drugs and the activation of procarcinogens. These

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findings provide further insight into individual differences in smoking behaviors, drug
efficacies, and cancer susceptibilities.

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Acknowledgments: We thank the Biomedical Research Core of Tohoku University
Graduate School of Medicine for technical support.

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Authorship Contributions
Participated in research design: Hosono, Kumondai, and Hiratsuka
Conducted experiments: Hosono and Kumondai
Contributed new reagents or analytic tools: Hirasawa, Maekawa, Yamaguchi, Mano,
and Hiratsuka
Performed data analysis: Hosono, Kumondai, Maekawa, Oda, and Hiratsuka
Wrote or contributed to the writing of the manuscript: Hosono, Kumondai, and
Hiratsuka

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