0090-9556/03/3108-1049-1053$20.00
DMD 31:1049-1053, 2003
ROLES OF HUMAN CYP2A6 AND 2B6 AND RAT CYP2C11 AND 2B1 IN THE 10-HYDROXYLATION OF (–)-VERBENONE BY LIVER MICROSOMES
Mitsuo Miyazawa,
Atsushi Sugie, and
Tsutomu Shimada
Department of Applied Chemistry, Faculty of Science and Engineering,
Kinki University, Kowakae, Higashiosaka, Osaka, Japan (M.M., A.S.); and Osaka
Prefectual Institute of Public Health, Osaka, Japan (T.S.)
(Received February 4, 2003;
Accepted April 25, 2003)
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Abstract
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(–)-Verbenone, a monoterpene bicyclic ketone, is a component of the
essential oil from rosemary species such as Rosmarinus officinalis
L., Verbena triphylla, and Eucalyptus globulus and is
used for an herb tea, a spice, and a perfume. In this study, (–)-verbenone was
found to be converted to 10-hydroxyverbenone by rat and human liver microsomal
cytochrome P450 (P450) enzymes. The product formation was determined by
high-performance liquid chromatography with UV detection at 251 nm. There was
a good correlation between activities of coumarin 7-hydroxylation and
(–)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human
samples, indicating that CYP2A6 is a principal enzyme in (–)-verbenone
10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed
(-)verbenone 10-hydroxylation at Vmax values of 15 and 21
nmol/min/nmol P450 with apparent Km values of 16 and 91
µM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze
(–)-verbenone 10-hydroxylation at all, suggesting that there were
species-related differences in the catalytic properties of human and rat CYP2A
enzymes in the metabolism of (–)-verbenone. In the rat, recombinant CYP2C11,
CYP2B1, and CYP3A2 catalyzed (–)-verbenone 10-hydroxylation with
Vmax and Km ratios (ml/min/nmol P450)
of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major
enzyme in (–)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1
catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat
CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone
10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are
the major catalysts in the metabolism of (–)-verbenone by liver microsomes and
that there are species-related differences in human and rat CYP2A enzymes and
sex-related differences in male and female rats in the metabolism of
(–)-verbenone.
Verbenone, a monoterpene bicyclic ketone, has been shown to be a component
of the essential oil from rosemary species such as Rosmarinus officinalis
L., Verbena triphylla, and Eucalyptus globulus
(Ravid et al., 1997
;
Giorgio et al., 2002).
(–)-Verbenone enantiomer has a spicy odor and camphoraceous fragrance and has
been used for an herb tea, a spice, and a perfume
(Ravid et al., 1997;
Giorgio et al., 2002). It has
not previously been reported whether verbenone enantiomers or their
metabolites have any biological and toxicological potencies in mammals when
these chemicals are ingested into animals and humans.
Our previous studies have demonstrated that several terpenes, 1,4-cineole,
1,8-cineole, and (+)- and (–)-limonenes are catalyzed by cytochrome P450
(P4501) enzymes to
their respective oxidation products in rat and human liver microsomes
(Miyazawa et al.,
2001a,b;
2002a,b).
1,4-Cineole and 1,8-cineole have been determined to be oxidized at the
2-position of the molecules both by rat and human CYP3A enzymes (Miyazawa et
al.,
2001a,b).
Limonene enantiomers are metabolized to their respective carveols and perillyl
alcohols in liver microsomes of mice, rats, guinea pigs, rabbits, dogs,
monkeys, and humans (Miyazawa et al.,
2002a,b).
There are species-related differences in the metabolism of carveols to carvone
in which dogs, rabbits, and guinea pigs catalyze this reaction, although other
species of animals examined do not
(Shimada et al., 2002b). It
has also been reported that there are sex-related differences in the
metabolism of limonene enantiomers in rats, in which male-specific CYP2C11
catalyzes limonene metabolism, although female-specific CYP2C12 does not
(Miyazawa et al., 2002a).
Recently we have established that human liver microsomes catalyze
10-hydroxylation of (–)-verbenone enantiomers by analysis with gas
chromatography-mass spectrometry (Miyazawa
et al., 2002c). Since P450 enzymes are likely to be involved in
the 10-hydroxylation of (–)-verbenone, we examined which P450 species are the
major enzymes in the metabolism of (–)-verbenone in liver microsomes of rats
and humans. (–)-Verbenone was incubated in the presence of an NADPH-generating
system with liver microsomes of rats treated with different chemical inducers
and with liver microsomes of different human liver samples. Recombinant rat
and human P450 enzymes expressed in Trichoplusia ni cells were also
used. The product formation was measured with HPLC using a C18
5-µm analytical column.
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Materials and Methods
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Chemicals. (–)-Verbenone and its 10-hydroxylated metabolite were
synthesized as described previously
(Miyazawa et al., 2002c).
NADP+, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase
were purchased from Sigma-Aldrich (St. Louis, MO). Other reagents and
chemicals used were obtained from sources as described previously or were of
the highest quality commercially available
(Wang et al., 1980; Shimada et
al., 1994,
2002a,
2002b; Miyazawa et al.,
2002a,b).
Enzymes and Antibodies. Human liver samples were obtained from organ
donors or patients undergoing liver resection as described previously (Shimada
et al., 1994,
1999). Liver microsomes were
prepared as described and suspended in 10 mM Tris-HCl buffer (pH 7.4)
containing 1.0 mM EDTA and 20% glycerol (v/v)
(Guengerich, 1994;
Shimada et al., 2002a).
Recombinant CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2E1, and 3A4
expressed in T. ni infected with a baculovirus containing rat P450
and NADPH-P450 reductase cDNA inserts were obtained from BD Gentest (Woburn,
MA); the P450 contents in these systems were tested as described in the data
sheets provided by the manufacturer.
Male Sprague-Dawley rats (weighing about 200 g) obtained from Nihon Clea
Co. (Osaka, Japan) were used throughout this study. Liver microsomes from
untreated rats and rats treated with PB (80 mg/kg, daily for 3 days),
ß-naphthoflavone (50 mg/kg, daily for 3 days), and pregnenolone
16
-carbonitrile (80 mg/kg, daily for 3 days) were prepared as described
and suspended in 10 mM Tris-HCl buffer (pH 7.4) containing 1.0 mM EDTA and 20%
glycerol (v/v) (Guengerich,
1994; Shimada et al.,
2002a). Rabbit anti-human CYP2A6 antibodies were prepared as
described (Yun et al.,
1991).
HPLC Analysis of (–)-Verbenone 10-Hydroxylation. Standard
incubation mixtures consisted of rat and human liver microsomes (0.10 mg of
protein/ml) or recombinant rat and human P450s (10 pmol) and 200 µM
(–)-verbenone (dissolved in 2 µl of dimethyl sulfoxide) in a final volume
of 0.50 ml of 100 mM potassium phosphate buffer (pH 7.4) containing an
NADPH-generating system (0.5 mM NADP+, 5 mM glucose 6-phosphate,
and 0.5 unit of glucose-6-phosphate dehydrogenase/ml). Incubations were
carried out at 37°C for 20 min and terminated by adding 1.5 ml of
CH2Cl2. The mixture was mixed vigorously and centrifuged
at 2,000 rpm for 4 min. The organic layer was evaporated to dryness under
nitrogen atmosphere, and the residues were dissolved in small amounts of 40%
methanol containing 20 mM NaClO4 (pH 2.5).
An aliquot (usually 25 µl) of the extracts described above was used for
HPLC analysis with a LC-CCPS system (Tosoh Co., Tokyo, Japan) with a
spectrofluorometer (FS-8020; Tosoh Co.). Separation was done with a
C18 5-µm analytical column (Mightsil RP-18, 150 x 4.6 mm;
Kanto Chemical Co., Tokyo, Japan) equipped with a C18 5-µm guard
column (Mightsil RP-18, 5–4.6 mm; Kanto Chemical Co.). The eluent
consisted of a mixture of 40% methanol containing 20 mM NaClO4 (pH
2.5). The flow rate was 1.0 ml/min, and the UV detection was done at 251 nm
(Fig. 1). Peak areas thus
obtained were integrated with a Chromatopac Instrument (C-R6A Chromatopac;
Shimadzu, Kyoto, Japan).
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FIG. 1. HPLC analysis of the (–)-verbenone 10-hydroxylation by liver
microsomes of human sample HL-J22 (A) and of PB-treated rats (B), and the
dependence of incubation time (C) and protein concentration (D) on the
formation of the hydroxylated metabolite was determined in liver microsomes of
PB-treated rats.
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Other Assays. P450 and protein contents were estimated by the
methods described elsewhere (Lowry et al.,
1951; Omura and Sato,
1964).
Statistical Analysis. Kinetic parameters for verbenone
10-hydroxylation by rat and human liver microsomes and recombinant rat and
human P450s were estimated using a computer program (KaleidaGraph program from
Synergy Software, Reading, PA) designed for nonlinear regression analysis of a
hyperbolic Michaelis-Menten equation.
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Results
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HPLC Analysis of (–)-Verbenone 10-Hydroxylation by Liver
Microsomes of Rats and Humans. (–)-Verbenone was incubated with liver
microsomes of human sample HL-J22 and of rats treated with PB, and the
products were extracted with dichloromethane and then analyzed with HPLC as
described under Materials and Methods
(Fig. 1). Human liver
microsomes produced small amounts of 10-hydroxyverbenone
(Fig. 1A), whereas liver
microsomes of PB-treated rats formed (–)-verbenone at much higher rates
(Fig. 1B). No other metabolites
of (–)-verbenone were detected in these liver microsomes on analysis with
HPLC. Formation of 10-hydroxyverbenone increased linearly with incubation time
up to 20 min (Fig. 1C) and with
liver microsomes of PB-treated rats up to 0.2 mg/ml of incubation
(Fig. 1D).
Role of Human CYP2A6 in the 10-Hydroxylation of
(–)-Verbenone. Liver microsomes of 16 human samples were used for
the determination of 10-hydroxylation of (–)-verbenone and 7-hydroxylation of
coumarin, and correlation of theses activities was compared
(Fig. 2). There was good
correlation between these activities (r = 0.89).
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FIG. 2. Correlation between (–)-verbenone 10-hydroxylation and
coumarin 7-hydroxylation in liver microsomes of 16 human samples.
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Recombinant human P450 enzymes expressed in T. ni cells were
tested for their catalytic activities to catalyze (–)-verbenone
(Fig. 3). Among 10 human P450
enzymes determined so far, CYP2A6 and 2B6 were found to have the highest
activities to hydroxylate (-)verbenone at turnover rates of 18 and 16
nmol/min/nmol P450, respectively. Activities (nmol/min/nmol P450) with other
recombinant human P450s were 0.39 for CYP3A4, 0.38 for CYP2D6, 0.28 for
CYP2C9, 0.26 for CYP1A1, 0.19 for CYP1B1, 0.16 for CYP1A2, 0.09 for CYP2E1,
and 0.02 for CYP2C19.
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FIG. 3. (–)-Verbenone 10-hydroxylation by recombinant human P450
enzymes expressed in T. ni cells.
Substrate concentration used was 200 µM, and incubation time was 20
min.
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Anti-CYP2A6 IgG significantly inhibited the (–)-verbenone 10-hydroxylation
catalyzed by human liver microsomes, HL-J22
(Fig. 4A).
(R)-(+)-Menthofuran, an inhibitor of CYP2A6
(Khojasteh-Bakht et al., 1998),
inhibited (–)-verbenone 10-hydroxylation catalyzed by human liver microsomes
(Fig. 4B) and by recombinant
CYP2A6 (Fig. 4C).
Triethylenethiophosphoramide (thioTEPA), an inhibitor of human CYP2B6
(Rae et al., 2002), inhibited
(-)verbenone 10-hydroxylation catalyzed by liver microsomes slightly
(Fig. 4B) and by recombinant
CYP2B6 significantly (Fig.
4D).
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FIG. 4. Effects of anti-CYP2A6 IgG (A) and (R)-(+)-menthofuran, coumarin,
and thioTEPA (B) on the (–)-verbenone 10-hydroxylation by liver microsomes of
HL-J22, and the effects of methofuran on CYP2A6-dependent verbenone
hydroxylation (C) and of thioTEPA on CYP2B6-dependent verbenone hydroxylation
(D) were also determined.
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Roles of Rat CYP2C11 and 2B1 in the 10-Hydroxylation of
(–)-Verbenone. (–)-Verbenone 10-hydroxylation activities were
determined in recombinant rat P450 enzymes expressed in T. ni cells
(Fig. 5). CYP2B1 was the
highest in the verbenone hydroxylation followed by CYP2C11, CYP2A2, and
CYP3A1. Other rat P450s were very low in the 10-hydroxylation of
(–)-verbenone. Particularly, female-specific CYP2C12 did not catalyze the
hydroxylation at all.
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FIG. 5. (–)-Verbenone 10-hydroxylation by recombinant rat P450 enzymes
expressed in T. ni cells.
Other details are the same as in the legend to
Fig. 3.
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Kinetic Analysis of (–)-Verbenone 10-Hydroxylation by Human and
Rat P450 Enzymes. Kinetic analysis of (–)-verbenone 10-hydroxylation
catalyzed by liver microsomes of human HL-J22 and C12 showed that apparent
Km values were 91 and 67 µM, and
Vmax values were 3.5 and 0.4 nmol/min/nmol P450,
respectively (Table 1). The
Km values of recombinant human CYP2A6 and 2B6 were 16 and
91 µM, respectively. The ratio of Vmax and
Km was about 4-fold higher in CYP2A6 than in CYP2B6. In
the rat, the Km values of (–)-verbenone 10-hydroxylation
by liver microsomes of untreated rats and of PB-treated rats were 198 and 61
µM, respectively. Liver microsomes of rats treated with
ß-naphthoflavone and pregnenolone-16-carbonitrile had high
Km values for (–)-verbenone 10-hydroxylation. The apparent
Km value of recombinant CYP2B6 was lower than that of
CYP2C11 in the verbenone 10-hydroxylation, and the Km
values of CYP3A1 and 3A2 were 900 and 1850 µM, respectively.
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TABLE 1 Kinetic analysis of the 10-hydroxylation of verbenone by rat and human
liver microsomes and by recombinant rat and human P450 enzymes expressed in
Trichoplusia ni cells
Data are means of duplicate determinations and S.E.M.
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Discussion
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Our results showed that human and rat liver microsomes catalyze
10-hydroxylation of (–)-verbenone, a monoterpene bicyclic ketone and a
component of the essential oil from rosemary species such as Rosmarinus
officinalis L., Verbena triphylla, and Eucalyptus
globulus (Ravid et al.,
1997; Giorgio et al.,
2002). CYP2A6 was identified to be a principal enzyme involved in
the reaction in human liver microsomes with the following lines of evidence.
First, there was good correlation between 10-hydroxylation of (–)-verbenone
and 7-hydroxylation of coumarin, a typical substrate of CYP2A6
(Shimada et al., 1994;
Pelkonen et al., 2000), in
liver microsomes of 16 human samples. Second, anti-CYP2A6, coumarin, and
(R)-(+)-menthofuran significantly inhibited (–)-verbenone
10-hydroxylation by human liver microsomes. Third, recombinant human CYP2A6
expressed in T. ni cells had very high turnover rates in the
hydroxylation of (–)-verbenone. Finally, the efficiency of catalysis
(Vmax/Km value) of CYP2A6 was found to
be 4-fold higher than that of CYP2B6, having apparent Km
values for CYP2A6 and 2B6 of 16 and 91 µM, respectively. Although
recombinant CYP2B6 had a high turnover rate for (–)-verbenone
10-hydroxylation, the contribution of this enzyme in human liver microsomes
may be minor, due to the fact that CYP2B6 has been shown to be present at low
levels in liver microsomes of human samples
(Shimada et al., 1994).
There were clear species-related differences in the metabolism of
(–)-verbenone by liver microsomes of humans and rats. As described above,
human CYP2A6 was a major enzyme, and CYP2B6 was catalyzed partially in the
10-hydroxylation of (–)-verbenone by liver microsomes. In contrast, rat CYP2A1
and 2A2 were devoid of catalytic activities in rat liver microsomes. Such
differences in the catalytic roles of CYP2A enzymes in rats and humans have
already been reported in the metabolism of testosterone and coumarin
(Yamazaki et al., 1994;
Pelkonen et al., 2000). Human
CYP2A6 catalyzed coumarin hydroxylation at high rates, whereas rat CYP2A
enzymes did not (Yamazaki et al.,
1994). It has also been reported that testosterone is catalyzed by
rat CYP2A enzymes at the 7-position, although human CYP2A6 does not
catalyze this reaction (Yamazaki et al.,
1994). Thus the present results suggest that (–)-verbenone is
another substrate that shows differences in the metabolism in rat and human
CYP2A enzymes. However, it remains unclear whether such species-related
differences in the metabolism of (–)-limonene by CYP2A enzymes affect the
biological and toxicological activities of this monoterpene compound in rats
and humans.
Species-related differences were also noted in the catalytic roles of rat
and human CYP2C enzymes in the 10-hydroxylation of (-)verbenone in liver
microsomes. Rat CYP2C11 catalyzed (–)-verbenone 10-hydroxylation at a rate of
10 nmol/min/nmol P450, whereas human CYP2C9 and 2C19 had turnover rates of
0.28 and 0.02 nmol/min/nmol P450, respectively. In addition, our present
results showed that there were sex-related differences in the roles of rat
CYP2C enzymes in the (–)-verbenone 10-hydroxylation, in that female-specific
CYP2C12 did not catalyze this reaction. Our recent studies have also shown
that male rat-specific CYP2C11, but not female rat-specific CYP2C12,
metabolizes (+)- and (–)-limonene enantiomers, monoterpene cyclic ethers, in
liver microsomes (Miyazawa et al.,
2001a,b,
2002b). Collectively, these
studies are of interest, since it has been suggested that CYP2C family enzymes
are considered to be diversely developed by catalyzing a variety of lipophilic
compounds to inert and polar metabolites, when these are ingested through
foods and air (Nebert and Gonzalez,
1987; Gonzalez,
1990).
We have previously reported that 1,4-cineole, 1,8-cineole, and (+)and
(–)-limonene enantiomers, monoterpene compounds present in nature, are
metabolized by different forms of P450 enzymes in several animal species
including rats and humans (Fig.
6) (Miyazawa et al.,
2001a,b;
2002b). 1,4-Cineole and
1,8-cineole are both principally oxidized at the 2-position by CYP3A4 in
humans and CYP3A1/2 in rats, and it has also been shown that 1,4-cineole is
partially catalyzed by CYP2A6 in humans (Miyazawa et al.,
2001a,b).
The role of CYP2A6 in the oxidation of 1,8-cineole has been reported to be
minor (Miyazawa et al.,
2001b). (+)- and (–)-Limonenes are metabolized by liver microsomes
to form respective carveols and perillyl alcohols by CYP2C9 and 2C19 in humans
and CYP2C11 in rats (Miyazawa et al.,
2002b; Shimada et al.,
2002b). Interestingly, rat CYP2B1 has high catalytic rates for the
metabolism of (+)- and (–)-limonene enantiomers, whereas the orthologous human
enzyme, CYP2B6, does not oxidize limonene metabolism capacity at all
(Miyazawa et al., 2002b;
Shimada et al., 2002b). These
results collectively indicated that natural terpene compounds in the
environment are differentially oxidized by different P450 enzymes in several
animal species and that the structures of these chemicals determine which
molecules are attacked by P450 enzymes
(Korzekwa and Jones, 1993;
Lewis, 1998;
Dai et al., 2000). Further work
will be required to determine how these terpene compounds cause biological and
toxicological responses in animal species, including humans.
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FIG. 6. Metabolism of (–)-verbenone, 1,4-cineole, 1,8-cineole, and
(+)-limonene by human and rat P450 enzymes.
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In conclusion, the present results showed that (–)-verbenone, a monoterpene
bicyclic ketone, was catalyzed by P450 enzymes to form 10-hydroxylated
verbenone in human and rat liver microsomes. Human CYP2A6 had the highest rate
in the (–)-verbenone 10-hydroxylation, followed by CYP2B6 in human liver
microsomes. In the rat, although CYP2A1/2 was inactive in catalyzing verbenone
hydroxylation, CYP2C11, a male-specific enzyme, was a major catalyst in
untreated male rats. Female-specific CYP2C12 did not catalyze verbenone
10-hydroxylation at a significant rate. Rat CYP2B1 was a major enzyme in the
metabolism of verbenone in liver microsomes of PB-treated rats.
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Footnotes
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This work was supported in part by grants from the Ministry of Education,
Science, and Culture of Japan, and the Ministry of Health and Welfare of
Japan.
1 Abbreviations used are: P450, cytochrome P450; HPLC, high-performance
liquid chromotography; PB, phenobarbital; thioTEPA,
triethylenethiophosphoramide. 
Address correspondence to: Dr. Mitsuo Miyazawa, Department of Applied
Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae,
Higashiosaka, Osaka 577-8502, Japan. E-mail;
miyazawa{at}apch.kindai.ac.jp
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Abstract
Materials and Methods
