Department of Applied Chemistry, Faculty of Science and
Engineering, Kinki University, Kowakae, Higashiosaka, Osaka, Japan
(M.M., M.S.); and Osaka Prefectural Institute of Public Health,
Nakamichi, Higashinari-ku, Osaka, Japan (T.S.)
Limonene, a monocyclic monoterpene, is present in orange peel and
other plants and has been shown to have chemopreventive activities.
(+)- and (
)-Limonene enantiomers were incubated with human liver
microsomes and the oxidative metabolites thus formed were analyzed
using gas chromatography-mass spectrometry. Two kinds of metabolites,
(+)- and ()-trans-carveol (a product by 6-hydroxylation) and (+)- and ()-perillyl alcohol (a product by
7-hydroxylation), were identified, and the latter metabolites were
found to be formed more extensively, the former ones with liver
microsomes prepared from different human samples. Sulfaphenazole, flavoxamine, and antibodies raised against purified liver cytochrome P450 (P450) 2C9 that inhibit both CYP2C9- and 2C19-dependent
activities, significantly inhibited microsomal oxidations of (+)- and
()-limonene enantiomers. The limonene oxidation activities correlated
well with contents of CYP2C9 and activities of tolbutamide methyl
hydroxylation in liver microsomes of 62 human samples, whereas these
activities did not correlate with contents of CYP2C19 and activities of
S-mephenytoin 4-hydroxylation. Of 11 recombinant human
P450 enzymes (expressed in Trichoplusia ni cells)
tested, CYP2C8, 2C9, 2C18, 2C19, and CYP3A4 catalyzed oxidations of
(+)- and ()-limonenes to respective carveols and perillyl alcohol.
Interestingly, human CYP2B6 did not catalyze limonene oxidations,
whereas rat CYP2B1 had high activities in catalyzing limonene
oxidations. These results suggest that both (+)- and ()-limonene
enantiomers are oxidized at 6- and 7-positions by CYP2C9 and CYP2C19 in
human liver microsomes. CYP2C9 may be more important than CYP2C19 in
catalyzing limonene oxidations in human liver microsomes, since levels
of the former protein are more abundant than CYP2C19 in these human
samples. Species-related differences exist in the oxidations of
limonenes in CYP2B subfamily in rats and humans.
 |
Introduction |
The monocyclic monoterpene (+)-
and ()-limonene enantiomers have been shown to be present in orange
peel and other plants and are used as fragrances in household products
and components of artificial essential oils (Crowell and Gould, 1994
).
The (+)-limonene isomeric form is more abundantly present in these
plants than the racemic mixture and ()-isomeric form (Haudenschild et
al., 2000). It has previously been shown that (+)-limonene has
chemopreventive activities in experimental animal models including rats
and mice (Crowell and Gould, 1994; el-Bayoumy, 1994). Some of the
hydroxylated metabolites of (+)-limonene, such as sobrerol, carveol,
and uroterpenol, have been reported to be more potent than the parent
compound in preventing mammary tumors caused by
7,12-dimethyldibenz[a]anthracene (Crowell et al., 1992).
To better understand the basis of mechanisms of chemopreventive action
of limonene, it is of interest to examine the metabolism of limonene in
experimental animals and humans.
Metabolism of limonene enantiomers has been studied extensively in
experimental animal models in rats, mice, rabbits, guinea pigs, and
dogs both in vivo and in vitro (Igimi et al., 1974; Kodama et al.,
1974, 1976; Regan and Bjeldanes, 1976; Watabe et al., 1981). Cytochrome
P450 (P4501)
enzymes in liver microsomes of these animal species have been shown to
oxidize limonene to several oxidation products such as 1,2- and
8,9-epoxides, carveol (a product by 6-hydroxylation), perillyl alcohol
(a product by 7-hydroxylation) (Watabe et al., 1980, 1981; Jager et
al., 1999). Recently we reported that limonene enantiomers are oxidized
to carveols and perillyl alcohols by CYP2C11 in liver microsomes of
untreated rats and by CYP2B1 in those of phenobarbital-treated rats
(Miyazawa et al., 2002). Interestingly, a female specific CYP2C12 did
not catalyze oxidations of (+)- and ()-limonene enantiomers by liver
microsomes at significant levels. However, it remains unclear which
P450 enzymes catalyze limonene oxidations in human liver microsomes.
In this study, we examined oxidations of (+)- and ()-limonene by P450
enzymes in liver microsomes prepared from different human samples. The
metabolites thus formed were analyzed on GC-MS. To determine which
P450s are the major enzymes in the oxidations of (+)- and ()-limonene
enantiomers, we used specific P450 inhibitors and antibodies raised
against purified human liver P450 enzymes and P450s isolated from
Escherichia coli membranes to which human P450 isoform cDNAs
have been introduced. Catalytic rates with 11 forms of human P450
enzymes expressed in Trichoplusia ni cells in the oxidation
of limonene enantiomers are also reported.
| |
Experimental Procedures |
Chemicals.
(+)- and ()-Limonene enantiomers, (+)- and
()-trans-carveols and (+)- and ()-perillyl alcohols,
were purchased from Wako Pure Chemical Co. (Osaka, Japan) and were used
without further purification; the purities of these compounds were
judged to be >99% on analysis with GC-MS.
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 of highest qualities commercially
available (Shimada et al., 1999, 2001; Miyazawa et al., 2001).
Enzymes.
Human liver samples were obtained from 27 Japanese and 35 Caucasians;
the latter samples were the generous gifts of Dr. F. P. Guengerich
(Vanderbilt University). All of the liver samples were collected from
portions of the livers without particular pathological changes (Inoue
et al., 2000; Shimada et al., 2001). Liver microsomes were prepared as
described and suspended in 10 mM Tris-Cl buffer (pH 7.4) containing 1.0 mM EDTA and 20% glycerol (v/v) (Guengerich, 1994; Shimada et al.,
1994). 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 Gentest Corp. (Woburn, MA); the P450 contents in these systems
were used as described in the data sheets provided by the manufacturer.
CYP2C9 was purified to electrophoretic homogeneity from human liver
microsomes as described previously (Shimada et al., 1986). Rabbit
antihuman CYP2C9 antibodies were prepared as described (Kaminsky et
al., 1981; Shimada et al., 1986). Rabbit antisera raised against
purified CYP2C8, 2C9, and 2C19 were the generous gifts from Nihon Nosan
Kogyo KK (Yokohama, Japan).
Oxidation of (+)- and ()-Limonene by Human Liver Microsomes and
by Human P450 Enzymes.
Oxidations of (+)- and ()-limonene enantiomers by human P450 enzymes
were determined as follows. Standard reaction mixture contained liver
microsomes (0.05 mg of protein/ml) or recombinant P450 (3 pmol/ml) and
200 µM (+)- or ()-limonene 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 units of
glucose 6-phosphate dehydrogenase/ml) (Shimada et al., 1986).
Incubations were carried out at 37°C for 30 min and terminated by
adding 1.0 ml of dichloromethane. The extracts (organic layer) were
collected by centrifugation at 3,000 rpm for 5 min and were used for
analysis with GC-MS for identification of the metabolites.
A Hewlett-Packard model 5890A gas chromatograph (Hewlett-Packard,
Atlanta, GA) equipped with a split injector was combined with a direct
coupling to Hewlett-Packard 5972 mass spectrometer. The metabolites
were separated by a TC-WAX FFS (GL Sciences, Tokyo, Japan) silica
capillary column (0.25 mm × ~60 m) using helium (at 1 ml/min)
as a carrier gas, as described previously (Miyazawa et al., 2002). A
GC-MS system equipped with Wiley 138K Mass Spectral Database software
(John Wiley & Sons Inc., New York, NY) was used for identification of products.
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 (+)- and ()-limonene oxidations by human P450
enzymes were estimated using a computer program (KaleidaGraph; Synergy
Software, Reading, PA) designed for nonlinear regression analysis.
| |
Results |
Oxidation of (+)- and ()-Limonene Enantiomers by Human Liver
Microsomes.
On incubation of (+)-limonene with human liver microsomes in the
presence of an NADPH-generating system, we detected two metabolites, namely (+)-trans-carveol (a product by 6-hydroxylation) and
(+)-perillyl alcohol (a product by 7-hydroxylation) (Fig.
1). Liver samples from two Japanese and
two Caucasians were used for analysis, and we found that human liver
microsomes were more active in oxidizing at 7-position than at
6-position (Fig. 2). Formation of these oxidative metabolites increased with incubation time up to 30 min and
protein concentration up to 0.5 mg/ml incubation mixture (Fig. 2, A and
B). Dependence of formation rates on substrate concentrations of (+)-
and ()-enantiomers of limonene by human liver microsomes were similar
(Fig. 2C). Using five human liver samples, we found that
Km and
Vmax values for the oxidation of limonenes to respective carveols and perillyl alcohol were very similar
(Table 1). Enzyme efficiencies
(Vmax/Km
ratio) were 3.5- and 4.0-fold higher in the formation of perillyl
alcohol than that of carveol.
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Fig. 1.
Metabolism of (+)-limonene by P450 enzymes.
It was suggested that ()-limonene is also metabolized through a
similar fashion by P450 enzymes.
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TABLE 1
Oxidation of (+)- and ()-limonene enantiomers to respective carveol
and perillyl alcohol derivatives by liver microsomes of five human
samples
Data for limonene oxidation activities are means of five human samples
and S.D.
|
|
Effects of Flavoxamine, Sulfaphenazole, Ketoconazole, and Anti-P450
Antibodies on Oxidations of Limonenes by Human Liver Microsomes.
Flavoxamine and sulfaphenazole, known inhibitors of CYP2C-dependent
catalytic activities (Guengerich and Shimada, 1991; Inoue et al.,
1997), significantly inhibited 6- and 7-hydroxylations of limonene
enantiomers catalyzed by human liver microsomes (Fig. 3). However, ketoconazole, a potent
inhibitor of CYP3A enzymes (Guengerich and Shimada, 1991), weakly
inhibited limonene oxidation activities by human liver microsomes.
Antibodies raised against purified human liver CYP2C9 that inhibit both
CYP2C9- and CYP2C19-dependent catalytic activities (Shimada et al.,
1986), were found to inhibit significantly limonene oxidation
activities catalyzed by liver microsomes of HL-104 (Fig. 4). We also used antibodies raised
against purified CYP2C8, 2C9, and 2C19 (generous gifts from Nihon Nosan
Kogyo KK) that were isolated from membranes of E. coli in
which respective P450 cDNAs have been introduced (Fig. 4). The data
sheets provided by the manufacturer suggested that both anti-CYP2C8 and
anti-CYP2C19 inhibited taxol hydroxylation and S-mephenytoin
4-hydroxylation, respectively, by more than 80% at 2 µl sera/100
µg of liver microsomal protein. However, anti-CYP2C9 sera was not so
potent to inhibit activities, since the antibodies inhibited by only
40% diclofenac 4'-hydroxylation catalyzed by human liver microsomes
(data not shown). Among these three antibodies examined, anti-CYP2C19
inhibited (+)-limonene oxidations more strongly than anti-CYP2C9 did,
whereas both antibodies were equally inhibitory for the oxidations of ()-limonene enantiomer by human liver microsomes. Anti-CYP2C8 slightly inhibited the oxidations of limonene enantiomers (Fig. 4).
Correlation between Contents of CYP2C9 and CYP2C19 and Activities
of Limonene Oxidations by Liver Microsomes of 62 Human Samples.
These above results suggest that CYP2C9 and 2C19 are the important
enzymes in the oxidations of limonene enantiomers by human liver
microsomes. Correlation between contents of CYP2C9 and CYP2C19 and
rates of formation of carveol and perillyl alcohol of (+)-limonene was
compared in liver microsomes of 62 human samples (Fig.
5). The mean levels (±S.D.) of CYP2C9
and CYP2C19 in 62 human samples were estimated to be 44 ± 26 pmol/mg of protein (19 ± 12% of total P450) and 3.2 ± 2.6 pmol/mg of protein (1.1 ± 0.7% of total P450), respectively. We
found that there were good correlations between CYP2C9 levels and
formation of carveol (r = 0.88) and (+)-perillyl alcohol (r = 0.76) in these human samples (Fig. 5).
Limonene oxidation activities were also found to correlate with CYP2C9
marker activities (tolbutamide methyl hydroxylation), but not with
CYP2C19 marker activities (S-mephenytoin 4-hydroxylation),
in these human samples (data not shown).
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Fig. 5.
Correlation between contents of CYP2C9 (A
and B) and CYP2C19 (C and D) and catalytic activities of liver
microsomes of 62 human samples toward oxidation of (+)-limonene.
In A and B, contents of CYP2C9 were compared with formation of
(+)-carveol and (+)-perillyl alcohol, respectively, in these human
samples; in C and D, contents of CYP2C19 were compared with formation
of (+)-carveol and (+)-perillyl alcohol, respectively, in these human
samples.
|
|
Oxidation of (+)- and ()-Limonenes by Recombinant (T.
ni) Human P450 Enzymes.
Oxidation of limonenes by recombinant systems containing 11 forms of
human P450s and NADPH-P450 reductase expressed in T. ni
cells (Table 2). CYP2C8, 2C9, 2C18, 2C19,
and 3A4 catalyzed limonene oxidations at significant levels whereas all
other forms of P450 including CYP1A1, 1A2, 1B1, 2A6, 2B6, and 2E1 were
not active. CYP2C19 was the highest in catalyzing oxidations of
limonenes, followed by CYP2C9, 3A4, 2C8, and 2C18. In all cases, the
rates of formation of perilly alcohols (by 7-hydroxylation) were higher than those of formation of carveols (by 6-hydroxylation).
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TABLE 2
Oxidative metabolism of (+)- and ()-limonene enantiomers to
respective carveol and perillyl alcohol derivatives by recombinant
human P450 enzymes together with NADPH-P450 reductase expressed in T. ni cells
Oxidations of (+)- and ()-limonene enantiomers by recombinant human
P450 enzymes were determined by the methods as described under
Experimental Procedures. Substrate concentrations used were
200 µM. Data are means of duplicate determinations and range.
|
|
Recently, we found that CYP2B1 is a principal enzyme in catalyzing
oxidations of limonenes by liver microsomes of phenobarbital-treated rats, although CYP2C11 is a major enzyme in liver microsomes of untreated male rats (Miyazawa et al., 2002). Since our present study
suggested that CYP2B6 did not have any considerable activities for the
oxidations of limonene enantiomers, we examined the effects of
concentrations of rat CYP2B1 and human CYP2B6 on the oxidation of
(+)-limonene in recombinant (T. ni cells) P450 systems. The results showed that CYP2B6 did not catalyze oxidations of (+)-limonene at considerable rates, whereas CYP2B1 catalyzed limonene oxidations at
higher rates for the formation of carveol than those of perillyl alcohol (data not shown).
Kinetic analysis were performed in recombinant CYP2C9, 2C19, and 3A4 in
the oxidations of (+)- and ()-limonene enantiomers (Fig.
6). Km
values for the formation of carveols and perillyl alcohols by these
P450 enzymes were around 0.3 mM, except that Km value for the formation of carveol
by CYP2C9 was somewhat higher (0.57 mM) (Table
3). Vmax
values were higher in CYP2C19 than those in CYP2C9 and 3A4 in the
oxidations of (+)- and ()-limonene enantiomers. As a result, enzyme
efficiencies
(Vmax/Km
ratio) were always higher in CYP2C19 than those of CYP2C9 and 3A4
(Table 3).
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Fig. 6.
Dependence on concentrations of oxidations
of (+)-limonene (A and B) and ()-limonene (C and D) to respective
(+)- and ()-carveol and perillyl alcohol by recombinant human CYP2C9
( ), CYP2C19 ( ), and CYP3A4 ( ).
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TABLE 3
Kinetic analysis of metabolism of (+)- and ()-limonenes to respective
carveols and perillyl alcohols by recombinant human P450 in T. ni cells
expressing CYP2C9, CYP2C19, and CYP3A4
|
|
| |
Discussion |
In this study, we found that (+)- and ()-limonene enantiomers
were oxidized to respective carveol (6-hydroxylation) and perillyl alcohol (7-hydroxylation) metabolites by CYP2C9 and CYP2C19 in human
liver microsomes. Although enzyme efficiencies
(Vmax/Km ratio) were much higher in CYP2C19 than those of CYP2C9 in the formation of carveols and perillyl alcohols from limonene enantiomers by recombinant P450 systems, CYP2C9 was found to be more active than
CYP2C19 in catalyzing limonene enantiomers by human liver microsomes
with the following lines of evidence. First, sulfaphenazole, a specific
CYP2C9 inhibitor, significantly suppressed the activities of oxidations
of limonene enantiomers catalyzed by human liver microsomes. Second,
anti-CYP2C9 IgG, which was obtained from rabbits immunized with
purified human liver CYP2C9 (Shimada et al., 1986), completely
inhibited limonene oxidations by liver microsomes. Third, antisera
raised against CYP2C9 (purified from membranes of E. coli)
inhibited very significantly the oxidations of (+)- and ()-limonene
enantiomers by human liver microsomes. It should, however, be mentioned
that antibodies raised against CYP2C19 (purified from membranes of
E. coli) inhibited more effectively than anti-CYP2C9 for the
oxidation of (+)-limonene by liver microsomes (Fig. 4). The data sheets
provided by the manufacturer (Nippon Nosan Kogyo Co.) suggested that
anti-CYP2C19 (at 2 µl sera) inhibited S-mephenytoin 4-hydroxylation by more than 80%, whereas anti-CYP2C9 sera (at 2 µl)
inhibited by only 40% diclofenac 4'-hydroxylation catalyzed by human
liver microsomes. Finally, limonene oxidation activities were found to
correlate with contents of CYP2C9, but not CYP2C19, in liver microsomes
of 62 human samples. The reasons why CYP2C9 is more active than CYP2C19
in the oxidations of limonenes by liver microsomes may be related to
higher contents of this P450 species in human livers. Our estimate
suggested that the mean levels of CYP2C9 in liver microsomes of 62 human samples were about 14-fold higher that those of CYP2C19, probably
resulting in more important roles of CYP2C9 than CYP2C19 in catalyzing
limonene oxidations by human liver microsomes.
Recombinant CYP3A4 was found to be one of the enzymes in catalyzing
oxidations of limonene enantiomers. Although the enzyme efficiencies
(Vmax/Km
ratio) for the oxidation of limonene enantiomers by recombinant
(T. ni cells) CYP3A4 were found to be similar to those by
recombinant CYP2C9, the contribution of this P450 isoform seemed be
minor in human liver microsomes (Table 3). These were supported
by the inability of ketoconazole, a potent inhibitor of CYP3A4
(Guengerich and Shimada, 1991), to inhibit limonene oxidations
catalyzed by human liver microsomes.
We have recently shown that there are sex-related differences in the
metabolism of limonenes by rat liver microsomes and that the
male-specific CYP2C11 has higher catalytic rates for the oxidation of
limonenes than female-specific CYP2C12 (Miyazawa et al., 2002). Such
differences may, in part, be related to the occurrence of male-specific
nephrotoxicity caused by limonenes in rats (Lehman-Mckeeman et al.,
1989; Borghoff et al., 1990; Hard, 1998). In fact, some of the limonene
metabolites are shown to be more potent to interact with
2µ-globulin than the parent compound (Lehman-Mckeeman et al.,
1989; Borghoff et al., 1990). In humans, however, it has been reported
that there are few sex-related differences in the contents and
catalytic properties of individual P450 enzymes (Shimada et al., 1994).
(+)-Limonene has been shown to have chemopreventive activities in
experimental animal models (Crowell et al., 1992; Crowell and Gould,
1994; el-Bayoumy, 1994). Crowell et al. (1992) have reported that some
of the hydroxylated metabolites of (+)-limonene, such as sobrerol,
carveol, and uroterpenol, are more potent than the parent compound in
preventing mammary tumors caused by
7,12-dimethyldibenz[a]anthracene. It has also been shown
that (+)-limonene itself does not induce P450 enzymes but induces phase
II enzymes such as glutathione S-transferase and
UDP-glucuronosyltransferase enzymes (Ariyoshi et al., 1975; el-Bayoumy,
1994). One of the mechanisms underlying chemoprevention of limonenes
may be related to inactivation of the ultimate carcinogens by inducing
phase II enzymes. It is not known at present whether the metabolites of
limonenes, such as carveol and perillyl alcohl, induce phase II enzymes
in mammalian tissues.
In this study, we found that CYP2B6 did not catalyze oxidations of
limonenes at significant rates. This is in contrast with the results of
rat studies in which CYP2B1, a homologous enzyme to CYP2B6, catalyzes
limonene oxidations at high rates and anti-CYP2B1 significantly
inhibits limonene oxidations by liver microsomes of
phenobarbital-treated rats (Miyazawa et al., 2002). Such
species-related differences in the metabolism of xenobiotic chemicals
by the same family of P450 enzymes are of interest when the biological
and pharmacological data are to be extrapolated from experimental animals to humans (Wrighton et al., 1995; Iwatsubo et al., 1997).
In summary, present results showed that both (+)- and ()-limonene
enantiomers are oxidized to respective carveol and perillyl alcohol
derivatives by CYP2C9 and 2C19 in human liver microsomes. Our results
also showed CYP2C9 plays more important roles than CYP2C19 in
catalyzing limonene oxidations. Although recombinant CYP3A4 catalyzed
limonene oxidations at considerable rates, contribution of this P450
isoform may be minor, since ketoconazole, a potent CYP3A4 inhibitor,
inhibited weakly limonene oxidations catalyzed by liver microsomes. The
exact roles of CYP2C enzymes in the biological significance of limonene
enantiomers when humans are exposed to these chemicals are unknown at present.
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.
Abbreviations used are:
P450, cytochrome P450;
GC-MS, gas chromatography-mass spectrometry.
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)-Limonenes to Respective Carveols and
Perillyl Alcohols by CYP2C9 and CYP2C19 in Human Liver Microsomes
Top
Abstract
Introduction
and 
) were
incubated with human liver microsomes in the presence of an
NADPH-generating system, and the carveols (open symbols) and perillyl
alcohols (closed symbols) formed were analyzed on GC-MS. In A,
concentrations of protein and limonenes were 0.2 mg/ml and 0.2 mM,
respectively; in B, incubation time and substrate concentration was 30 min and 0.2 mM, respectively; and in C, incubation time and protein
concentration were 30 min and 0.2 mg/ml, respectively.
) by liver microsomes of a human sample HL-104.
