Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes

Article abstract of DOI:10.1007/s002040050588

Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the K(m) values by these three P450 enzymes were determined to be 11.0, 105, and 132 μM, respectively, and the V(max) values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 μM) substrate concentration. CyP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 μM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 μM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 μM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti- CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 μM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 μM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes.

Full text of DOI:10.1007/s002040050588

Arch Toxicol (1999) 73: 65±70
Ó Springer-Verlag 1999
TOXICOKINETICS AND METABOLISM
Hiroshi Yamazaki á Kiyoshi Inoue á Masafumi Hashimoto
Tsutomu Shimada
Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation
by human liver microsomes
Received: 27 October 1998 / Accepted: 11 January 1999
Abstract Nicotine C-oxidation by recombinant human roles for nicotine C-oxidation at lower substrate con-
cytochrome P450 (P450 or CYP) enzymes and by human centration and both CYP2A6 and 2B6 play roles at
liver microsomes was investigated using a convenient higher substrate concentrations in human liver micro-
high-performance liquid chromatographic method. Ex- somes.
periments with recombinant human P450 enzymes in
baculovirus systems, which co-express human nicotin- Key words Nicotine á CYP2A6 á CYP2B6 á Human
amide adenine dinucleotide phosphate (reduced form) P450 á Liver microsomes
(NADPH)-P450 reductase, revealed that CYP2A6 had
the highest nicotine C-oxidation activities followed by
CYP2B6 and CYP2D6; the Km values by these three Introduction
P450 enzymes were determined to be 11.0, 105, and
1
8
32 lM, respectively, and the Vmax values to be 11.0, Nicotine has been shown to be one of the major
.2, and 8.6 nmol/min per nmol P450, respectively. constituents in tobacco smoke and to be oxidized by
CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed cytochrome P450 (P450 or CYP) enzymes to a nicotine-
nicotine C-oxidation only at high (500 lM) substrate 1¢(5¢)-iminium ion; this intermediate is further oxidized
concentration. CYP1B1, 2C18, 3A5, and 4A11 had no by cytosolic aldehyde oxidase to form cotinine (Flam-
measurable activities even at 500 lM nicotine. In liver mang et al. 1992; Benowitz et al. 1996; Messina et al.
microsomes of 16 human samples, nicotine C-oxidation 1997). Flammang et al. (1992) have reported that of 12
activities were correlated with CYP2A6 contents at human P450 enzymes expressed in cultured human
1
0 lM substrate concentration, whereas such correla- hepatoma cells (HepG2), CYP2B6 is the most active in
tion coecients were decreased when the substrate catalyzing nicotine C-oxidation activities. These authors
concentration was increased to 500 lM. Contribution of also showed that several other forms including CYP2A6,
CYP2B6 (as well as CYP2A6) was demonstrated by 2C8, 2C9, 2E1, 2F12, and 4B1 have considerable activ-
experiments with the e€ects of orphenadrine (and also ities, although lower than CYP2B6, for nicotine C-oxi-
coumarin and anti-CYP2A6) on the nicotine C-oxida- dation. Very recently, Messina et al. (1997) have found
tion activities by human liver microsomes at 500 lM that CYP2A6 must be the most important enzyme in
nicotine. CYP2D6 was found to have minor roles since catalyzing nicotine C-oxidation by human liver micro-
quinidine did not inhibit microsomal nicotine C-oxida- somes, based on the correlation with activities of cou-
tion at both 10 and 500 lM substrate concentrations. marin 7-hydroxylation in di€erent human liver
These results support the view that CYP2A6 has major microsomal preparations and on the chemical- and im-
muno-inhibition of the activities catalysed by liver mic-
rosomes. However, it is still unclear whether or not other
P450 enzymes are signi®cantly involved in the oxidation
of nicotine by liver microsomes.
In this study, we further examined the roles of P450
enzymes in the oxidation of nicotine to cotinine by liver
microsomes using liver cytosol fraction as a source of
aldehyde oxidase activity. The assay method for the
detection of cotinine as reported by Messina et al. (1997)
was modi®ed. Fourteen forms of recombinant human
P450 enzymes in baculovirus systems, which co-express
H. Yamazaki* á K. Inoue á M. Hashimoto á T. Shimada (&)
Osaka Prefectural Institute of Public Health,
3-69 Nakamichi 1-chome, Higashinari-ku,
Osaka 537, Japan
e-mail: shimada@iph.pref.osaka.jp
Tel.: (+81)-6-672-1321; Fax: (+81)-6-672-2393
*
Present address: Division of Drug Metabolism,
Faculty of Pharmaceutical Sciences, Kanazawa University,
3-1 Takara-machi, Kanazawa 920-0934, Japan
1

66
human nicotinamide adenine dinucleotide phosphate Osaka). The elution was conducted with a mixture of 15% CH
3
CN
v/v) containing 20 mM sodium phosphate bu€er, pH 4.0 and
(
(
reduced form) (NADPH)-P450 reductase, were also
1
mM octane sulfonic acid at a ¯ow rate of 0.6 ml/min; detection
used to determine which P450 enzymes are very impor-
tant in the oxidation reaction. Kinetic analysis for the
was by UV absorbance at 259 nm. Coumarin 7-hydroxylation ac-
tivities were determined as described previously (Yun et al. 1991;
oxidation of nicotine by liver microsomes and by re- Yamazaki et al. 1994).
combinant P450 enzymes and the e€ects of chemical
P450 inhibitors and anti-CYP2A6 antibodies on liver
Other assays
microsomal nicotine C-oxidation activities were also
examined.
Concentrations of P450 and protein were estimated by the methods
of Omura and Sato (1964) and Lowry et al. (1951), respectively.
The contents of human P450 proteins in liver microsomes were
determined by coupled sodium dodecyl sulfate-polyacrylamide gel
electrophoresis/immunochemical development (Western blotting;
Guengerich et al. 1982). Kinetic parameters for substrate oxida-
tions by recombinant P450 enzymes and human liver microsomes
were estimated using a computer program (KaleidaGraph program
Materials and methods
Reagents
Nicotine, cotinine, and ketamine were purchased from Sigma from Synergy Software, Reading, Pa; USA) designed for nonlinear
Chemical Co. (St. Louis, Mo., USA) and were used without further regression analysis.
puri®cation. Other chemicals and reagents used in this study were
obtained from sources described previously or were of the highest
quality commercially available (Shimada et al. 1997).
Results
Enzyme preparation
HPLC analysis for the detection of nicotine oxidation
by human liver microsomes and recombinant
human P450 enzymes
Recombinant CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18,
2C19, 2D6, 2E1, 3A4, 3A5 and 4A11 were expressed in microsomes
of Trichoplusia ni cells infected with a baculovirus containing hu-
man P450 and NADPH-P450 reductase cDNA inserts. These were
obtained from Gentest Co. (Woburn, MA Mass., USA); the P450
contents in these systems were used as described in the data sheets
provided by the manufacturer. Human liver samples were obtained
Nicotine was incubated with human liver microsomes or
by recombinant CYP2A6 in the presence of a liver cy-
tosol fraction (as a source of aldehyde oxidase activity).
from organ donors or patients undergoing liver resection as de- The product cotinine was separated from nicotine and
scribed previously (Yamazaki et al. 1997; Inoue et al. 1997). For
preparation of microsomal and cytosol fractions, the livers were
dition of liver cytosol fraction to the incubation mixture
homogenized with 4 vol of 0.1 M TRIS-HCl bu€er, pH 7.4, con-
an internal standard ketamine by HPLC (Fig. 1). Ad-
taining 0.1 M KCl and 1 mM ethylenediaminetetraacetic acid
(EDTA) in a Potter-type homogenizer. The homogenate was cen-
trifuged at 9000 g for 15 min, and the microsomal and cytosol
fractions were obtained from the resultant supernatant by centrif-
ugation at 78 000 g for 90 min. The microsomal pellet was washed
once with the same bu€er and ®nally suspended in 0.01 M TRIS-
HCl bu€er, pH 7.4 containing 1 mM EDTA and 20% glycerol.
CYP2A6 was puri®ed to electrophoretic homogeneity from
human liver microsomes as described (Yun et al. 1991) and rabbit
anti-CYP2A6 antibodies were prepared as described (Kaminsky
et al. 1981). Rabbit anti-monkey CYP2B17 (Ohmori et al. 1993)
and rat CYP2D1 (Imaoka et al. 1991) antibodies were the generous
gifts from Dr Shigeru Ohmori of Chiba University Hospital and
Dr Yoshihoko Funae of Osaka City University Medical School,
respectively.
Enzyme assays
Nicotine C-oxidation activities were determined by the method of
Messina et al. (1997) with slight modi®cations. The standard in-
cubation mixture (®nal volume of 0.25 ml) contained liver micro-
somes (0.5 mg protein/ml) or recombinant human P450 (20 pmol/
ml), liver cytosol (0.4 mg/ml), 50 mM potassium phosphate bu€er
(
pH 7.4), an NADPH-generating system consisting of 0.5 mM
+
NADP , 5 mM glucose 6-phosphate, 0.5 Unit of glucose 6-phos- Fig. 1 High-performance liquid chromatography (HPLC) analysis of
phate dehydrogenase/ml, and nicotine (10±500 lM). Incubations nicotine C-oxidation by liver microsomes of human sample HL-18 (A)
were carried out at 37 °C for 30 min and terminated by adding and recombinant CYP2A6 (B). Standard incubation mixtures
1.5 ml of CH
2
Cl
2
and 25 ll of 2 M NaCl containing 1 M Na
2
CO
3
.
containing liver microsomes (0.5 mg protein/ml) or recombinant
Ketamine was added as an internal standard to this solution at ®nal CYP2A6 (20 pmol/ml) in the presence of liver cytosol (0.4 mg/ml)
concentration of 40 lM. The organic phase was taken to dryness were used as described in the Materials and methods. Incubations
under a nitrogen stream at 37 °C. The residue was dissolved in were done at 37 °C for 30 min and terminated by adding 1.5 ml of
2
2 2 2 3
00 ll of 0.01 N HCl. Product formation was determined by high- CH Cl containing 33 mM NaCl and 16 mM Na CO . Ketamine was
performance liquid chromatography (HPLC) with a 4.6 ´ 150 mm used as an internal standard by adding ®nal concentration of 40 lM.
Nucleosil octylsilyl (C
8
) reverse-phase column (Chemco Scienti®c, Other details were as described in the Materials and methods

6
7
containing human liver microsomes caused an increase P450 forms are more active in catalyzing nicotine C-
in the formation of cotinine in a concentration depen- oxidation at substrate concentrations of 50 and 500 lM
dent manner. The maximal cotinine formation was at- (Fig. 4). Since Messina et al. (1997) have reported that
tained when liver cytosol was added at 0.4 mg protein/ the apparent Km value for nicotine C-oxidation by hu-
ml incubation mixture (Fig. 2). Cotinine formation from man liver microsomes (n = 31) is 64.9‹32.7 lM, we
nicotine in standard reaction mixture containing human selected the substrate concentration of 50 lM and the
liver microsomes and cytosol increased linearly with tenfold higher concentration of 500 lM in this experi-
time up to 60 min and with microsomal protein con- ment. At 50 lM nicotine, CYP2A6 should the highest
centration up to 0.12 mg/ml (Fig. 3).
activity in converting nicotine to cotinine, followed by
CYP2D6 and 2B6. Other forms of human P450 enzymes
gave very low activities at 50 lM nicotine. When the
substrate concentration was increased to 500 lM, sev-
eral P450 enzymes including CYP1A1, 1A2, 2C8, 2C9,
2C19, 2E1, and 3A4 as well as CYP2A6, 2B6, and 2D6,
but not CYP1B1, 2C18, 3A5, and 4A11, catalyzed nic-
Characterization of nicotine C-oxidation
by recombinant human P450 enzymes
and liver microsomes
Fourteen forms of recombinant human P450 enzymes otine C-oxidation to varying extents.
expressed in baculovirus system co-expressing human
The above results suggested that major P450 enzymes
NADPH-P450 reductase were used to compare which involved in nicotine C-oxidation may be CYP2A6, 2B6,
and 2D6. Correlations between the contents of
CYP2A6, CYP2B6, and CYP2D6 and activities of nic-
otine C-oxidation by liver microsomes of 16 human
samples were examined (Fig. 5). Two substrate concen-
trations, 10 lM and 500 lM, were used. At low sub-
strate concentration, nicotine C-oxidation activities were
correlated (r = 0.75) with the contents of CYP2A6. At
5
00 lM nicotine, the correlation coecient between
nicotine oxidation and CYP2A6 content was decreased
r = 0.60). None of the CYP2B6 and 2D6 contents
(
showed good correlations with nicotine C-oxidations
activities in these liver microsomes at either of the
10 lM and 500 lM concentrations. Kinetic analysis of
the nicotine C-oxidation by recombinant CYP2A6, 2B6,
and 2D6 showed that K values for these enzymes were
m
11, 105, and 132 lM. Vmax values were 11, 8.2, and
8.6 nmol/min per nmol P450, respectively (Table 1).
Liver microsomes of human sample HL-18 gave two Km
Fig. 2 E€ects of liver cytosol on C-oxidation of nicotine by human values of 15 and 125 lM for nicotine C-oxidation.
liver microsomes (human sample HL-18). Standard incubation
mixture was used except that liver cytosol proteins ranged from 0 to
0.8 mg protein/ml
Fig. 3 E€ects of incubation time (A) and concentrations of micro- Fig. 4A, B E€ects of substrate concentrations on nicotine C-
somal proteins (B) on nicotine C-oxidation by human liver oxidation by recombinant human P450 enzymes. Standard incubation
microsomes (human sample HL-18) in the presence of liver cytosol mixtures containing recombinant P450 enzymes (20 pmol/ml) and
fraction (0.4 mg protein/ml). Microsomal protein concentration in A liver cytosol (0.4 mg/ml) were used except that two nicotine
was 0.5 mg/ml and incubation time in B was 30 min. Data are shown concentrations of 50 lM (A) and 500 lM (B) were used. Data are
of duplicate determinations
means of duplicate determinations

68
Fig. 5 Relationship between
nicotine C-oxidation activities
and CYP2A6 (A), CYP2B6 (B),
and CYP2D6 (C) contents in
liver microsomes of 16 human
samples. Nicotine C-oxidation
was determined at 10 lM (open
circles) and 500 lM (closed cir-
cles) substrate concentrations.
Correlation coecients (r) are
given
E€ects of chemical P450 inhibitors and
anti-CYP2A6 antibodies on nicotine C-oxidation
by human liver microsomes
eases including lung cancer in humans (Hecht et al.
1981; Hecht 1994, 1996). Tobacco-related nitrosamines,
such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
E€ects of coumarin, orphenadrine, and quinidine, and (NNAL), and N¢-nitrosonornicotine (NNN), have been
anti-CYP2A6 immunoglobulin G (IgG) on nicotine C- reported to be the constituents in tobacco smoke causing
oxidation activities catalysed by liver microsomes of human cancers (Ho€mann and Hecht, 1985; Hecht and
HL-13 and HL-18 were determined at substrate con- Ho€mann 1988; Hecht 1996). These chemicals require
centrations of 10 lM and 500 lM nicotine (Table 2). At metabolic activation by P450 enzymes in order to evoke
low substrate concentration, coumarin and anti- their carcinogenic potentials in mammalian cells;
CYP2A6 IgG were very e€ective in inhibiting nicotine CYP2A6, CYP2E1, and CYP2D6 have been reported to
C-oxidation by liver microsomes. Both orphenadrine be involved in the activation process (Yamazaki et al.
and quinidine did not inhibit nicotine metabolism at 1992; Hecht 1994, 1996; Crespi et al. 1991; Tiano et al.
10 lM substrate concentration. However, when 500 lM 1993, 1994). Since these P450 enzymes have been re-
nicotine was used, orphenadrine and coumarin inhibited ported to show genetic polymorphisms in humans, it is
nicotine C-oxidation by 52 and 33%, respectively, in believed that the genetic defects in P450 genes may in-
sample HL-13 and by 56 and 59%, respectively, in ¯uence the susceptibilities of individuals towards various
sample HL18. Quinidine did not inhibit the activities types of cancers.
even at 500 lM nicotine. Anti-CYP2A6 IgG inhibited
the microsomal nicotine C-oxidation activities at bacco smoke and to have pharmacological and physio-
00 lM although the inhibitory e€ects were less than logical e€ects involving the central and peripheral
Nicotine has also been shown to be present in to-
5
those seen at 10 lM nicotine.
nervous systems (Ho€mann and Hecht 1985; Messina
et al. 1997; Pianezza et al. 1998). This chemical has been
reported to be oxidized by P450 enzymes to form nico-
tine-1¢(5¢) imminium ion and this intermediate metabo-
lite is further oxidized by a cytosolic aldehyde oxidase to
Discussion
Several carcinogenic chemicals have been identi®ed in form the stable metabolite cotinine in mammals (Wil-
tobacco smoke and these carcinogens have been sug- liams et al. 1990a, b). In this study, we used liver cytosol
gested to be one of the major causes of respiratory dis- fraction as a source of aldehyde oxidase activity and
found that 0.4 mg of cytosol protein was required to
convert the nicotine-1¢(5¢) imminium ion to cotinine.
Table 1 Kinetic analysis of nicotine C-oxidation by recombinant The slightly modi®ed HPLC method for the detection of
CYP2A6, 2B6, and 2D6 enzymes and liver microsomes sample of
HL-18. Nicotine oxidation activities were determined at di€erent
nicotine concentrations between 3.2 and 500 lM. Data presented
are means ‹ SE
nicotine and cotinine reported by Messina et al. (1997)
(C8 column instead of C6 column, detection at 259 nm
instead of 210 nm, and 15 instead of 20% CH CN for
3
the elution) gave good separation using this method.
The limit of detection in this assay system was de®ned to
be 100 pmol cotinine/ml of incubation mixture and the
eciency of extraction of the cotinine was ꢀ90%.
The present results supported the view that CYP2A6
is one of the major enzymes involved in nicotine C-ox-
idation catalyzed by human liver microsomes according
to the following lines of evidence. Of fourteen forms of
K
m
(lM)
V
max
(
nmol/min per nmol P450)
CYP2A6
CYP2B6
CYP2C6
HL-18
11‹2
105‹25
132‹31
15‹3
11.0‹2.1
8.2‹2.1
8.6‹3.3
0.19‹0.1
0.29‹0.3
1
25‹22

6
9
Table 2 E€ects of coumarin,
orphenadrine, and a quinidine,
and anti-CYP2A6 im-
munoglobulin G (IgG) on ni-
cotine oxidation catalyzed by
human liver microsomes at
substrate concentrations of
Addition
Concentration
Nicotine C-oxidation
HL-13
HL-18
(
lM)
(nmol/min per mg protein)
None
±
0.055 ‹ 0.005
0.013 ‹ 0.003*
0.045 ‹ 0.005
0.057 ‹ 0.006
0.084 ‹ 0.008
Coumarin
Orphenadrine
Quinidine
25
300
5
0.011 ‹ 0.002*
0.078 ‹ 0.009
0.082 ‹ 0.010
1
5
0 lM (upper panel) and
00 lM (lower panel). Nicotine
C-oxidation activities were de-
termined at nicotine concentra-
tions of 10 lM or 500 lM.
Data are means of triplicate
determinations ‹SD
(mg IgG/nmol P450)
Preimmune IgG
Anti-CYP2A6
Anti-CYP2A6
2
1
2
0.055 ‹ 0.002
0.015 ‹ 0.002*
0.009 ‹ 0.002*
0.085 ‹ 0.003
0.025 ‹ 0.003*
0.012 ‹ 0.002*
(
lM)
(nmol/min per mg protein)
None
±
0.171 ‹ 0.022
0.114 ‹ 0.025*
0.082 ‹ 0.011*
0.167 ‹ 0.026
0.224 ‹ 0.028
Coumarin
Orphenadrine
Quinidine
25
0.091 ‹ 0.012*
0.098 ‹ 0.019*
0.242 ‹ 0.030
300
5
(mg IgG/nmol P450)
Preimmune IgG
Anti-CYP2A6
Anti-CYP2A6
2
1
2
0.181 ‹ 0.023
0.120 ‹ 0.012*
0.096 ‹ 0.022*
0.232 ‹ 0.033
0.105 ‹ 0.013*
0.092 ‹ 0.032*
*
P < 0.01, as compared with control (None group)
recombinant human P450 enzymes examined, CYP2A6 microsomes at both 10 and 500 lM nicotine concen-
was found to be the most active in catalyzing nicotine C- trations. Recent in vivo studies by Benowitz et al. (1996)
oxidation at low substrate concentration. CYP2B6 and have also shown that CYP2D6 is not a major P450 form
2D6 were also active in nicotine C-oxidation, but had involved in the metabolism of nicotine and cotinine.
lower activity than CYP2A6. Kinetic analysis showed In reconstituted systems containing 15 forms of rat
that CYP2A6 gave the lowest Km value (11 lM) com- P450 enzymes, CYP2B1 has been reported to have the
pared with CYP2B6 (105 lM) and CYP2D6 (132 lM) highest activities for nicotine oxidation, followed by
for nicotine C-oxidation activities. Using liver micro- CYP2C11, CYP1A2, CYP2B2, and CYP2D1 (Naka-
somes from 16 human samples, contents of CYP2A6, yama et al. 1993). The authors also found that neither
but not CYP2B6 and 2D6, were found to be well cor- CYP2A1 nor 2A2 catalyzed conversion of nicotine to
related with nicotine C-oxidation activities by liver cotinine in reconstituted systems. Marked species-relat-
microsomes at 10 lM nicotine concentration. In addi- ed di€erences in testosterone 7a-hydroxylation and
tion, nicotine C-oxidation activities catalyzed by human coumarin 7-hydroxylation activities by CYP2A enzymes
liver microsomes were inhibited by coumarin and anti- in rat and man have been reported in several laborato-
CYP2A6 antibodies very signi®cantly at low substrate ries (Yamazaki et al. 1994; Pearce et al. 1992; Fern-
concentration.
andez-Salguero and Gonzalez 1995). Nicotine C-
The results presented in this study also support the oxidation is presumably another case for species-related
suggestion that CYP2B6 as well as CYP2A6 is involved di€erences in the catalytic roles of CYP2A enzymes in
in nicotine C-oxidation by human liver microsomes rat and human liver microsomes. It is interesting to note
particularly at high substrate concentrations. The Km in this context that rabbit CYP2A enzymes catalyze
value (125 lM) for the low-anity component in human nicotine oxidation in nasal and lung tissues (Williams
liver microsomes (HL-18) was similar to that (105 lM) et al. 1990a, b).
of recombinant CYP2B6 (Table 1). The inhibition in
In conclusion, the present results support the sug-
human liver microsomes of nicotine C-oxidation by or- gestion that CYP2A6 is a major enzyme involved in
phenadrine, a relatively selective inhibitor of CYP2B6 nicotine C-oxidation catalyzed by human liver micro-
(Reidy et al. 1989; Stevens et al. 1997), supports the somes. Recombinant CYP2B6 was also found to be in-
above suggestion. At di€erent substrate concentrations volved in nicotine C-oxidation, particularly at high
in recombinant P450 enzyme systems, CYP2D6 was also substrate concentration. While recombinant CYP2D6
found to have nicotine C-oxidation activities, having Km catalyzed nicotine C-oxidation at signi®cant levels, the
and Vmax values of 132 lM and 8.6 nmol/min per nmol contribution of this enzyme may be minor because a
P450; these values were very similar to those catalyzed potent inhibitor of CYP2D6, quinidine, did not
by recombinant CYP2B6. However, contribution of a€ect the microsomal nicotine C-oxidation activities.
CYP2D6 to nicotine oxidation may be minor, because CYP2A6 and 2B6 are found to be expressed in respira-
quinidine, a potent inhibitor of CYP2D6 (Guengerich tory organs as well as in liver and these two P450 forms
and Shimada 1991; Ching et al. 1995; Strobl et al. 1993), may have roles for tobacco-related biological e€ects in
did not inhibit the activities catalyzed by human liver humans.

70
Acknowledgements This work was supported partly by grants from Nakayama H, Okuda H, Nakashima T, Imaoka S, Funae Y (1993)
the Ministry of Education, Science, and Culture of Japan, the
Ministry of Health and Welfare of Japan, and the Osaka Prefect-
ural Government.
Nicotine metabolism by rat hepatic cytochrome P450s. Bio-
chem Pharmacol 45: 2554±2556
Ohmori S, Shirakawa C, Motohashi K, Yoshida H, Abe H, Nak-
amura T, Horie T, Kitagawa H, Asaoka K, Rikihisa T, Ka-
nakubo Y, Kitada M (1993) Puri®cation from liver microsomes
from untreated cynomolgus monkeys of cytochrome P450
closely related to human cytochrome P450 2B6. Mol Pharmacol
References
43: 183±190
Benowitz NL, Jacob P, Perez-Stable E (1996) CYP2D6 phenotype Omura T, Sato R (1964) The carbon monoxide-binding pigment of
and the metabolism of nicotine and cotinine. Pharmacogenetics
: 239±242
Ching MS, Blake CL, Ghabrial H, Ellis SW, Lennard MS, Tucker Pearce R, Greenway D, Parkinson A (1992) Species di€erence and
liver microsomes. I. Evidence for its hemoprotein nature. J Biol
Chem 239: 2370±2378
6
GT, Smallwood RA (1995) Potent inhibition of yeast-expressed
CYP2D6 by dihydroquinidine, quinidine, and its metabolites.
Biochem Pharmacol 50: 833±837
interindividual variation in liver microsomal cytochrome P450
2A enzymes: e€ects on coumarin, dicoumarol, and testosterone
oxidation. Arch Biochem Biophys 298: 211±225
Crespi CL, Penman BW, Gelboin HV, Gonzalez FJ (1991) A Pianezza ML, Sellers EM, Tyndale RF (1998) Nicotine metabolism
tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-
defect reduces smoking. Nature 393: 750
3-pyridyl)-1-butanone, is activated by multiple human cyto- Reidy GF, Mehta I, Murray M (1989) Inhibition of oxidative drug
(
chrome P450s including the polymorphic human cytochrome
P4502D6. Carcinogenesis 12: 1197±1201
Fernandez-Salguero P, Gonzalez FJ (1995) The CYP2A gene
metabolism by orphenadrine: in vitro and in vivo evidence for
isozyme-speci®c complexation of cytochrome P-450 and inhi-
bition kinetics. Mol Pharmacol 35: 736±743
subfamily: species di€erences, regulation, catalytic activities and Shimada T, Mimura M, Inoue K, Nakamura S, Oda H, Ohmori S,
role in chemical carcinogenesis. Pharmacogenetics 5: 123±128
Flammang AM, Gelboin HV, Aoyama T, Gonzalez FJ, McCoy
GD (1992) Nicotine metabolism by cDNA-expressed human
cytochrome P-450s. Biochem Arch 8: 1±8
Yamazaki H (1997) Cytochrome P450-dependent xenobiotic-
oxidizing activities of liver microsomes in various animal spe-
cies including rats, guinea pigs, dogs, monkeys, and humans.
Arch Toxicol 71: 401±408
Guengerich FP, Shimada T (1991) Oxidation of toxic and carcin- Stevens JC, White RB, Hsu SH, Martinet M (1997) Human liver
ogenic chemicals by human cytochrome P-450 enzymes. Chem
CYP2B6-catalyzed hydroxylation of RP 73401. J Pharmacol
Res Toxicol 4: 391±407
Exp Ther 282: 1389±1395
Guengerich FP, Wang P, Davidson NK (1982) Estimation of iso- Strobl GR, Kruedener SV, Stockigt J, Guengerich FP, Wol€ T
zymes of microsomal cytochrome P-450 in rats, rabbits, and
humans using immunochemical staining coupled with sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. Biochem-
istry 21: 1698±1706
(1993) Development of a pharmacophore for inhibition of hu-
man liver cytochrome P-450 2D6: Molecular modeling and in-
hibition studies. J Med Chem 36: 1136±1145
Tiano HF, Hosokawa M, Chulada P, Smith PB, Wang RL,
Gonzalez F, Crespi CL, Langenbach R (1993) Retroviral
mediated expression of human cytochrome P450 2A6 in
C3H/10T1/2 cells confers transformability by 4-(methylnitros-
amino)-1-(3-pyridyl)-1-butanone (NNK). Carcinogenesis 14:
1421±1427
Hecht SS (1994) Metabolic activation and detoxi®cation of to-
bacco-speci®c nitrosamines. A model for cancer prevention
strategies. Drug Metab Rev 26: 373±390
Hecht SS (1996) Recent studies on mechanisms of bioactivation
and detoxi®cation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK), a tobacco-speci®c lung carcinogen. Crit Rev Tiano HF, Wang R-L, Hosokawa M, Crespi C, Tindall KR,
Toxicol 26: 163±181
Langenbach R (1994) Human CYP2A6 activation of 4-(meth-
ylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): mutational
speci®city in the gpt gene of AS52 cells. Carcinogenesis 15:
2859±2866
Hecht SS, Ho€mann D (1988) Tobacco-speci®c nitrosamines, an
important group of carcinogens in tobacco and tobacco smoke.
Carcinogenesis 9: 875±884
Hecht SS, Carmella S, Mori H, Ho€man DA (1981) A study of Williams DE, Ding X, Coon MJ (1990a) Rabbit nasal cytochrome
tobacco carcinogenesis. XX. Role of catechol as a major car-
cinogen in the weakly acidic fraction of smoke condensate.
J Natl Cancer Inst 66: 163±169
P-450 NMa has high activity as a nicotine oxidase. Biochem
Biophys Res Commun 166: 945±952
Williams DE, Shigenaga MK, Castagnoli NJr (1990b) The role of
cytochromes P-450 and ¯avin-containing monooxygenase in the
metabolism of (S)-nicotine by rabbit lung . Drug Metab Dispos
18: 418±428
Ho€mann D, Hecht SS (1985) Nicotine-derived N-nitrosamines
and tobacco-related cancer. Current status and future direc-
tions. Cancer Res 45: 935±944
Imaoka S, Fujita S, Funae Y (1991) Age-dependent expression of Yamazaki H, Inui Y, Yun CH, Guengerich FP, Shimada T (1992)
cytochrome P-450s in rat liver. Biochim Biophys Acta 1097:
87±192
Inoue K, Yamazaki H, Imiya K, Akasaka S, Guengerich FP,
Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for
metabolic activation of N-nitrosodialkylamines and tobacco-
related nitrosamines in human liver microsomes. Carcinogene-
sis 13: 1789±1794
1
Shimada T (1997) Relationship between CYP2C9 and 2C19
genotypes and tolbutamide methyl hydroxylation and S-me- Yamazaki H, Mimura M, Sugahara C, Shimada T (1994) Catalytic
phenytoin 4¢-hydroxylation activities in livers of Japanese and
Caucasian populations. Pharmacogenetics 7: 103±113
Kaminsky LS, Fasco MJ, Guengerich FP (1981) Production and
roles of rat and human cytochrome P450 2A enzymes in tes-
tosterone 7a- and coumarin 7-hydroxylations. Biochem Phar-
macol 48: 1524±1527
application of antibodies to rat liver cytochrome P-450. Meth- Yamazaki H, Inoue K, Turvy CG, Guengerich FP, Shimada
ods Enzymol 74: 262±272
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein
measurement with the Folin phenol reagent. J Biol Chem 193:
T (1997) E€ects of freezing, thawing, and storage of hu-
man liver samples on the microsomal contents and activi-
ties of cytochrome P450 enzymes. Drug Metab Dispos 25:
168±174
2
65±275
Messina ES, Tyndale RF, Sellers EM (1997) A major role for Yun CH, Shimada T, Guengerich FP (1991) Puri®cation and
CYP2A6 in nicotine C-oxidation by human liver microsomes.
J Pharmacol Exp Ther 282: 1608±1614
characterization of human liver microsomal cytochrome P-450
2A6. Mol Pharmacol 40: 679±685