pON~"~Nv

l".0~'.M~t,"
ELSEVIER

Environmental Toxicology and Pharmacology 2 (1996) 331-337

I~l'it~''

ETAP

Species difference in the inhibition of pentobarbital metabolism by

empenthrin
Ryozo Tsuji
a

a,*,

Naohiko Isobe

a,

Yasuyuki Kurita b Koji Hanai

Hajime Kawasaki a

a,

Yoshiyasu Yabusaki c,

Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98, Kasugade-naka, Konohana-ku, Osaka 554, Japan
b Agricultural Chemicals Research Laboratory, Sumitomo Chemical Co., Ltd., 4-2-1, Takatsukasa, Tarazuka, Japan
c Biotechnology Laboratory, Sumitomo Chemical Co., Ltd., 4-2-1, Takatsukasa, Tarazuka, Japan

Received 22 March 1996; revised 6 August 1996; accepted 12 August 1996

Abstract
Empenthrin, synthetic pyrethroid, prolonged the pentobarbital-induced sleeping time in mice, but not in rats, guinea pigs or hamsters.
Empenthrin did not delay the clearance of pentobarbital from serum in dogs. In addition, empenthrin dose-dependently inhibited in vitro
metabolism of pentobarbital in mice, but not in rats, guinea pigs, hamsters or rabbits. Lineweaver-Burk plots indicated that the inhibition
was competitive in mice. Microsomal fractions of recombinant yeast expressing human cytochrome P-450 (CYP)s were used to determine
the inhibitory effect of empenthrin on pentobarbital metabolism in humans. CYP2B6 and CYP2D6 were responsible for biotransformation
of pentobarbital to a pentobarbital alcohol identified as 5-ethyl-5-(l'-methyl-3'-hydroxybutyl) barbituric acid. The structure of pentobarbital fit the criteria for a CYP2D6 substrate on computational analysis. Empenthrin did not inhibit the pentobarbital metabolism
catalyzed by these two CYPs. These findings suggest that the inhibition of pentobarbital metabolism by empenthrin in mice does not
occur in other species including humans.
Keywords: Pentobarbital; Empenthrin; Species difference

1. Introduction

Our previous studies have revealed that pyrethroids

modify pentobarbital-induced sleeping time in mice and
that there is a clear relationship between the chemical
structure of pyrethroids and their effect on pentobarbitalinduced sleeping time (Tsuji et al., 1996a). The group of
pyrethroids, which possess an ethynyl group on the acid or
alcohol moiety, prolong pentobarbital-induced sleeping
time. Pentobarbital is known to be almost completely
hydroxylated by the cytochrome P-450 (CYP) hepatic
microsomal enzymes (Brodie et al., 1953; Freudenthal and
Carroll, 1973). The prolongation of pentobarbital-induced
sleeping time by empenthrin, a typical pyrethroid with an
ethynyl group, is due to the inhibition of pentobarbitalmetabolizing enzymes by empenthrin (Tsuji et al., 1996b).

* Corresponding author. Tel.: + 81-6-466-5343; Fax: + 81-6-466-5443.

In contrast, in rats, empenthrin affects neither pentobarbital-induced sleeping time nor pentobarbital
metabolism in vitro (Tsuji et al., 1996b), although the
acute oral toxicity of empenthrin in mice is nearly equal to
or higher than that in rats (Kaneko et al., 1992). These
findings indicate the existence of a species difference
between mice and rats in the inhibition effect of empenthrin on pentobarbital metabolism. However, it is not
clear whether this inhibition by empenthrin is specific to
mice. In this study, the effect of empenthrin on pentobarbital-induced sleeping time was examined in guinea
pigs and hamsters in addition to mice and rats. In addition,
the effects of empenthrin on biotransformation of pentobarbital in vitro were examined in other species including
humans. It is well known that mammals possess numerous
isozymes of CYP in hepatic microsomes (Nebert et al.,
1991). There are many reports describing the interaction of
drugs and isozymes of CYP. Several human CYP genes
have been expressed in yeast using conventional gene
engineering techniques for use in study of metabolic activ-

1382-6689/96/$15.00 Copyright 1996 Elsevier Science B.V. All rights reserved.

PII S 1382-6689(96)00066-X

332

R. Tsuji et al./ Environmental Toxicology and Pharmacology 2 (1996) 331-337

ity in human (Brian et al., 1989; Imaoka et al., 1996).

However, there is no report available describing isozymes
that metabolize pentobarbital in animal or humans. In this
study we found isozymes of CYP that metabolize pentobarbital in humans using microsomes of recombinant yeast
expressing human CYPs, and attempted to determine
whether empenthrin inhibits pentobarbital metabolism in
humans, as it does in mice.

2. Materials and methods

2.1. Animals

Male 6-week-old ICR mice, 7-week-old SD rats, 7week-old hamsters and 10-week-old guinea pigs, which
were purchased from Charles River Japan (Shiga, Japan),
were used after a 1 week period of acclimation. Male
3-month-old rabbits and dogs of both sexes, 8 months of
age and older, were purchased from Kitayama Labes
(Kyoto, Japan) and White Eagle Laboratories (Pennsylvania, USA), respectively, were used after a 2 week and a
1 month period of acclimation, respectively. They were
housed in rooms with an ambient temperature and relative
humidity and 12 h light cycle. Animals had free access to
food and water, except for dogs. Dogs were given limited
access to food but had free access to water.
2.2. Chemicals

Empenthrin [(RS)- 1-ethynyl-2-methyl-2-pentenyl(1R)cis, trans-chrysanthemate] (Fig. 1) was synthesized at Sumitomo Chemical Co. (Osaka, Japan). Pentobarbital sodium
and pentobarbital sodium injection (Nembutal ) were purchased from Nacalai Tesque (Kyoto, Japan) and Dainippon
Pharmaceutical Co. (Osaka, Japan), respectively. Pentobarbital alcohol, 5-ethyl-5-(l'-methyl-3'-hydroxybutyl) barbituric acid, was purified from urine of rats injected with
pentobarbital sodium by HPLC. HPLC was performed
with a pump instrument (LC-10AS, Shimadzu, Kyoto,
Japan), a ODS column (ODS-AM12S05, 20 mm (i.d.)X
250 mm, 5 /xm, YMC, Tokyo, Japan), and UV detector (at
210 nm, UV-SPD-10A Shimadzu). A mixture of acetonitrile and water (gradient from 20 : 80 to 70 : 20) was used
as a mobile phase. The flow rate was 9.9 ml/min. The
chemical structure of pentobarbital alcohol was identified
using secondary ion mass spectra (SI-MS) (M-80B mass

Fig. 1. The chemical structure of empenthrin.

spectrometer, Hitachi, Tokyo, Japan) and ~H- and 13CNMR spectra (JEOL GSX-270 spectrometer, JEOL, Tokyo,
Japan). NADPH was purchased from Oriental Yeast Co.
(Osaka, Japan). All other chemicals and solvents were of
analytical grade.
2.3. Measurement o f sleeping time

For measurement of pentobarbital-induced sleeping

time, pentobarbital sodium was injected (mice and hamsters: 45 m g / k g ; rats: 35 m g / k g ; guinea pigs: 25 m g / k g )
intraperitoneally 2 h after oral administration of empenthrin (0, 100 or 300 m g / k g ) dissolved in corn oil.
Sleeping time was defined as the time interval from loss to
reappearance of the righting reflex.
2.4. Determination o f pentobarbital concentration in dog
serum

Pentobarbital sodium (50 m g / k g ) was injected into

dogs 2 h after oral administration of empenthrin (0 or 1
g / k g in one capsule). Blood was collected 10 and 30 min,
and 1, 2 and 4 h after administration of pentobarbital.
Serum was mixed with acetonitrile and methanol in the
ratio of 1 : 1 : 1. After centrifugation at 10000 X g for 10
min, the supernatant was subjected to HPLC. HPLC was
performed with a pump instrument (LC-10AS, Shimadzu),
a ODS column (A-212, 6 mm (i.d.) 150 mm, 5 /~m,
Sumika Chemical Analysis Service, Osaka, Japan), and a
UV detector (at 210 nm, UV-SPD-10A, Shimadzu). A
mixture of acetonitrile and 0.05 M phosphate buffer (1 : 1)
was used as a mobile phase. The flow rate was 1 ml/min.
The retention time of pentobarbital was about 6.4 min.
2.5. Pentobarbital metabolism in vitro

Animals were killed by decapitation, and isolated livers

were homogenized with a Poter-Elevehjem glass-Teflon
homogenizer in ice-cold 1.15% KC1 (20%, w / v ) . The
homogenate was centrifuged at 10000 X g for 10 min. The
supernatant was further centrifuged at 105 000 X g for 60
min to obtain a microsomal pellet. The pellet was resuspended in 0.05 M phosphate buffer, pH 7.4, to a concentration of 4 - 8 mg protein/ml. Protein concentration was
determined using bicinchonic acid (Smith et al., 1985).
In the species difference study, the biotransformation
rate of pentobarbital was determined by measuring the
decrease in substrate concentration. The assay mixture
contained microsomal preparation (1.2 mg protein/ml), 3
mM NADPH, 50 mM phosphate buffer (pH 7.4) and 20
/xM pentobarbital in a final volume of 1 ml. Empenthrin
was dissolved in acetone and added to the incubation
mixture at a rate of 10 /~l/ml to prepare final concentrations of 0.2-20 /zM. Reaction was carried out at 37C
with shaking for 20 min (mice and hamsters) or 30 min
(rats, guinea pigs and rabbits). To terminate the incubation,

R. Tsuji et aL / Environmental Toxicology and Pharmacology 2 (1996) 331-337

2 ml of a 1 : 1 mixture of ice-cold acetonitrile and methanol
was added. After centrifugation at 10000 X g for 10 min,
the concentration of pentobarbital in the supernatant was
measured by HPLC as described above. In the kinetic
study, the biotransformation rate of pentobarbital was determined by measuring the concentration of pentobarbital
alcohol. The assay mixture contained microsomal preparation (1.0 mg protein/ml), 3 mM NADPH, 50 mM phosphate buffer (pH 7.4) and 40, 80, 160 or 320 /xM pentobarbital in a final volume of 1 ml. Empenthrin was dissolved in acetone and added to the incubation mixture at a
rate of 1 0 / z l / m l to prepare final concentrations of 0.2-20
/zM. Reaction was carried out at 37C with shaking for 20
min. To terminate the incubation, 1 ml of a 1 : 1 mixture of
ice-cold acetonitrile and methanol was added. After centrifugation at 10000 X g for 10 min, the concentration of
pentobarbital alcohol in the supernatant was measured by
HPLC as described above without mobile phase. A mixture of acetonitrile and 0.05 M phosphate buffer (gradient
from 20:80 to 70:20) was used as a mobile phase. The
flow rate was 1 ml/min. The retention time of pentobarbital alchohol was about 7.2 min.
The rate of reaction was determined using LineweaverBurk plots. The mean of three determinations was used to
average and plot each point.

333

each isomer were generated with the systematic search

method in SYBYL. Bonds 1, 2, 3 and 4 (Fig. 7) were
defined as rotatable and were incremented by 60, 60, 120
and 120 degrees, respectively. If there were atoms the
distance between which was less than the sum of scaled
van der Waals radii, the conformation was considered to
be very unstable and excluded. The scaling factor was set
to 0.6. The geometries of each initial conformation were
optimized using the AM1 method (Dewar et al., 1985) in
the MOPAC 93 semiempirical molecular orbital package
(MOPAC93, J.J.P. Stewart and Fujitsu, Tokyo, Japan).
Energy of the most stable conformer was also calculated at
the H F / 6 - 3 1 G * and HF(SCRF = DIPOLE)/6-31G* levels for each isomer using the AM1 optimized geometries
and the Gaussian 94 ab initio molecular orbital package
(Gaussian 94, Revision A.1, Gaussian, Pittsburgh, PA,
1995). At the latter level, solvent effects were considered.
Dielectric constant of the solvent water was set to 78.4.
2.8. Statistical analysis
The significance of differences between the control and
treated groups was determined by analysis of variance
(ANOVA) and the least significant difference (LSD)
method at 5% and 1% levels.

2.6. Human pentobarbital metabolism in vitro

3. Results

The microsomal fractions of yeast expressing human

CYP (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19,
2D6, 2El and 3A4, Sumitomo Chemical Co., Osaka,
Japan) were used. The biotransformation rate for pentobarbital was determined by measuring the concentration of
pentobarbital alchohol. The assay mixture contained microsomal preparation (50 pmol), 3 mM NADPH, 50 mM
phosphate buffer (pH 7.4) and 50 /zM pentobarbital in a
final volume of 0.5 ml. In the study of the effect of
empenthrin, empenthrin was dissolved in acetone and added
to the incubation mixture at a rate of 10 / z l / m l to prepare
a final concentration of 20 /xM. Reaction was carried out
at 37C with light shaking for 30 min. To terminate the
incubation, 1 ml of a 1 : 1 mixture of ice-cold acetonitrile
and methanol was added. Pentobarbital alcohol in the
supernatant was measured by HPLC as described above.

Fig. 2 shows the effect of empenthrin on pentobarbitalinduced sleeping time in males of several species. A single
dose of 100 m g / k g or higher of empenthrin prolonged the
pentobarbital-induced sleeping time in mice in a dose-dependent manner. However, in rats, hamsters and guinea
pigs, administration of empenthrin did not change the
pentobarbital-induced sleeping time even at doses as high
as 300 m g / k g (Fig. 2).

2.7. Computational analysis

There was no significant difference in the serum concentration of pentobarbital between controls and empenthrin-treated dogs (Fig. 3).

The coordinates of c~-methylamobarbital (Smit and

Kanters, 1974) obtained from the Cambridge Structural
Database (Allen et al., 1983) were converted to the initial
geometries of the eight isomers pentobarbital (Fig. 6) using
the SYBYL program (version 6.1, TRIPOS Associates, St.
Louis, USA). Only the neutral forms of pentobarbital were
considered because the p K a value of pentobarbital is 8.18
(Slater et al., 1994) and about 86% of pentobarbital exists
in the neutral forms at pH 7.4. The initial conformations of

3.1. Effects of empenthrin on pentobarbital-induced sleeping time

3.2. Effect of empenthrin on pentobarbital concentration in

dog serum

3.3. In vitro pentobarbital metabolism

Empenthrin inhibited the biotransformation of pentobarbital by hepatic microsomes isolated from mice in a
dose-dependent manner from 0.2 /zM to 20 /zM, but did
not affect the enzyme activity of rats, guinea pigs, hamsters or rabbits at concentrations up to 20 /zM (Fig. 4).
Linerweaver-Burk plots of pentobarbital hydroxylation

R. Tsuji et al. / Enuironmental Toxicology and Pharmacology 2 (1996) 331-337

334
900 -

**

800 O

700 -

O
o

800-

100

L~." . . . . . . . . . . . . . . . . . . .

~;E-'"

~'~'~"=' ..............A

,.-~-.-..-r~.S--_='~"~,:';:-"-': --=. . . . . . . . . . Q

500 -

400-

a.
O3

300-

en

5o

200 -

r-==-

100-~
0 ~

Mice

Rats

G u i n e a pigs

0.2

20

Hamsters
Concentration of empenthrin (~tM)

Fig. 2. Effects of empenthrin on pentobarbital-induced sleeping time in

mice, rats, guinea pigs and hamsters. Pentobarbital (mice and hamsters:
45 m g / k g ; rats: 35 m g / k g ; guinea pigs: 25 m g / k g ) was injected
intraperitoneally 2 h after oral administration of empenthrin (0 (open bar),
100 (hatched bar) or 300 m g / k g (filled bar). Each bar represents the
mean _+S.E. for 8-10 animals. Sleeping times of the control animals were
38.7+3.2 (mice), 72.6+6.0 min (rats), 110.0_+3.0 (guinea pigs) and
40.4-1-6.2 min (hamsters). *, P < 0.05; * *, P < 0.01: significantly
different from control.

:=.

Fig. 4. The effect of empenthrin on the rate of biotransformation of

pentobarbital by liver microsomes. Each value (mice ([3), rats ( a ) ,
guinea pigs (O), hamsters (O), rabbits ( )) represents the mean _ S.E.
for 5-7 experiments as a percentage of control activities. The control
values were 100 + 4.8 (mice), 85.8 + 8.6 (rats), 73.9 + 6.7 (guinea pigs),
266+7.1 (hamsters) and 86.15:5.1 (rabbits) ( p m o l / m g protein/min,
mean 5: S.E.). The data for mice and rats were quoted from Tsuji et al.,
1996b. *, P < 0.05; * *, P < 0.01: significantly different from control.

3020

o=

i 208

~
O

15-

_~

10-

s-

10o.
E
0
0.25 01.5

-2

-t0

Time after administration (h)

1;
1/[Pentobarbital]

Fig. 3. Effect of empenthrin on derease in serum pentobarbital. Pentobarbital (50 m g / k g ) was injected intravenously 2 h after oral administration
of empenthrin (0 ( ) or 1 g / k g (O). Each value represents the mean +
S.E. for four animals.

2;

3o

(raM q )

Fig. 5. Linerweaver-Burk plot of the effect of empenthrin on pentobarbital metabolism in mice. Each point represents the mean of three
separate determinations. Symbols are 0 (O), 3 ( a ) and 10 ( m ) M
empeuthrin.

Table 1
Relative energies of the most stable conformers (zlE, kcal/mol), r[N a - C r ] and r[N b - C r ] of the most stable conformers (/k), and the Bolzmann
average of r[N a - C r ] and r[N b -- C r] at 298.15 K (r[N a - C r ] a v . and r[N b -- Cr]av, ,~) calculated for isomers of pentobarbital
Isomer

AE(AM1)

AE(HF) a

AE(SCRF) b

r[N a _ C r ]

r[N b _ C r ]

r[N a - -

1
2a
2b
3a
3b
4a
4b
5

0.0
19.8
19.6
15.9
15.7
35.5
35.4
34.5

0.0
23.5
23.3
19.3
18.4
38.8
38.2
40.5

0.0
22.9
22.8
18.2
17.0
37.8
37.4
37.5

4.97
4.95
4.85
4.97
4.82
4.97
4.82
4.96

4.82
4.82
4.97
4.83
4.96
4.85
4.96
4.82

5.36
5.49
5.22
5.54
5.13
5.46
5.18
5.42

Cr]av.

r[N b _ Cr]av.

5.16
5.29
5.37
5.32
5.32
5.27
5.39
5.21

" AE(HF)s were calculated at the HF/6-31G * level using the AM1 optimized geometries.
b AE(SCRF)s were calculated at the HF(SCRF = DIPOLE)/6-31G * level using the AM1 optimized geometries. Dielectric constant was set to 78.4.

335

R. Tsuji et al. / Environmental Toxicology and Pharmacology 2 (1996) 331-337

E 15-

o
"6
E
e~

-~ 10E

g
N

5-

2~
e~
e~
o

2B6

2D6

Fig. 6. The effect of empenthrin on the pentobarbital hydroxylation

activity of yeast expressing human CYP microsome. Each value (0 ([]),
20 #xM ( ) ) represents the mean+ S.E. for three experiments.
Fig. 8. The most stable conformer 1 of pentobarbitalat the AMI level.
activity are shown in Fig. 5. Vmax and K m were 370
/ z m o l / m g p r o t e i n / m i n and 66 #zM, respectively, and the
K i for empenthrin was 1.6 ~ M . These findings indicated
that inhibition by empenthrin in mice was competitive.
Pentobarbital was biotransformed to pentobarbital alchohol by human CYP2D6 and CYP2B1, but not by
CYP1A1, C Y P 1 A 2 , CYP2A6, CYP2C8, CYP2C9,
CYP2C18, CYP2C19, CYP2E1 or CYP3A4. The pento-

ell3

CH 3

CH 3

\,

~C 4 H2

j..H

barbital hydroxylation activities of CYP2B6 and 2D6 were

5.4 + 0.53 and 15.1 + 0.92 p m o l / p m o l C Y P / 3 0 min, respectively.
Empenthrin did not inhibit biotransformation of pentobarbital by CYP2D6 or CYP2B6 (Fig. 6).

/ o C 5 3 i~CH3

~ C C " ~ H c....

CH~.zC4H2
~ : O ,~'3 tCH3/

H.

t~H

C....

C....

H CHz

_'O , ~ 3 ,ICH3/
,~H
C "''~
C'"'

.'o

"

u"

7""

""

CH3

"

2a (2b)
CH 3

( / C rH 2

( / C rH 2

'o ~ 3 ,,,CHd c ..... H \

, "~c.=

NN

:O ~

) o

,,CH~

, c....
%
CH2
H' ciH~

ell2

\'o CH/ 3

4a (4b)

CH2

3a (3b)

CH3

..o--~,, 0 ~ ;

\'o/

CH3

CH 3

\\

Fig. 7. Isomersof pentobarbitaland the difinitionof rotatable bonds 1-4, N" and N b (amide nitrogenatoms), and C r (oxidationsite).

336

R. Tsuji et al./ Environmental Toxicology and Pharmacology2 (1996) 331-337

3.4. Computational analysis

Following geometry optimizations using the AM1

method, 62, 53, 53, 55, 53, 58, 58 and 53 conformers were
obtained for the isomers of pentobarbital 1, 2a, 2b, 3a, 3b,
4a, 4b and 5, respectively. Isomer 1 is predicted to be
much more stable than the other isomers at all levels of
calculations (Table 1). In the most stable conformer of
isomer 1, r[N a - C r ] (distance between the amide nitrogen
atom N a and the oxidation site cr; see Fig. 7) and r[N b C r ] were 4.97 and 4.82 ,~, respectively (Fig. 8). Assuming
the Boltzmann distribution at 298.15 K, the average of
r[N a - C r ] and r[N b - C r ] for all conformers of isomer 1
are 5.36 and 5.16 ,~, respectively. These values are also
about 5 A for the other isomers (Table 1).

4. Discussion
In this study, prolongation of pentobarbital-induced
sleeping time by empenthrin was observed in mice, but not
in rats, guinea pigs or hamsters. It is widely accepted that
change in pentobarbital-induced sleeping time reflects
change in the hepatic hydroxylation activity of pentobarbital (Andre et al., 1984). Empenthrin delayed the
clearance of pentobarbital from serum in mice, but not in
rats (Tsuji et al., 1996b). Empenthrin inhibited pentobarbital metabolism by liver microsomes of mice, but not
that of rats, guinea pigs, hamsters or rabbits. In dogs,
empenthrin had no effect on the clearance of serum pentobarbital. This data indicated that empenthrin did not effect
pentobarbital metabolism in dogs. These findings suggest
that the inhibition of pentobarbital metabolism by empenthrin is specific to mice. There are few reports describing species difference in the inhibition of pentobarbital
metabolism, although many substances are known to inhibit pentobarbital metabolism and prolong the sleeping
time of mice (Andre et al., 1984; Ishikawa et al., 1991).
The CYP isozymes were tested to determine their preferential activity onto pentobarbital biotransformation using
microsomal fraction of yeast expressing human CYP. In
humans, pentobarbital is metabolized primarily by oxidation of the penultimate carbon (omega-l) of the methylbutyl side chain to give a 3'-hydroxy metabolite (Brodie et
al., 1953; Cooper and Brodie, 1957; Maynert, 1965). This
hydroxylated metabolite was produced by CYP2B6 and by
CYP2D6.
Substances metabolized by CYP2D6 have a common
structural features that there are one or more basic nitrogen
atoms (Wolff et al., 1986; Meyer et al., 1986), and a site of
hydroxylation can be positioned about 5 or 7 ,~ away from
basic nitrogen atom (Guengerich et al., 1986: Wolff et al.,
1986; Meyer et al., 1986). In addition, a hydrophobic
domain is near the site of hydroxylation (Guengerich et al.,
1986; Meyer et al., 1986). In the present study, the distances between the oxidation site and two nitrogen atoms

in pentobarbital were found to be about 5 A. Conformational analysis of pentobarbital indicated that the structure
fits the above criteria and that pentobarbital is a 5 ,~
substrate. However, although the substrate specificities of
CYP 2D6 exhibit a coplanar conformation near the oxidation site and have negative molecular electrostatic potentials (MEPs) in a part of this planar domain (Koymans et
al., 1992), pentobarbital is not agreement with these findings.
In contrast, empenthrin does not meet the above criteria,
since it has no nitrogen atom. This finding indicates that
empenthrin is not catalyzed by CYP2D6.
Empenthrin did not inhibit the metabolism of pentobarbital metabolism by CYP2B6 and CYP2D6. Therefore
it appears unlikely that empenthrin inhibits pentobarbital
metabolism in humans.
The maximum prolongation of pentobarbital-induced
sleeping time by empenthrin was observed 2 - 4 h after its
oral administration, and its effects disappeared 24 h after
administration (Tsuji et al., 1996b). The concentration of
empenthrin reached maximum 2 h after oral administration
and decreased thereafter (Isobe et al., 1992). These findings indicate that time-course changes in the effect of
empenthrin on pentobarbital-induced sleeping time are
clearly correlated with the concentration of empenthrin in
blood. The present finding that the inhibition of empenthrin of pentobarbital biotransformation in mice was
competitive supports this conclusion.
The finding of competitive interaction of two drugs in
metabolism by liver microsomes may suggest that the
metabolism of the drugs is catalyzed by the same CYP
isozymes. Hydroxylation of empenthrin at the isobutenyl
or omega-1 position of the penten group was observed in
an in vivo metabolism study using rats (Isobe et al., 1992),
and the reaction was considered to be catalyzed by hepatic
microsomal fraction. A limited member of P-450s are
responsible for oxidation of specific sites in pyrethroid
molecules in humans (Miyamoto et al., 1995). For example, cypermethrin and permethrin undergo oxidation by
CYP2C19 and CYP2C19, 2C9, 1A2, 2C18, and 2D6,
respectively (Miyamoto et al., 1995). The same substrates
are catalyzed by different CYP isozymes in different
species (Shimada et al., 1989a,b). Therefore the species
difference in inhibition of pentobarbital metabolism by
empenthrin, occurs because pentobarbital and empenthrin
can be catalyzed by the same CYP isozymes in mice and
by different CYP isozymes in other species. Of course, it
is also possible that empenthrin specifically affects pentobarbital metabolizing enzyme(s) in mice. Further work is
now in progress.

Acknowledgements
The authors thank Mr. H. Fujimoto and Mr. H. Matsunaga for purification and identification of pentobarbital

R. Tsuji et al. / Environmental Toxicology and Pharmacology 2 (1996) 331-337

alcohol, and Mr. S. Yamada for skilful technical assistance.

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