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Stereochemical considerations on the inhibition of hepatic epoxide hydrolase by some pesticides and their epoxides
273
Toxicology Letters, 30 (1986) 273-278 Elsevier
TOXLett.
1554
STEREOCHEMICAL HEPATIC EPOXIDE
CONSIDERATIONS ON THE INHIBITION OF HYDROLASE BY SOME PESTICIDES AND THEIR
EPOXIDES
(Pesticides;
epoxides;
metabolism;
D. COVAa,
A. ARNOLDIb,
epoxide
R. COLOMBO’
hydrolase)
and L. ROSSINI”
‘Department of Pharmacology, CNR Center of Cytopharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, bInstitute of General Biochemistry, University of Milan, Via Celoria 2, 20133 Milan, and ‘Department of Biomedical Sciences and Technology, University of Milan, St. Raphael Hospital, Via Olgettina 60, Milan (Italy) (Received
October
25th,
1985)
(Accepted
January
29th,
1986)
SUMMARY The present
studies
some pesticides of reactive
epoxide
Some pesticides
substrates
the steric factors
epoxide
intermediates
and their parent
ring. The results by substituents
identify
by hepatic
indicate
while tri-substituted
of epoxide
were carried
interactions
of this enzyme
and non-competitive
are virtually
in the hydration
intermediates
out regarding
with the hepatic
inactive
depends
Mono-
inhibitors
on inhibiting
of the hydration
the hepatic
epoxide
hydrolase.
on the oxirane
on the steric hindrance
and di-substituted
of
the formation
epoxide
were selected on the basis of the steric hindrance
ring of these pesticides.
hydrolase
epoxides
involved Investigations
and their different epoxides
that the inhibition
on the oxirane
of the epoxide
hydrolase.
oxiranes
produced are good
of styrene hydrolase
oxide, activity.
INTRODUCTION
The importance of epoxidation in the metabolism of pesticides has become apparent only in recent years with the evidence that unstable epoxides are intermediates in oxidative hydroxylation. Generally these reactions are catalyzed by epoxide hydrolase, a microsomal enzyme converting epoxides into diols, which are less toxic than most of their parent compounds [l-3]. We examined some pesticides and their epoxide intermediates reported in Fig. 1; specifically we tested at first vinclozolin epoxide, a mono-substituted oxirane formed during the bio-transformation of vinclozolin, an antifungal compound 0378-4274/86/s 03.50 0 Elsevier Science Publishers
B.V. (Biomedical
Division)
274
Parent
Monosubstftuled
Epoxfdes
gem-Dlsubstttuted
compounds
/O\
R-CH-CH2
Epoxldes
Rotenone
Fig. 1. Epoxides
tested and structures
of their parent
compounds.
derived from 3,5-dichloro aniline [4,5]. We also tested the effect on epoxide hydrolase activity of di-substituted oxiranes such as the two diastereoisomeric epoxides formed during the oxidative metabolism of the isopropenyl side-chain of rotenone [6]. Rotenone is the main component of the class of Rotenoids, the active principle of plants of the genus Derris, having insecticidal properties. We further examined cis- and trans-phenothrins, two synthetic pyrethroids, in which only the acidic residue contains a double bond susceptible of oxidation [7], to produce a tri-substituted epoxide (Fig. 1). Here we report our recent findings concerning the correlation between the structure of the epoxide intermediates of these pesticides differently substituted on the oxirane ring and their activity on hepatic epoxide hydrolase. MATERIALS
AND METHODS
6’,7’-Epoxyrotenone A and B were prepared from rotenone by oxidation with 3-chloroperbenzoic acid by a procedure already described [8]. 3-(3,5-dichloro(vinclozolin epoxide) was phenyl)-5-oxyranyl-5-methyl-2,Coxazolidinedione prepared by oxidation of vinclozolin with 3-chloroperbenzoic acid at room temperature. The pure epoxide was obtained by preparative thin layer chromatography. l-R,&,1 ‘-RS-phenothrin epoxides were prepared by oxidation of l-R,&,
215
phenothrin with 3-chloroperbenzoic acid and the mixture of the diastereoisomeric epoxides was obtained from flash chromatography [9]. 1-R,Wns, 1’ -S and 1-RJrans, 1 ‘-R-phenothrin epoxides were prepared by a similar procedure starting from 1-R,trans,phenothrin. Careful flash chromatography was carried out to separate 1’ -S-epoxide, first eluted, and 1 ‘-R-epoxide. In vitro studies were carried out with liver microsomes prepared from Charles River male rats weighing approx. 150-180 g according to the method of Kato [lo]. The activity of epoxide hydrolase was determined by utilizing styrene epoxide as a substrate. The enzymatic reaction was carried out as follows: 4.3 ml buffer was added to 0.1 ml of an acetone solution of the compounds to obtain a final concentration ranging from 0.01 to 0.05 mM for vinclozolin and its epoxide intermediate; rotenone and the two diastereoisomeric epoxyrotenones A and B were added at concentrations ranging from 0.01 to 0.05 mM; cis- and trans-phenothrins and their epoxides from 0.1 to 2.5 mM. At the same time 0.5 ml of a liver microsomal suspension containing about 5 mg of protein were added. After 15 min pre-incubation at 37”C, TABLE
I
EFFECT
OF
SOME
PESTICIDES
AND
THEIR
EPOXIDES
ON
EPOXIDE
HYDROLASE
ACTIVITY Compound
added
Activity
Inhibitor
to
substrate
ratio
70”
pb
100
None Vinclozolin Vinclozolin
epoxide
Rotenone Epoxyrotenone
A
Epoxyrotenone
B
cis-Phenothrin I-R,cis-1 ‘-RS-phenothrin
epoxide
1
99.5
0.1
51.4
0.25
24.9
0.5
13.6
< 0.001
I 0.1
99.7 67.2
< 0.001
0.2 0.5
39.0 25.8
< 0.001
0.1
43.3
0.2
21.8
0.5
13.8
2.5
101.2
ns
2.5
104.1
ns
ns
ns
truns-phenothrin I-R,truns-1 ‘-S-phenothrin
2.5
101.5
ns
epoxide
2.5
98.7
ns
I-R,truns-1
epoxide
2.5
loo.5
ns
‘-R-phenothrin
a Basal specific min/mg
protein
b Significant
activity (mean
effect
of microsomal value
f
with P
epoxide
hydrolase:
S.E. of 4 individual (ns, not significant).
7.94 + 0.23 nmol of phenylethyleneglycol/
experiments).
216
0.1 ml of an acetone
solution
of styrene
epoxide was added to obtain
a final concen-
tration ranging from 0.05 to 1 mM. The reaction was stopped after 5 min by the addition of 1 ml of 0.6 M NaOH. The samples were immediately extracted with 10 ml of ethyl acetate and the extracts dried under vacuum at room temperature. Phenylethyleneglycol, the diol formed during the enzymatic reaction from styrene epoxide, was determined after esterification with n-butylboronic acid, according to the method of Belvedere et al. [l 11. Protein concentration was determined by the method of Lowry et al. [12]. RESULTS
Table I summarizes the effects of the pesticides and their epoxide intermediates on the activity of hepatic epoxide hydrolase. Vinclozolin epoxide has a marked inhibitory effect, reducing the activity to about 50% at an inhibitor-to-substrate ratio of 0.1. When this ratio rises up to 0.5, only 13.6% of styrene epoxide is transformed into the diol. Epoxyrotenones A and B also have a marked inhibitory effect on the activity of the enzyme, even if they are weaker inhibitors than vinclozolin epoxide. However, their effect is still significant, since at an inhibitor-to-substrate ratio of 0.1 the activity of the enzyme is only about 33% and 60070, respectively. The parent compounds vinclozolin, rotenone, cis-phenothrin, trans-phenothrin and the diastereoisomeric epoxides of these latter pyrethroids did not show any influence on the activity of epoxide hydrolase. As regards the two compounds having the greater effect on the epoxide hydrolase activity, Fig. 2 shows the Dixon plot in which the reciprocal of the rate of epoxide hydrolase activity at four substrate concentrations was plotted against vinclozolin epoxide and epoxyrotenone B concentrations. These plots revealed that the mode of inhibition by vinclozolin epoxide (I) and epoxyrotenone B (II) is non-competitive, with a Ki of 1 to 2 X 10M5 M. L v
II
d ./ c
_I,G ii
/
005 InhIbItor
concentrat,on
lnhlbltor
Fig. 2. Dixon plot of the inhibitory tivity of epoxide
hydrolase.
effect of vinclozolin
The concentrations
epoxide
of the substrate,
01
025
mi-4
concentrotlon
(I) and epoxyrotenone styrene
epoxide,
B (11) on the ac-
were: for vinclozolin
epoxide: 0.05 mM (0); 0.10 mM (A); 0.25 mM (A); 0.5 mM (0); for epoxyrotenone B: 0.1 mM (A); 0.25 mM (A); 0.5 mM (0); 1 mM (0). V is given as nmol of phenylethyleneglycol formed/mg protein/min.
271
DISCUSSION
The compounds tested, selected on the basis of the steric hindrance on the oxirane ring, indicate that mono-substituted oxiranes, such as vinclozolin epoxide, are strong inhibitors of the hydration of styrene epoxide. Both l,l-di-substituted OXiranes, such as epoxyrotenones A and B, are inhibitors of epoxide hydrolase, although they are weaker inhibitors than vinclozolin epoxide. Tri-substituted oxdo not significantly inhibit the epoxide iranes, such as phenothrin epoxides, hydrolase activity, and the lack of inhibition may be related to the unfavourable steric interaction with epoxide hydrolase. These findings on the inhibition of epoxide hydrolase by epoxide intermediates of some pesticides are interesting for two reasons at least. First, inhibition studies can provide informations about the nature of the active site and the mechanism of action of the enzyme. Second, selective inhibitors may be useful to investigate and clarify the long-term toxicity and co-toxicity of these reactive intermediates in mammals. Indeed, the inhibition of epoxide hydrolase by epoxide metabolites at such low concentrations in the case of long exposures could interfere with the biotransformation process of some toxic agents by affecting the degradation of their epoxides into metabolites without toxic activity. ACKNOWLEDGEMENT
Research work supported paper No. 152.
by CNR,
Italy - Special Grant
I.P.R.A.
Subproject
3,
REFERENCES 1 D. Jerina, oxepin
J. Daly,
system
B. Witkop,
P. Zaltmann-Nirenberg
in the metabolism
of aromatic
and S. Udenfriend,
substrates,
Arch.
Role of the arene oxide-
Biochem.
Biophys.,
128 (1968)
176-186. 2 D. Jerina,
H. Ziffer and J. Daly, The role of arene oxide-oxepin
substrates,
IV. Stereochemical
Am. Chem.
Sot.,
3 K.C. Leibmann
considerations
on dihydrodiol
system in the metabolism formation
of aromatic
and dehydrogenation,
J.
92 (1970) 1056-1068.
and E. Ortiz,
Oxidation
of indene
by liver microsomes,
Mol. Pharmacol.,
4 (1968)
201-208. 4 D. Cova, E. Chiesara
and R. Rizzi, Some aspects
of the metabolic
fate of Vinclozolin,
Toxicol.
Lett.,
S.I. No. 1 (1980) 147-151. 5 E. Chiesara, A. Arnoldi, D. Cova and R. Rizzi, Detection of mutagenicity epoxide intermediate, Arch. Toxicol., Suppl. 5 (1982) 345-349. 6 T. Unai, rotenone 7 L.O.
H.M.
Cheng,
derivatives,
Ruzo,
chrysantemates
I.H.
I. Yamamoto Agr.
Smith and E.J. Phenothrin
and E.J. Casida,
Biol. Chem., Casida,
Chemical
in Vinclozolin
and biological
0demethylation
and its of
37 (1973) 1937-1943. Pyrethroid
and Tetramethrin,
biochemistry:
J. Agric.
Food
photooxidation
Chem.,
reactions
30 (1982) 110-122.
of the
278
8 D. Cova and A. Arnoldi, activity, 9 K.
Arch.
Ueda,
L.C.
pyrethroids,
Effect
Toxicol.,
Suppl.
Gaughan
J. Agric.
Food
and
E.J.
Chem.,
10 R. Kato and M. Takayanagi, and male sex hormone
of diastereoisomeric
Epoxyrotenones
on hepatic
epoxide
hydrolase
6 (1983) 254-259. Casida,
Differences
on microsomal
Photodecomposition
of
Resmethrin
and
related
22 (1974) 212-219. among
the action
drug-metabolizing
of Phenobarbital,
systems
Methylcholanthrene
of rat liver, Jap.
J. Pharmacol.,
16 (1966) 380-386. I1 G. Belvedere, method activities, 12 O.H. phenol
J. Pachecka,
L. Cantoni,
for the determination J. Chromatog.,
Lowry, reagent,
N.J.
E. Mussini and M. Salmona,
of microsomal
styrene
A specific gas-chromatographic
monooxygenase
and styrene
epoxide
hydratase
I18 (1976) 387-389.
Rosebrough,
J. Biol. Chem.
A.L.
Farr and R.J.
193 (1951) 265-271.
Randall,
Protein
measurement
with the Folin
Toxicology Letters, 30 (1986) 273-278 Elsevier
TOXLett.
1554
STEREOCHEMICAL HEPATIC EPOXIDE
CONSIDERATIONS ON THE INHIBITION OF HYDROLASE BY SOME PESTICIDES AND THEIR
EPOXIDES
(Pesticides;
epoxides;
metabolism;
D. COVAa,
A. ARNOLDIb,
epoxide
R. COLOMBO’
hydrolase)
and L. ROSSINI”
‘Department of Pharmacology, CNR Center of Cytopharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, bInstitute of General Biochemistry, University of Milan, Via Celoria 2, 20133 Milan, and ‘Department of Biomedical Sciences and Technology, University of Milan, St. Raphael Hospital, Via Olgettina 60, Milan (Italy) (Received
October
25th,
1985)
(Accepted
January
29th,
1986)
SUMMARY The present
studies
some pesticides of reactive
epoxide
Some pesticides
substrates
the steric factors
epoxide
intermediates
and their parent
ring. The results by substituents
identify
by hepatic
indicate
while tri-substituted
of epoxide
were carried
interactions
of this enzyme
and non-competitive
are virtually
in the hydration
intermediates
out regarding
with the hepatic
inactive
depends
Mono-
inhibitors
on inhibiting
of the hydration
the hepatic
epoxide
hydrolase.
on the oxirane
on the steric hindrance
and di-substituted
of
the formation
epoxide
were selected on the basis of the steric hindrance
ring of these pesticides.
hydrolase
epoxides
involved Investigations
and their different epoxides
that the inhibition
on the oxirane
of the epoxide
hydrolase.
oxiranes
produced are good
of styrene hydrolase
oxide, activity.
INTRODUCTION
The importance of epoxidation in the metabolism of pesticides has become apparent only in recent years with the evidence that unstable epoxides are intermediates in oxidative hydroxylation. Generally these reactions are catalyzed by epoxide hydrolase, a microsomal enzyme converting epoxides into diols, which are less toxic than most of their parent compounds [l-3]. We examined some pesticides and their epoxide intermediates reported in Fig. 1; specifically we tested at first vinclozolin epoxide, a mono-substituted oxirane formed during the bio-transformation of vinclozolin, an antifungal compound 0378-4274/86/s 03.50 0 Elsevier Science Publishers
B.V. (Biomedical
Division)
274
Parent
Monosubstftuled
Epoxfdes
gem-Dlsubstttuted
compounds
/O\
R-CH-CH2
Epoxldes
Rotenone
Fig. 1. Epoxides
tested and structures
of their parent
compounds.
derived from 3,5-dichloro aniline [4,5]. We also tested the effect on epoxide hydrolase activity of di-substituted oxiranes such as the two diastereoisomeric epoxides formed during the oxidative metabolism of the isopropenyl side-chain of rotenone [6]. Rotenone is the main component of the class of Rotenoids, the active principle of plants of the genus Derris, having insecticidal properties. We further examined cis- and trans-phenothrins, two synthetic pyrethroids, in which only the acidic residue contains a double bond susceptible of oxidation [7], to produce a tri-substituted epoxide (Fig. 1). Here we report our recent findings concerning the correlation between the structure of the epoxide intermediates of these pesticides differently substituted on the oxirane ring and their activity on hepatic epoxide hydrolase. MATERIALS
AND METHODS
6’,7’-Epoxyrotenone A and B were prepared from rotenone by oxidation with 3-chloroperbenzoic acid by a procedure already described [8]. 3-(3,5-dichloro(vinclozolin epoxide) was phenyl)-5-oxyranyl-5-methyl-2,Coxazolidinedione prepared by oxidation of vinclozolin with 3-chloroperbenzoic acid at room temperature. The pure epoxide was obtained by preparative thin layer chromatography. l-R,&,1 ‘-RS-phenothrin epoxides were prepared by oxidation of l-R,&,
215
phenothrin with 3-chloroperbenzoic acid and the mixture of the diastereoisomeric epoxides was obtained from flash chromatography [9]. 1-R,Wns, 1’ -S and 1-RJrans, 1 ‘-R-phenothrin epoxides were prepared by a similar procedure starting from 1-R,trans,phenothrin. Careful flash chromatography was carried out to separate 1’ -S-epoxide, first eluted, and 1 ‘-R-epoxide. In vitro studies were carried out with liver microsomes prepared from Charles River male rats weighing approx. 150-180 g according to the method of Kato [lo]. The activity of epoxide hydrolase was determined by utilizing styrene epoxide as a substrate. The enzymatic reaction was carried out as follows: 4.3 ml buffer was added to 0.1 ml of an acetone solution of the compounds to obtain a final concentration ranging from 0.01 to 0.05 mM for vinclozolin and its epoxide intermediate; rotenone and the two diastereoisomeric epoxyrotenones A and B were added at concentrations ranging from 0.01 to 0.05 mM; cis- and trans-phenothrins and their epoxides from 0.1 to 2.5 mM. At the same time 0.5 ml of a liver microsomal suspension containing about 5 mg of protein were added. After 15 min pre-incubation at 37”C, TABLE
I
EFFECT
OF
SOME
PESTICIDES
AND
THEIR
EPOXIDES
ON
EPOXIDE
HYDROLASE
ACTIVITY Compound
added
Activity
Inhibitor
to
substrate
ratio
70”
pb
100
None Vinclozolin Vinclozolin
epoxide
Rotenone Epoxyrotenone
A
Epoxyrotenone
B
cis-Phenothrin I-R,cis-1 ‘-RS-phenothrin
epoxide
1
99.5
0.1
51.4
0.25
24.9
0.5
13.6
< 0.001
I 0.1
99.7 67.2
< 0.001
0.2 0.5
39.0 25.8
< 0.001
0.1
43.3
0.2
21.8
0.5
13.8
2.5
101.2
ns
2.5
104.1
ns
ns
ns
truns-phenothrin I-R,truns-1 ‘-S-phenothrin
2.5
101.5
ns
epoxide
2.5
98.7
ns
I-R,truns-1
epoxide
2.5
loo.5
ns
‘-R-phenothrin
a Basal specific min/mg
protein
b Significant
activity (mean
effect
of microsomal value
f
with P
epoxide
hydrolase:
S.E. of 4 individual (ns, not significant).
7.94 + 0.23 nmol of phenylethyleneglycol/
experiments).
216
0.1 ml of an acetone
solution
of styrene
epoxide was added to obtain
a final concen-
tration ranging from 0.05 to 1 mM. The reaction was stopped after 5 min by the addition of 1 ml of 0.6 M NaOH. The samples were immediately extracted with 10 ml of ethyl acetate and the extracts dried under vacuum at room temperature. Phenylethyleneglycol, the diol formed during the enzymatic reaction from styrene epoxide, was determined after esterification with n-butylboronic acid, according to the method of Belvedere et al. [l 11. Protein concentration was determined by the method of Lowry et al. [12]. RESULTS
Table I summarizes the effects of the pesticides and their epoxide intermediates on the activity of hepatic epoxide hydrolase. Vinclozolin epoxide has a marked inhibitory effect, reducing the activity to about 50% at an inhibitor-to-substrate ratio of 0.1. When this ratio rises up to 0.5, only 13.6% of styrene epoxide is transformed into the diol. Epoxyrotenones A and B also have a marked inhibitory effect on the activity of the enzyme, even if they are weaker inhibitors than vinclozolin epoxide. However, their effect is still significant, since at an inhibitor-to-substrate ratio of 0.1 the activity of the enzyme is only about 33% and 60070, respectively. The parent compounds vinclozolin, rotenone, cis-phenothrin, trans-phenothrin and the diastereoisomeric epoxides of these latter pyrethroids did not show any influence on the activity of epoxide hydrolase. As regards the two compounds having the greater effect on the epoxide hydrolase activity, Fig. 2 shows the Dixon plot in which the reciprocal of the rate of epoxide hydrolase activity at four substrate concentrations was plotted against vinclozolin epoxide and epoxyrotenone B concentrations. These plots revealed that the mode of inhibition by vinclozolin epoxide (I) and epoxyrotenone B (II) is non-competitive, with a Ki of 1 to 2 X 10M5 M. L v
II
d ./ c
_I,G ii
/
005 InhIbItor
concentrat,on
lnhlbltor
Fig. 2. Dixon plot of the inhibitory tivity of epoxide
hydrolase.
effect of vinclozolin
The concentrations
epoxide
of the substrate,
01
025
mi-4
concentrotlon
(I) and epoxyrotenone styrene
epoxide,
B (11) on the ac-
were: for vinclozolin
epoxide: 0.05 mM (0); 0.10 mM (A); 0.25 mM (A); 0.5 mM (0); for epoxyrotenone B: 0.1 mM (A); 0.25 mM (A); 0.5 mM (0); 1 mM (0). V is given as nmol of phenylethyleneglycol formed/mg protein/min.
271
DISCUSSION
The compounds tested, selected on the basis of the steric hindrance on the oxirane ring, indicate that mono-substituted oxiranes, such as vinclozolin epoxide, are strong inhibitors of the hydration of styrene epoxide. Both l,l-di-substituted OXiranes, such as epoxyrotenones A and B, are inhibitors of epoxide hydrolase, although they are weaker inhibitors than vinclozolin epoxide. Tri-substituted oxdo not significantly inhibit the epoxide iranes, such as phenothrin epoxides, hydrolase activity, and the lack of inhibition may be related to the unfavourable steric interaction with epoxide hydrolase. These findings on the inhibition of epoxide hydrolase by epoxide intermediates of some pesticides are interesting for two reasons at least. First, inhibition studies can provide informations about the nature of the active site and the mechanism of action of the enzyme. Second, selective inhibitors may be useful to investigate and clarify the long-term toxicity and co-toxicity of these reactive intermediates in mammals. Indeed, the inhibition of epoxide hydrolase by epoxide metabolites at such low concentrations in the case of long exposures could interfere with the biotransformation process of some toxic agents by affecting the degradation of their epoxides into metabolites without toxic activity. ACKNOWLEDGEMENT
Research work supported paper No. 152.
by CNR,
Italy - Special Grant
I.P.R.A.
Subproject
3,
REFERENCES 1 D. Jerina, oxepin
J. Daly,
system
B. Witkop,
P. Zaltmann-Nirenberg
in the metabolism
of aromatic
and S. Udenfriend,
substrates,
Arch.
Role of the arene oxide-
Biochem.
Biophys.,
128 (1968)
176-186. 2 D. Jerina,
H. Ziffer and J. Daly, The role of arene oxide-oxepin
substrates,
IV. Stereochemical
Am. Chem.
Sot.,
3 K.C. Leibmann
considerations
on dihydrodiol
system in the metabolism formation
of aromatic
and dehydrogenation,
J.
92 (1970) 1056-1068.
and E. Ortiz,
Oxidation
of indene
by liver microsomes,
Mol. Pharmacol.,
4 (1968)
201-208. 4 D. Cova, E. Chiesara
and R. Rizzi, Some aspects
of the metabolic
fate of Vinclozolin,
Toxicol.
Lett.,
S.I. No. 1 (1980) 147-151. 5 E. Chiesara, A. Arnoldi, D. Cova and R. Rizzi, Detection of mutagenicity epoxide intermediate, Arch. Toxicol., Suppl. 5 (1982) 345-349. 6 T. Unai, rotenone 7 L.O.
H.M.
Cheng,
derivatives,
Ruzo,
chrysantemates
I.H.
I. Yamamoto Agr.
Smith and E.J. Phenothrin
and E.J. Casida,
Biol. Chem., Casida,
Chemical
in Vinclozolin
and biological
0demethylation
and its of
37 (1973) 1937-1943. Pyrethroid
and Tetramethrin,
biochemistry:
J. Agric.
Food
photooxidation
Chem.,
reactions
30 (1982) 110-122.
of the
278
8 D. Cova and A. Arnoldi, activity, 9 K.
Arch.
Ueda,
L.C.
pyrethroids,
Effect
Toxicol.,
Suppl.
Gaughan
J. Agric.
Food
and
E.J.
Chem.,
10 R. Kato and M. Takayanagi, and male sex hormone
of diastereoisomeric
Epoxyrotenones
on hepatic
epoxide
hydrolase
6 (1983) 254-259. Casida,
Differences
on microsomal
Photodecomposition
of
Resmethrin
and
related
22 (1974) 212-219. among
the action
drug-metabolizing
of Phenobarbital,
systems
Methylcholanthrene
of rat liver, Jap.
J. Pharmacol.,
16 (1966) 380-386. I1 G. Belvedere, method activities, 12 O.H. phenol
J. Pachecka,
L. Cantoni,
for the determination J. Chromatog.,
Lowry, reagent,
N.J.
E. Mussini and M. Salmona,
of microsomal
styrene
A specific gas-chromatographic
monooxygenase
and styrene
epoxide
hydratase
I18 (1976) 387-389.
Rosebrough,
J. Biol. Chem.
A.L.
Farr and R.J.
193 (1951) 265-271.
Randall,
Protein
measurement
with the Folin