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Vol. 29, Issue 2, 200-205, February 2001
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 1-chome, Higashinari-ku, Osaka, Japan (T.S.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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1,8-Cineole, the monoterpene cyclic ether known as eucalyptol, is
one of the components in essential oils from Eucalyptus polybractea. We investigated the metabolism of 1,8-cineole by liver microsomes of rats and humans and by recombinant cytochrome P450
(P450 or CYP) enzymes in insect cells in which human P450 and
NADPH-P450 reductase cDNAs had been introduced. 1,8-Cineole was found
to be oxidized at high rates to
2-exo-hydroxy-1,8-cineole by rat and human liver
microsomal P450 enzymes. In rats, pregenolone-16
-carbonitrile (PCN)
and phenobarbital induced the 1,8-cineole 2-hydroxylation activities by
liver microsomes. Several lines of evidence suggested that CYP3A4 is a
major enzyme involved in the oxidation of 1,8-cineole by human liver
microsomes: 1) 1,8-cineole 2-hydroxylation activities by liver
microsomes were inhibited very significantly by ketoconazole, a CYP3A
inhibitor, and anti-CYP3A4 immunoglobulin G; 2) there was a good
correlation between CYP3A4 contents and 1,8-cineole 2-hydroxylation
activities in liver microsomes of eighteen human samples; and 3) of
various recombinant human P450 enzymes examined, CYP3A4 had the highest
activities for 1,8-cineole 2-hydroxylation; the rate catalyzed by
CYP3A5 was about one-fourth of that catalyzed by CYP3A4. Kinetic
analysis showed that Km and
Vmax values for the oxidation of 1,8-cineole
by liver microsomes of human sample HL-104 and rats treated with
PCN were 50 µM and 91 nmol/min/nmol P450 and 20 µM and 12 nmol/min/nmol P450, respectively. The rates observed using human liver
microsomes and recombinant CYP3A4 were very high among other CYP3A4
substrates reported so far. These results suggest that 1,8-cineole, a
monoterpenoid present in nature, is one of the effective
substrates for CYP3A enzymes in rat and human liver microsomes.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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1,8-Cineole
(Fig. 1), known as eucalyptol, is
one of the components present in essential oils from Eucalyptus
polybractea. This component has characteristic fresh and
camphoraceous fragrance and pungent taste and is used for
pharmaceutical preparations as an external applicant, a nasal spray, a
disinfectant, an analgesic, or a food flavoring. It is also for
cosmetics. Furthermore, it has been reported that 1,8-cineole is used
to treat cough, muscular pain, neurosis, rheumatism, asthma, and
urinary stone (Geremia, 1955
; Margret, 1999). The biotransformation of
1,8-cineole had not been studied extensively until several alcohol and
ketone metabolites such as 2-exo-hydroxy-1,8-cineole,
(±)-3-endo-hydroxy-1,8-cineole, (±)-3-exo-hydroxy-1,8-cineole, 2-oxo-1,8-cineole, and
3-oxo-1,8-cineole were identified in rabbit urine (Miyazawa et al.,
1989). These oxidation products of 1,8-cineole, which have not usually
been formed as chemical reaction products, have been reported by us to
be produced by microorganisms, such as Gromellera cingulata and Aspergillus niger (Nishimura et al., 1982; Miyazawa et
al., 1991). However, the metabolite of 1,8-cineole in humans has not yet been reported.
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A variety of components in numerous species of plants have been
reported to be oxidized by multiple forms of cytochrome P450 (P450 or
CYP1) in laboratory animals and humans (Gonzalez
and Nebert, 1990; Nelson et al., 1993; Khojasteh-Bakht et al., 1999).
P450 enzymes have been shown to detoxify and/or toxify these compounds
to more polar, and sometimes more reactive, metabolites (Thomassen et al., 1988; Gonzalez, 1989; Guengerich and Shimada, 1991; Guengerich, 1992; Khojasteh-Bakht et al., 1999). For example, pulegone, the major
constituent of pennyroyal oil and a hepatotoxin in humans (Anderson et
al., 1996), is catalyzed by P450 enzymes to a proximate hepatotoxic
metabolite, menthofuran, by P450 enzymes (Thomassen et al., 1988;
McClanahan et al., 1989). Several human P450 enzymes, such as CYP2E1,
CYP1A2, and CYP2C19, have recently been shown to catalyze oxidation of
pulegone (Khojasteh-Bakht et al., 1999).
The aim of the current study was to investigate metabolism of
1,8-cineole by human liver microsomes and by recombinant human P450
enzymes expressed in insect cells in which both human P450 and
NADPH-P450 reductase cDNAs had been introduced (Inoue et al., 1999;
Shimada et al., 1999). We also determined the 1,8-cineole 2-hydroxylation activities by liver microsomes of untreated rats and
rats treated with several P450 inducers.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals.
Ketoconazole, NADP+, glucose 6-phosphate, and
glucose-6-phosphate dehydrogenase were purchased from Sigma Chemical
Co. (St. Louis, MO). 1,8-Cineol was purchased from Taiyo Koryo Co.
(Osaka, Japan). 2-exo-Hydroxy-1,8-cineole was isolated and
purified from cultured medium on incubation of 1,8-cineole with
G. cingulata (Miyazawa et al., 1991). Other reagents and
chemicals used in this study were obtained from sources as described
previously or of the highest quality commercially available (Shimada
et al., 1994, 1998).
Enzyme Preparation.
Human liver samples were obtained from organ donors or patients
undergoing liver resection as described previously (Mimura et al.,
1993; Shimada et al., 1994). 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).
Preparation of Rat Liver Microsomes.
Male Sprague-Dawley rats (weighing about 200 g) obtained from
Nihon Clea Co. (Osaka, Japan) were treated with 
-naphthoflavone and
isosafrole (50 mg/kg, daily for 3 days), phenobarbital (80 mg/kg, daily
for 3 days), or pregenolone-16-carbonitrile (PCN, 100 mg/kg, daily
for 3 days). Ethanol was given to rats in drinking water for 6 days at
a final concentration of 15% (v/v). Rats were starved overnight before
being killed, and liver microsomes were prepared.
1,8-Cineole 2-Hydroxylation Assay.
1,8-Cineole 2-hydroxylation activities by P450 enzymes were determined
as follows. Standard reaction mixture contained rat and human liver
microsomes (0.025 mg of protein/ml) or recombinant P450 (50 pmol/ml)
with 200 µM 1,8-cineole in a final volume of 0.50 ml of 100 mM
potassium phosphate buffer (pH 7.4) containing an NADPH-generating
system consisting of 0.5 mM NADP+, 5 mM glucose
6-phosphate, and 0.5 units of glucose-6-phosphate dehydrogenase/ml
(Shimada et al., 1996). Incubations were carried out at 37°C for 30 min, terminated by adding 1.0 ml of ethyl acetate, and mixed
vigorously. The extracts (organic layer) were collected by
centrifugation at 3000 rpm for 10 min and transferred to an insert for
analysis by electron impact (EI)-MS.
Other Assays.
P450 contents were estimated spectrally by the original method (Omura
and Sato, 1964). The contents of human CYP3A proteins in human liver
microsomes were estimated by coupled SDS-polyacrylamide gel
electrophoresis/immunochemical development ("Western blotting") (Guengerich et al., 1982). Rabbit anti-serum raised against
purified human liver CYP3A4 was prepared as described previously
(Mimura et al., 1993). The intensities of the immunoblots were measured with an Epson (Tokyo, Japan) GT-8000 Scanner equipped with
NIH Image/Gel Analysis program adapted for Macintosh
computers. Protein concentrations were estimated by the method of Lowry
et al. (1951).
Statistical Analysis. Kinetic parameters for 1,8-cineole 2-hydroxylation by rat and human P450 enzymes were estimated using a computer program (KaleidaGraph, Synergy Software, Reading, PA) designed for nonlinear regression analysis. Substrate concentrations used for the analysis of 1,8-cineole 2-hydroxylation activities were 60, 120, 160, and 200 µM.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification of 2-Hydroxylated Metabolite of 1,8-Cineole on
Incubation with Human Liver Microsomes.
1,8-Cineole was incubated with liver microsomes of human sample HL-104
in the presence of an NADPH-generating system. We detected only one
metabolite in this assay condition using GC-MS equipped with EI-MS as
described under Materials and Methods (Fig.
2). The formation of
2-exo-hydroxylated metabolite of 1,8-cineole was suggested
with the following lines of evidence from analysis with GC and GC-MS:
1) the molecular mass of the metabolite was increased from 154 to
170; 2) dehydration peak
(M+ 
H2O) was
changed from 170 to 152 by this fragment, indicating that a
hydroxyl group was introduced into the molecule at the C-2 position of
1,8-cineole (Nishimura et al., 1982); and 3) the position and relative
stereochemistry of a hydroxyl group were determined by relative
abundance of mass fragments and retention time with GC. These values
were consistent with those of authentic 2-exo-hydroxy-1,8-cineole, which has been isolated from
biotransformation products of 1,8-cineole with G. cingulata
(Miyazawa et al., 1991).
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Metabolism of 1,8-Cineole to 2-exo-Hydroxy-1,8-Cineole by Human and Rat Liver Microsomes. On incubation of 1,8-cineole with human liver microsomes in the presence of an NADPH-generating system, the formation of 2-exo-hydroxy-1,8-cineole metabolite was increased with increasing incubation time, P450 levels in liver microsomes, and substrate concentrations (Fig. 3, A, B, and C, respectively).
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1,8-Cineole 2-Hydroxylation by Recombinant P450 Enzymes. Ten forms of human P450 enzymes expressed in insect cells were used to determine which P450 enzymes are the major catalysts for the 1,8-cineole 2-hydroxylation in humans (Table 4). CYP3A4 had the highest activity for the 1,8-cineole 2-hydroxylation, followed by CYP3A5, CYP2B6, and CYP2A6. Other P450 enzymes including CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 had very low activities or activities below the limit of detection.
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Kinetic Analysis of 1,8-Cineole 2-Hydroxylation by Rat and Human Liver Microsomes and by Recombinant CYP3A4. Kinetic analysis of the 1,8-cineole 2-hydroxylation activities catalyzed by liver microsomes of human sample HL-104 and of rats treated with PCN and by recombinant CYP3A4 were determined (Fig. 5). The Km value for 1,8-cineole 2-hydroxylation by liver microsomes of rats treated with PCN was 20 µM, and those of liver microsomes of HL-104 and recombinant CYP3A4 were 50 and 90 µM, respectively. Vmax values were highest in HL-104 (91 nmol/min/nmol P450) followed by recombinant CYP3A4 (48 nmol/min/nmol P450) and PCN-treated rats (12 nmol/min/nmol P450).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Various monoterpenoids, including 1,8-cineole, have been reported
to be biotransformed by several microorganisms (Miyazawa, 1997). We
have previously shown that 1,8-cineole is metabolized by G. cingulata, a pathogenic fungus, to (+)-2-hydroxy-1,8-cineole as a
major metabolite, as well as by some minor metabolites (Miyazawa et
al., 1991). Additional studies have indicated that A. niger converts 1,8-cineole to three alcohols and two ketones, such as 2-exo-hydroxy-1,8-cineole,
(±)-3-endo-hydroxy-1,8-cineole,
(±)-3-exo-hydroxy-1,8-cineole, 2-oxo-1,8-cineole, and
3-oxo-1,8-cineole (Nishimura et al., 1982). In mammalian species, it
has been reported that 1,8-cineole is metabolized to four alcohols,
namely 2-exo-hydroxy-1,8-cineole, 2-endo-hydroxy-1,8-cineole,
3-exo-hydroxy-1,8-cineole, and
3-endo-hydroxy-1,8-cineole, in rabbits (Miyazawa et al.,
1989). However, it remains unclear whether 1,8-cineole is metabolized
in humans.
The following lines of evidence suggested that 1,8-cineole is actually oxidized at the 2-position forming 2-hydroxy-1,8-cineole by CYP3A4 in human liver microsomes. First, on incubation of 1,8-cineole with human liver microsomes, 2-hydroxylated metabolite of 1,8-cineole was found to be formed on analysis with GC-MS-EI. Second, ketoconazole, an inhibitor of CYP3A-catalyzed reaction, and anti-CYP3A4 IgG significantly inhibited the 1,8-cineole 2-hydroxylation activities catalyzed by human liver microsomes, and there was good correlation between CYP3A4 contents and 1,8-cineole 2-hydroxylation activities in liver microsomes of 18 human samples examined. Finally, of 10 recombinant human P450 enzymes tested, CYP3A4 had the highest rate for the 1,8-cineole 2-hydroxylation activities. CYP3A5 was found to have activities of 1,8-cineole 2-hydroxylation at rate of one-fourth of that of CYP3A4 in recombinant systems. It should be mentioned that in the present assay condition we did not detect oxidation products other than 2-exo-hydroxy-1,8-cineole in rat and human liver microsomes.
In this study, we found that ketoconazole more potently inhibited 1,8-cineole 2-hydroxylation activities catalyzed by CYP3A4/5 enzymes in human liver microsomes than the CYP3A enzymes in rat liver microsomes. Mechanisms underlying such different inhibitory action of ketoconazole in rat and human CYP3A enzymes are not known at present.
Kinetic analysis suggested that the Vmax
value for 1,8-cineole 2-hydroxylation catalyzed by liver microsomes of
human sample HL-104 was extremely high (91 nmol/min/nmol P450). This
human sample has been shown to have a high level of CYP3A enzymes in liver microsomes; immunoblotting analysis determined the level of CYP3A
proteins in this sample to be about 60% of total P450 (Shimada et al.,
1994, 1999). The Vmax value for 1,8-cineole
2-hydroxylation by recombinant CYP3A4 expressed in insect cells was
determined to be 48 nmol/min/nmol P450; the value was similar to, but
lower than, that of liver microsomes of HL-104.
Recombinant human CYP2B6 and CYP2A6 had activities for 1,8-cineole
2-hydroxylation at rates of 2.3 and 1.9 nmol/min/nmol P450, respectively. The levels of these proteins in human liver microsomes have been determined to be about 1 and 4%, respectively, of total P450
(Shimada et al., 1994), indicating that these P450 forms may have minor
roles in the oxidation of 1,8-cineole in human liver microsomes.
It has been reported that there are at least four CYP3A enzymes in
rats, and among them CYP3A1 and -3A2 are shown to be the major enzymes
in rat liver microsomes (Guengerich and Shimada, 1991; Maurel, 1996).
Both CYP3A1 and -3A2 have been reported to be induced by several
chemical inducers, such as PCN and phenobarbital, although the latter
chemical has also been known to be a typical inducer of CYP2B enzymes
in rat livers (Maurel, 1996; Yamazaki et al., 1996). The 1,8-cineole
2-hydroxylation activities by liver microsomes were induced in rats by
both PCN and phenobarbital, suggesting that CYP3A enzymes are involved
in the oxidation of 1,8-cineole in rats. The
Km value for 1,8-cineole 2-hydroxylation by
liver microsomes of rats treated with PCN was lower than that of liver
microsomes of HL-104, whereas the
Vmax/Km ratio
of 1,8-cineole 2-hydroxylation was found to be 3-fold higher in HL-104
than in PCN-treated rats.
In conclusion, the present study showed that 1,8-cineole is highly catalyzed by CYP3A enzymes to form 2-exo-hydroxy-1,8-cineole in rat and human liver microsomes. CYP3A4 is suggested to be a principal enzyme in catalyzing 1,8-cineole 2-hydroxylation by human liver microsomes; the rate catalyzed by liver microsomes of 18 human samples was 26.9 ± 16.9 nmol/min/nmol P450. In rats, 1,8-cineole 2-hydoxylation was also suggested to be catalyzed by CYP3A enzymes that are induced by PCN and phenobarbital in liver microsomes. The rate catalyzed by liver microsomes of PCN-treated rats was about 10 nmol/min/nmol P450 for 1,8-cineole 2-hydroxylation.
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Footnotes |
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Received May 10, 2000; accepted October 10, 2000.
This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, the Ministry of Health and Welfare of Japan, and the Developmental and Creative Studies from Osaka Prefectural Government.
Send reprint requests 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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Abbreviations |
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Abbreviations used are:
P450, general term for
cytochrome P450;
CYP, individual isoform of P450;
IgG, immunoglobulin
G;
PCN, pregnenolone-16-carbonitrile;
GC, gas chromatography;
EI, electron impact;
MS, mass spectrometer.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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), by recombinant (insect cell) CYP3A4 (
), and by liver
microsomes of rats treated with PCN (
).