Stereochemical aspects in the 4-vinylcyclohexene biotransformation with rat liver microsomes and purified cytochrome P450s: Diepoxide formation and hydrolysis

Article abstract of DOI:10.1021/tx025573z

The stereochemical course of the biotransformation of 1,2-monoepoxides of 4-vinylcyclohexene (2 and 3) by liver microsomes from control and induced rats and by purified P4502B1 and P4502E1 has been determined. The epoxidation of monoepoxides cis-4-vinylcyclohexene 1,2-epoxide (2) and trans-4-vinylcyclohexene 1,2-epoxide (3) gives the corresponding eight isomeric diepoxides cis-4-vinylcyclohexene diepoxide (9) and trans-4-vinylcyclohexene diepoxide (10). The stereoselectivity of this process is affected by P450 induction. Phenobarbital is able to enhance the yield of epoxidation to give preferentially diepoxide (1R,2S,4R,7R)-trans-10b. This enantiomer is also formed as nearly the sole product by P450-catalyzed epoxidation of (1R,2S,4R)-trans-3b, the monoepoxide that, as a consequence of the selective formation from 4-vinylcyclohexene and/or reduced elimination by epoxide hydrolase, tends to accumulate in rat. Also, the P4502B1 but not 2E1, in a reconstituted system, is able to perform the epoxidation of (1R,2S,4R)-trans-3b to produce selectively the same diepoxide. Diepoxides cis-9 and trans-10 are biotransformed by mEH catalyzed hydrolysis. Although the hydrolysis of diepoxides 9 is characterized by a lower substrate enantioselection, the reaction of diepoxides 10 occurs with a good substrate enantioselectivity favoring the hydrolysis of the epoxides (1R,2S,4R,7S)-trans-10b and (1S,2R,4S,7S)-trans-10a. Diepoxide (1R,2S,4R,7R)-trans-10b is therefore the isomer primarily formed by P450-catalyzed oxidation of monoepoxide trans-3, and it is also the compound showing the lower propensity to undergo mEH-catalyzed hydrolysis. On the basis of this result, the ovotoxicity of 4-vinylcyclohexene is expected to be due to the stereoisomer diepoxide (1R,2S,4R,7R)-trans-10b, whose biological reactivity, via cross-linking, may be strongly different to the other isomer diepoxides, being dependent by its specific conformation.

Full text of DOI:10.1021/tx025573z

56
Chem. Res. Toxicol. 2003, 16, 56-65
Ster eoch em ica l Asp ects in th e 4-Vin ylcycloh exen e
Biotr a n sfor m a tion w ith Ra t Liver Micr osom es a n d
P u r ified Cytoch r om e P 450s: Diep oxid e F or m a tion a n d
Hyd r olysis
Cinzia Chiappe,*,† Antonietta De Rubertis,‡ Giandomenico Piegari,†
Giada Amato,‡ and Pier Giovanni Gervasi*,‡
Dipartimento di Chimica Bioorganica e Biofarmacia, Universita` di Pisa, via Bonanno 33,
56126 Pisa, Italy, and Istituto di Fisiologia Clinica, area della Ricerca CNR, Via G. Moruzzi,
56100 Pisa, Italy
Received J une 12, 2002
The stereochemical course of the biotransformation of 1,2-monoepoxides of 4-vinylcyclohexene
(2 and 3) by liver microsomes from control and induced rats and by purified P4502B1 and
P4502E1 has been determined. The epoxidation of monoepoxides cis-4-vinylcyclohexene 1,2-
epoxide (2) and trans-4-vinylcyclohexene 1,2-epoxide (3) gives the corresponding eight isomeric
diepoxides cis-4-vinylcyclohexene diepoxide (9) and trans-4-vinylcyclohexene diepoxide (10).
The stereoselectivity of this process is affected by P450 induction. Phenobarbital is able to
enhance the yield of epoxidation to give preferentially diepoxide (1R,2S,4R,7R)-trans-10b. This
enantiomer is also formed as nearly the sole product by P450-catalyzed epoxidation of
(1R,2S,4R)-trans-3b, the monoepoxide that, as a consequence of the selective formation from
4-vinylcyclohexene and/or reduced elimination by epoxide hydrolase, tends to accumulate in
rat. Also, the P4502B1 but not 2E1, in a reconstituted system, is able to perform the epoxidation
of (1R,2S,4R)-trans-3b to produce selectively the same diepoxide. Diepoxides cis-9 and trans-
10 are biotransformed by mEH catalyzed hydrolysis. Although the hydrolysis of diepoxides 9
is characterized by a lower substrate enantioselection, the reaction of diepoxides 10 occurs
with a good substrate enantioselectivity favoring the hydrolysis of the epoxides (1R,2S,4R,7S)-
trans-10b and (1S,2R,4S,7S)-trans-10a . Diepoxide (1R,2S,4R,7R)-trans-10b is therefore the
isomer primarily formed by P450-catalyzed oxidation of monoepoxide trans-3, and it is also
the compound showing the lower propensity to undergo mEH-catalyzed hydrolysis. On the
basis of this result, the ovotoxicity of 4-vinylcyclohexene is expected to be due to the stereoisomer
diepoxide (1R,2S,4R,7R)-trans-10b, whose biological reactivity, via cross-linking, may be
strongly different to the other isomer diepoxides, being dependent by its specific conformation.
system, whereas the microsomal epoxide hydrolase
(mEH)1-catalyzed hydrolysis of the epoxide intermediates
is less efficient in mice than in rats (3, 8). Furthermore,
previous studies have shown that the P4502A and 2B
isozymes, which are the major enzymes implied in the
epoxidation of VCH (9, 10), are inducible by VCH
exposure in mice but not in rats (7). Thus, the higher
activity of the P450 monooxygenase involved in the
metabolic activation of VCH in mice along with the lower
activity involved in the detoxication of epoxide intermedi-
ates may account for the high susceptibility of this
species. VCH is a chiral compound, and its epoxidation
by P450 system may form eight monoepoxides, which,
in turn, may be further oxidized to eight diepoxides.
Recently, stereochemical aspects of VCH biotransforma-
tion and regioselectivity in the formation of endocyclic
cis- and trans-VCH-1,2-epoxides in rodents and human
have been reported (11, 12). It was demonstrated that
in rats both endocyclic and exocyclic monoepoxides
undergo hydrolization by mEH with diastereo- and
In tr od u ction
4-Vinylcyclohexene (VCH) is a colorless liquid formed
by spontaneous dimerization of 1,3-butadiene during the
rubber process (1). VCH is also an intermediate in the
epoxide resin formation and in polymer manufacturing
(1). Various studies have shown that a chronic exposure
to VCH causes in rodents toxic effects, especially in the
ovary (2, 3). Mice are reported to be the most susceptible
species in VCH toxicity. A prolonged exposure of VCH
provokes in mice, but not in rats, a premature ovarian
failure, and ovarian neoplasms (3). These differences are
likely to be related to biotransformation of VCH to
epoxide metabolites, VCH-1,2-epoxide and VCH-7,8-
epoxide, and in particular to the mutagenic and carci-
nogenic 4-vinylcyclohexene diepoxide, thought to be the
ultimate ovitoxic metabolite (2). The activities of the main
enzymatic systems engaged in the metabolism of VCH
were studied in various species (4-7). Mice have a
greater capacity than rats to oxidize VCH by P450
* To whom correspondence should be addressed. E-mail: cinziac@
1
farm.unipi.it.
Abbreviations: mEH, microsomal epoxide hydrolase; PB, pheno-
† Universita` di Pisa.
‡ Istituto di Fisiologia Clinica.
barbital; DEX, dexamethasone; PYR, pyrazole; TCPO, trichloropropene
oxide; GC, gas chromatography.
10.1021/tx025573z CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/06/2002

Stereochemistry of 4-Vinylcyclohexene Biotransformation
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 57
enantioselectivity (12). It was observed that the endo-
cyclic epoxides, (1R,2S,4R)-trans-3b and (1S,2R,4R)-cis-
2b, whose formation was favored in phenobarbital-
induced microsomes, had also a major resistance to the
enzymatic hydrolysis. Therefore, these epoxides are
expected to accumulate in the liver and undergo further
oxidation in a stereochemical manner to specific reactive
diepoxides. In the present work, we have investigated the
stereochemical and mechanistic aspect of the second step
of VCH metabolism. By using liver microsomes from rats
pretreated with phenobarbital (PB), pyrazole (PYR),
dexamethasone (DEX), and purificated rat P4502B1 or
P4502E1, we have examined the stereochemistry of the
oxidation of endocyclic cis- and trans-VCH-1,2-epoxides
to the isomeric diepoxides. Subsequently, we have studied
the stereoselectivity of the mEH-catalyzed hydrolysis of
the racemic diepoxides to epoxydiols.
F igu r e 1. Gas chromatographic separation of a mixture (ca.
2:8) of monepoxides (1S,2R,4R)-cis-2 and (1R,2S,4R)-trans-3
arising from enzymatic resolution of the commercial mixture
of (()-cis-2 and (()-trans-3. Analysis conditions are outlined in
the Materials and Methods.
46.7 (epoxide CH2); 46.8 (epoxide CH); 47.0 (epoxide CH); 71.2
(CHOH); 71.5 (CHOH); 73.8(CHOH); 74.0 (CHOH).
(1R,2S,4R)-tr a n s-4-Vin ylcycloh exen e 1,2-Ep oxid e (3b)
was prepared by enzymatic kinetic resolution of a 1:1 mixture
of monoepoxides cis-2 and trans-3 using a bovine liver microso-
mal preparation. An ethanolic stock solution (0.2 mL) of the
mixture of (()-cis-2 and (()-trans-3 (160 mg) was added to 6
mL of bovine liver microsomal preparation, containing 5 mg of
protein/mL, at pH 7.4 (Tris-HCl), and the mixture was incubated
with shaking at 37 °C. After 6 h, a saturating amount of NaCl
was added to precipitate the microsomal proteins and the
incubation mixture was extracted with hexane (3 × 10 mL) to
remove the unreacted epoxide. The organic phase was dried
Ma ter ia ls a n d Meth od s
Ca u tion ! The following chemicals are hazardous and should
be handled carefully: 4-vinyl cyclohexene, 4-vinylcyclohexene 1,2-
epoxide, 4-vinylcyclohexene 7,8-epoxide, 4-vinylcyclohexene diep-
oxide.
Ma ter ia ls. DEX, PYR, and â-NF were obtained from common
commercial sources. PB is a control drug and was sold by Fluka
(Buchs, Switzerland) only for research purpose. 4-Vinyl cyclo-
hexene (1), 4-vinylcyclohexene 1,2-epoxide (1:1 mixture of cis-2
and trans-3) and 4-vinylcyclohexene diepoxide (mixture of four
cis-9 isomers and four trans-10 isomers) were purchased from
Aldrich Chemical Co (Milwaukee, WI). TCPO was obtained from
EGA-Chemie (Steinheim-Albuch, Germany). (S)-4-Vinylcyclo-
hexene (1a ), (()-4-vinylcyclohexene 7,8-epoxide (4), (()-4-vinyl-
cyclohexene 7,8-epoxide (5), (()-4-vinylcyclohexene 1,2-diol (6),
(()-4-vinylcyclohexene 7,8-diol (7) and (()-4-vinylcyclohexene
7,8-diol (8), (4S,7S)-4-vinylcyclohexene 7,8-diol (8), (4S,7R)-4-
vinylcyclohexene 7,8-diol (7), (4S,7S)-4-vinylcyclohexene 7,8-
epoxide (5), and (4R,7S)-4-vinylcyclohexene 7,8-epoxide (4) were
prepared as previously reported (12).
(MgSO4) evaporated under reduced pressure to give
a
2:8 mixture of the two monepoxides (1S,2R,4R)-cis-2b and
(1R,2S,4R)-trans-3b or pure (1R,2S,4R)-trans-3b. As shown in
Figure 1, GC analysis was conducted on a 50 m chiral CP-
Cyclodex B (CHROMPACK) column (helium flow of 50 kPa, and
with an evaporator and detector set at 200 °C, at 90 °C).
(1S,2R,4S,7S)-, (1S,2R,4S,7R)-, (1R,2S,4S,7S)-, a n d
(1R,2S,4S,7R)-4-Vin ylcycloh exen e Diepoxide (cis-9a, tr a n s-
10a ). To a solution of m-chloroperbenzoic acid (5 mmol) in
chloroform (15 mL) (S)-4-vinylcyclohexene (1a , 2 mmol) was
added and the mixture was allowed to stay for 2 days at room
temperature and 4 h at 4 °C. The solution was filtered, and 650
mg of KF, previously activated by heating at 120 °C in vacuo (1
mmHg) for 2 h, was added. The mixture was stirred at room
temperature for 1 h; then, the insoluble complexes were filtered
off, and the solvent was removed under reduced pressure to give
a mixture of (1S, 2R, 4S, 7S)-, (1S, 2R, 4S,7R)-, (1R,2S,4S,7S)-,
and (1R,2S,4S,7R)-4-vinylcyclohexene diepoxides (cis-9a and
trans-10a ). The crude product was analyzed by GC (Figure 2;
trace b) on a 25 m Chiraldex G-TA (ASTEC) chiral column at
98 °C (helium flow of 50 kPa, with an evaporator and detector
set at 200 °C).
Mixtu r e of (1R,2S,4S,7R)-cis-4-Vin ylcycloh exen e Diep -
oxid e (9a ) a n d (1S,2R,4S,7R)-tr a n s-4-Vin ylcycloh exen e
Diep oxid e (10a ). The mixture of (1R,2S,4S,7R)-cis-9a and
(1S,2R,4S,7R)-trans-10a was synthesized by epoxidation with
m-chloroperbenzoic acid from (4S,7R)-4-vinylcyclohexene 7,8-
epoxide (4a ), prepared as previously reported (12), according to
the above-reported procedure. The crude product was analyzed
by GC under conditions reported above. Figure 2, trace d.
Mixtu r e of (1R,2S,4S,7S)-cis-4-Vin ylcycloh exen e Diep -
oxid e (9a ) a n d (1S,2R,4S,7S)-tr a n s-4-Vin ylcycloh exen e
Diep oxid e (10a ). To a solution of m-chloroperbenzoic acid (2.5
mmol) in chloroform (10 mL) was added (4S,7S)-4-vinylcyclo-
hexene 7,8-epoxide (5a , 1 mmol), prepared as previously re-
ported (12), and the mixture was allowed to stay for 2 days at
room temperature and 4 h at 4 °C. The solution was filtered,
and 300 mg of KF, previously activated by heating at 120 °C in
vacuo (1 mmHg) for 2 h, was added. The mixture was stirred
at room temperature for 1 h; then the insoluble complexes were
filtered off, and the solvent was removed under reduced pressure
4-Vin ylcycloh exen e 1,2-Ep oxid e-7,8-d iols (11). To a solu-
tion of m-chloroperbenzoic acid (9 mmol) in chloroform (8 mL)
a 1:1 mixture of diols 7 and 8 (3 mmol) was added, and the
mixture was allowed to stay for 16 h at room temperature under
stirring and 4 h at 4 °C. The solution was filtered, and 400 mg
of KF, previously activated by heating at 120 °C in vacuo
(1 mmHg) for 2 h, was added. The mixture was stirred at room
temperature for 1 h; then the insoluble complexes were filtered
off, and the solvent was removed under reduced pressure to give
a mixture of eight diastereoisomeric epoxydiols 11. The crude
product was analyzed by NMR.
1H NMR (CDCl3) δ (ppm): 1.4-2.0 (m, 28H); 2.1-2.5 (m, 8H,
epoxide CH-CH); 3.1-3.7 (m, 12H, CHOH and CH2OH). 13C
NMR and DEPT (CDCl3) δ (ppm): 20.5 (CH2); 21.5(CH2); 23.1
(CH2); 23.1(CH2); 23.3 (CH2); 24.3 (CH2); 24.3 (CH2); 24.5 (CH2);
26.0 (CH2); 26.5 (CH2); 27.2 (CH2); 28.0 (CH2); 33.3 (CH); 33.9
(CH); 36.2 (CH); 36.8 (CH); 50.8 (epoxide CH); 51.4 (epoxide CH);
51.6 (epoxide CH); 51.7 (epoxide CH); 51.8 (epoxide CH); 52.7
(epoxide CH); 52.8(epoxide CH); 53.0 (epoxide CH); 67.8-68.2
(4CH2OH); 79.5 (CHOH); 79.6 (CHOH); 80.1 (CHOH);
80.2(CHOH).
4-Vin ylcycloh exen e 1,2-Diol-7,8-ep oxid es (12). The mix-
ture of four diastereoisomeric epoxydiols 12 was synthesized by
epoxidation with m-chloroperbenzoic acid from (()-4-vinyl-
cyclohexene 1,2-diol (6), according to the above-reported proce-
dure. The crude product was analyzed by NMR.
1H NMR (CDCl3) δ (ppm): 1.4-2.0 (m, 14H); 2.5 (m, 2H,
epoxide CH); 2.7 (m, 2H, epoxide CH); 2.9 (m, 2H, epoxide CH);
3.4 (m, 2H, CHOH); 3.6 (m, 2H, CHOH). 13C NMR and DEPT
(CDCl3) δ (ppm): 24.5 (CH2); 25.1 (CH2); 28.4 (CH2); 28.7 (CH2);
30.9 (CH2); 33.2 (CH2); 36.5 (CH); 36.7 (CH); 46.5 (epoxide CH2);
to give
a ca. 1:1 mixture of (1R,2S,4S,7S)-cis-9a and

58 Chem. Res. Toxicol., Vol. 16, No. 1, 2003
Chiappe et al.
were purified from rat liver as previously described (16).
En zym a tic In cu ba tion s. (1) Deter m in a tion of th e P 450
Activity a n d of th e En a n tiom er ic Excesses of th e P r od -
u cts. Incubation mixtures (10 mL) containing 100 mM potas-
sium phosphate buffer (pH 7.4), 3 mg/mL of hepatic microsomal
proteins, NADPH-regenerating system, consisting of 0.5 mM
NADP+, 5 mM glucose 6-phosphate, and 0.5 units/mL glucose
6-phosphate dehydrogenase and trichloropropene oxide (TCPO,
1 mM) to inhibit the mEH, were preincubated at 37 °C for 5
min, and the reaction were initiated by the addition of a proper
amount of the 1:1 mixture of epoxides 2 and 3 or of the
enantiomerically pure epoxide (1R,2S,4R)-trans-3b. The sub-
strate concentration was 10 mM to saturate the P450 enzymes
(8). After 60 min, a saturating amount of NaCl was added to
precipitate the microsomal proteins. The reaction products were
extracted with dichloromethane (2 × 5 mL) and analyzed by
GC after addition of appropriate amounts of 2-cyclohexen-1-one
as an internal standard. The enantiomeric composition of
diepoxides was determined by GC on a chiral 30 m Chiraldex
G-TA (ASTEC) column (helium flow of 50 kPa and with an
evaporator and detector set at 200 °C, and column at 98 °C).
The absolute configurations of the excess enantiomers of
diepoxides cis-9 and trans-10 were determined by comparison
of the retention times of each enantiomer with those of enan-
tiomerically pure compounds obtained by epoxidation of chiral
monopoxides prepared by enzymatic resolution or asymmetric
synthesis.
The epoxidations of the enantiomerically pure epoxide
(1R,2S,4R)-trans-3b were also measured in a reconstituted
system (0.5 mL) containing 0.1 nmol of purified rat P4502B1
or 2E1, 0.3 nmol of P450 reductase, 30 µg of dilauroylphos-
phatidylcholine and the substrate at concentration 10 mM. The
reactions were carried out at 37 °C for 60 min after the addition
of 1 mM NADPH and the products were extracted and analyzed
as described above.
(2) Deter m in a tion of th e Su bstr a te En a n tioselectivity
of th e m EH-Ca ta lyzed Hyd r olysis of Diep oxid es cis-9 a n d
tr a n s-10. Aliquots (20 µL) of an ethanolic stock solution of the
commercial mixture of diepoxides cis-9 and trans-10 were added
to 2 mL of a microsomal preparation (Control) containing 3 mg
of protein/mL in a such way to obtain a 20 mM substrate
concentration, and the reaction mixtures were incubated at 37
°C. At prefixed times (between 1 and 5 h), a saturating amount
of NaCl was added to precipitate the microsomal proteins and
the incubation mixtures were extracted with dichloromethane
(3 × 1 mL) to remove the unreacted epoxides followed by brief
centrifugation to separate the emulsion. 2-cyclohexen-1-one was
added as a standard to the combined extracts, which were
analyzed directly by GC on 30 m Chiraldex G-TA (ASTEC)
column (helium flow of 50 kPa, and with an evaporator and
detector set at 200 °C, and column at 98 °C).
F igu r e 2. Gas chromatographic separation of diepoxides cis-9
and trans-10. (a) commercial mixture of diepoxides cis-9 and
trans-10. The continuous line connects the enantiomer couples
determined on the basis of the peaks areas (1.6; 2.3; 4.7; 5.8).
(b) mixture of four diepoxides having configuration (4S), arising
from epoxidation of 1a . (c) Mixture of the two diepoxides having
configuration (4S,7S), arising from epoxidation of (4S,7S)-5a .
(d) Mixture of the two diepoxides having configuration (4S,7R),
arising from epoxidation of (4S,7R)-5a . (e) Mixture of the four
diepoxides arising from epoxidation of a 2:8 mixture of monep-
oxides (1S,2R,4R)-trans-3 and (1R,2S,4R)-trans-3. Peaks 1 and
5 correspond to diepoxides having configuration (1R,2S,4R),
peaks 3 and 7 correspond to diepoxides having configuration
(1S,2R,4R). On the basis of the information arising from these
analyses the following configurations may be attributed to the
eight diepoxides: (1R,2S,4R,7R)-trans-10b (1); (1R,2S,4S,7S)-
cis-9a (2); (1S,2R,4R,7R)-cis-9b (3); (1R,2S,4S,7R)-cis-9a
(4); (1R,2S,4R,7S)-trans-10b (5); (1S,2R,4S,7S)-trans-10a (6);
(1S,2R,4R,7S)-cis-9b (7); (1S,2R,4S,7R)-trans-10a (8). Analysis
conditions are outlined in the Materials and Methods.
To determine the nature of the products the commercial
mixture of epoxides cis-9 and trans-10 as neat liquid (40-60
mg) was added to 20 mL of microsomal preparation and the
reaction mixtures were incubated with shaking at 37 ° C for
the time necessary to obtain a practically complete conversion
(6 h). The incubations were then extracted with ethyl acetate
(3 × 10 mL), and the organic phase was dried and evaporated
under reduced pressure. The product mixture was analyzed by
NMR.
(1S,2R,4S,7S)-trans-10a . The crude product was analyzed by
GC under conditions reported above. Figure 2, trace c.
Mixtu r e of (1R,2S,4R,7R)-tr a n s-4-Vin ylcycloh exen e Diep-
oxid e (10b) a n d (1R,2S,4R,7S)-tr a n s-4-Vin ylcycloh exen e
Diep oxid e (10b). The mixture of (1R,2S,4R,7R)-trans-10b and
(1R,2S,4R,7S)-10b was synthesized by epoxidation with m-
chloroperbenzoic acid from (1R,2S,4R)-trans-4-vinylcyclohexene
1,2-epoxide (3b) according to the above-reported procedure. The
crude product was analyzed by GC under conditions reported
above.
Blank experiments carried out with boiled microsomal prepa-
rations show that the spontaneous hydrolysis does not contrib-
ute to the diols formation under the incubation conditions.
An im a l Micr osom a l P r ep a r a tion s a n d En zym es. Male
Sprague-Dawley rats were purchased from Charles River
(Calco, Como, Italy). Male rats were treated ip for 3 days with
PB (80 mg/kg daily), DEX (50 mg/kg daily), or PYR (200 mg/kg
daily), and microsomes were obtained from the liver as previ-
ously described (13). Microsomal protein concentrations were
assayed by using the method of Lowry et al. (14); the total P450
concentration was measured according to Omura and Sato (15).
P4502B1 and P4502E1 and NADPH cytochrome P450 reductase
Resu lts a n d Discu ssion
The stereochemical course of formation of 4-vinylcy-
clohexene diepoxides by epoxidation of the exocyclic
double bond of a mixture of monoepoxides 2 and 3 or of
the pure (1R,2S,4R)-3b was investigated using various
microsomal preparations from male rats and in re-
constituted monooxygenase systems containing either

Stereochemistry of 4-Vinylcyclohexene Biotransformation
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 59
Ta ble 1. Ster eoch em ica l Cou r se for th e Oxid a tion on th e
Exocyclic Dou ble Bon d of a 1:1 Mixtu r e of 2 a n d 3 by
Micr osom es fr om Ma le Con tr ol a n d P B-Tr ea ted Ra ts
P4502B1 or 2E1. This study was performed with male
rats since level of overall VCH metabolism was found
higher in male than in female rat liver microsomes
according to our previous published paper (12). Micro-
somal preparations from male rats were also used to
study the stereochemistry of hydrolysis of 4-vinylcyclo-
hexene diepoxides. In both cases, as a convenient ana-
lytical tool, the inclusion gas chromatography, which
enables a time-dependent enantiomer screening of diep-
oxides in the nmol range, was employed for the deter-
mination of the enantiomeric ratios (Figure 2) and for
the absolute configurations. To use this method to
determine the absolute configuration of the formed or
residual diepoxides, the synthesis of reference substances
with unequivocal stereochemistries was necessary.
Syn th esis of Refer en ce Diepoxides an d GC An aly-
sis on a Ch ir a l Colu m n . The four diastereoisomeric
diepoxides cis-9a and trans-10a , having (S) configuration
at C-4, were prepared by the epoxidation of (S)-4-
vinylcyclohexene 1a (12) with m-chloroperbenzoic acid.
Analogously, mixtures of two diasteroisomeric ep-
oxides, (1S,2R,4S,7R)-trans-10a + (1R,2S,4S,7R)-cis-9a ,
(1S,2R,4S,7S)-trans-10a + (1R,2S,4S,7S)-cis-9a , (1R,2S,-
4R,7S)-trans-10b + (1R,2S,4R,7R)-trans-10b, were pre-
pared by epoxidation with peracid of the corresponding
monoepoxides (4S,7R)-4, (4S,7S)-5, and (1R,2S,4R)-trans-
3b.
CTR
nmol (%)
PB
diepoxides
nmol (%)d
(1R,2S,4R,7S)-10b
(1R,2S,4R,7R)-10b
(1S,2R,4S,7R)-10a
(1S,2R,4S,7S)-10a
(1S,2R,4R,7R)-9b
(1S,2R,4R,7S)-9b
(1R,2S,4S,7R)-9a
(1R,2S,4S,7S)-9a
a
b
h
g
c
d
e
f
3.3 ( 0.6 (4.5)
27 ( 3 (36.9)
16 ( 2 (21.9)
3.6 ( 1 (4.9)
11 ( 2 (15.0)
3.2 ( 0.8 (4.4)
ndc
10 ( 2* (4.9)
66 ( 7* (32.7)
27 ( 3* (13.3)
14 ( 2* (6.9)
39 ( 4* (19.3)
8 ( 1* (3.9)
5 ( 0.8* (2.47)
33 ( 5* (16.3)
9 ( 2 (12.3)
a
Each value represents the mean ((SD) of at least three
b
determinations. a, b, c, d, e, f, g, and h refer pathways reported
in Scheme 5. c nd ) not detected. The asterisk (*) indicates values
d
significantly different from control (p < 0.01).
The oxidation of a 2:8 mixture of the diastereosiomeric
epoxides (1S,2R,4R)-cis-2b and (1R,2S,4R)-trans-3b, ob-
tained by partial enzymatic kinetic resolution, gave a
mixture of four diepoxides, (1S,2R,4R,7R)-cis-9b, (1S,2R,-
4R,7S)-cis-9b, (1R,2S,4R,7S)-trans-10b, and (1R,2S,4R,-
7R)-trans-10b, characterized by a ca. 2:8 ratio between
the isomers 9 and 10.
F igu r e 3. Enantiomeric composition of diepoxides 9 and 10
arising by oxidation of a 1:1 mixture of monoepoxides cis-2
and trans-3 catalyzed by microsomes from PB-treated rats.
Peaks: 1 (1R,2S,4R,7R)-trans-10b; 2, (1R,2S,4S,7S)-cis-9a ; 3,
(1S,2R,4R,7R)-cis-9b; 4, (1R,2S,4S,7R)-cis-9a ; 5, (1R,2S,4R,7S)-
trans-10b; 6, (1S,2R,4S,7S)-trans-10a ; 7, (1S,2R,4R,7S)-cis-9b;
8, (1S,2R,4S,7R)-trans-10a .
The commercial mixture of four couples of enantiomers
of diepoxides cis-9 and trans-10 give, when analyzed by
GC on a chiral column, as expected eight peaks (Figure
2, trace a). On the basis of the areas of the peaks four
couples of enantiomers can be evidenced. In particular,
peaks 1 and 6 having the same area correspond to a pair
of enantiomer. Analogously, peaks 2 and 3, 4 and 7, and
finally 5 and 8 correspond to the other three couples of
enantiomers. On the basis of the retention times of the
four diepoxides arising by exhaustive oxidation of (4S)-
1a (Figure 2, trace b), it was possible to attribute the
configuration (4S) to diastereoisomers corresponding to
the peaks 2, 4, 6, and 8. Analogously, considering the
starting material, configuration (4S,7S) was attributed
to peaks 2 and 6 (Figure 2, trace c), (4S,7R) to peaks 4
and 8 (Figure 2, trace d), (1R,2S,4R) to peaks 1 and 5,
and (1S,2R, 4R) to peaks 3 and 7 (Figure 2, trace e). On
the basis of this information, taking into account the
enantiomeric couples (Figure 2, traces a), the absolute
configuration was assigned to each peak, as reported in
Figure 2.
E p oxid a t ion of 1,2-Mon oep oxid es of 4-Vin yl-
cycloh exen e 2 a n d 3 b y R a t Liver Micr osom es.
The oxidation of the exocyclic double bond of mono-
epoxides cis-2 and trans-3 can give four pairs of enanti-
omers. cis- and trans-4-vinylcyclohexene 1,2-epoxides
possess a prochiral carbon-carbon double bond and three
chiral centers. The enzymatic epoxidation of these com-
pounds is therefore a competitive process which can occur
with substrate and/or product enantio- and diastereo-
selectivity. The correct determination of stereoselectivity
in the epoxidation requires the complete inhibition of
epoxide hydrolase, since the enzymatic hydrolysis of the
starting monoepoxides or formed diepoxides may repre-
sent an efficient competing enantioselective process.
The stereochemical course of oxidation of a 1:1 mixture
of monoepoxides cis-2 and trans-3 to the corresponding
diepoxides cis-9 and trans-10 was investigated by incu-
bating the substrate (10 mM) in the presence of TCPO
(1 mM), an efficient inhibitor of epoxide hydrolase (17),
by using liver microsomes obtained from untreated rats
or rats pretreated with PB, PYR, and DEX, classical
inducers of 2B1/2, 2E1, and 3A1/2, respectively (16, 18).
At prefixed times (60 min), the reactions were stopped
by adding a saturating amount of NaCl, and the incuba-
tion mixtures were extracted with dichloromethane and
analyzed by GC on a chiral column, after addition of
2-cyclohexen-1-one as an internal standard, to evaluate
the chemical yields. An incubation time of 60 min was
necessary to obtain a quantity of products enough to be
reproducibly detected by GC analysis. The use of the
chiral column allowed the determination of the diaste-
reoisomeric product ratios among the formed diepoxides
and, for each diastereoisomer, the enantiomeric ratio and
the absolute configuration of the enantiomer in excess,
by the comparison of the retention time of the formed
products with those of samples of known configuration
prepared as reported above.
Table 1 reports the diastereo- and enantiomeric com-
position of diepoxides cis-9 and trans-10 obtained by
oxidation of the mixture of monoepoxides cis-2 and
trans-3 by liver microsomes from male control and PB-
induced rats. Figure 3 shows the GC trace of the products
obtained by oxidation using microsomes from PB-induced

60 Chem. Res. Toxicol., Vol. 16, No. 1, 2003
Chiappe et al.
Sch em e 1
Sch em e 2
rats. It must be remarked that both the chemical yield
of epoxidation and the stereoselection of the process
strictly depend on the induction of the P450 isoformes
involved in these reactions. The use of PB-induced
microsomes compared to control microsomes considerably
increases the yields, indicating the involvement of the
2B1 isoform. On the other hand, when DEX- or PYR-
induced microsomes are used, the stereochemical course
of epoxidation was qualitatively the same of that obtained
with control microsomes for the formation of the diep-
oxides (1S,2R,4S,7R)-10a , (1R,2S,4R,7R)-10b, (1S,2R,4R,-
7R)-9b, and (1S,2S,4S,7S)-9a whereas the quantities of
the other formed diepoxides were lower of the detection
limit (<2 nmol). These results suggest that the P450
isoforms 2E1 and 3A1/2 induced in PYR- and DEX-
microsomes, respectively, have a minor role in the
epoxidation of vinylcyclohexene 1,2-monoepoxides. The
stereochemical selectivity depends on the P450 isozyme-
(s) responsible for the reactions and, hence, upon the
inducer. It must be remarked that the formation of the
eight diepoxides arises from the competition among the
processes a-h, reported in Scheme 5.
With control microsomes the exocyclic double bond
oxidation proceeds with a moderate substrate diastereo-
selectivity (70:30), given by (a + b + g + h) vs (c + d +
e + f), in favor of trans-3 epoxides, which is however
drastically reduced (55:45) when PB induced microsomes
are used. Also the substrate enantioselectivity in the
exocyclic double bond oxidation with control microsomes
is low (60:40), given by (a + b) vs (g + h) or (c + d) vs (e
+ f), in favor of the (1R,2S,4R)-enantiomer in the case of
the epoxide trans-3, and 55: 45 in favor of the (1S,2R,4R)-
enantiomer in the case of the epoxide cis-2, but this
selectivity is unaffected by induction.
At variance, the product diastereoselectivity for each
individual enantiomer, given by a vs b; c vs d; e vs f; g vs

Stereochemistry of 4-Vinylcyclohexene Biotransformation
Sch em e 3
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 61
Sch em e 4
h, is high and is only moderately inducer dependent. The
epoxidation of the exocyclic double bond of (1R,2S,4R)-
trans-3b occurs, independently of the inducer, with a very
high face selectively (13:87) to give preferentially the
diepoxide (1R,2S,4R,7R)-trans-10b. Analogously, that of
(1S,2R,4R)-cis-2b enantiomer is characterized by a selec-
tive attack on the re face (78:22) to give the enantiomer
(1S,2R,4R,7R)-cis-9b. The epoxidation of (1R,2S,4S)-cis-
2a enantiomer takes place with a complete product
diastereoselectivity to give diepoxides (1R,2S,4S,7S)-cis-
9a . The induction generally increases the chemical yields,
but reduces the product diastereoselectivity. A reduction
of the selectivity toward the attack on the re face may
be observed also in the epoxidation of (1S,2R,4S)-trans-
3a to give preferentially the diepoxide (1S,2R,4S,7R)-
trans-10a , which lowered from 83:17, with control mi-
crosomes, to 66:34 with PB-induced preparations.
Finally, the results reported in Table 1 may be dis-
cussed in terms of product enantioselectivity, arising from
the differentiation between the two enantiotopic faces of
the double bond of the two enantiomers, given by a vs h;
b vs g; c vs f; e vs d. With control microsomes, the
enantiomeric couple of diepoxides (1R,2S,4R,7S)-trans-
10b and (1S,2R,4S,7R)-trans-10a , arising from the oxi-
dation of the exocyclic double bond of 3, is formed in a
very high selectivity to give preferentially (84:16) the
(1S,2R,4S,7R)-10a enantiomer. This selectivity is re-
duced (73:27) with PB-induced microsomes. A high
selectivity characterized also the formation of diepoxides
(1R,2S,4R,7R)-trans-10b and (1S,2R,4S,7S)-trans-10a
occurring with the preferential (82:18) formation of
diepoxide (1R,2S,4R,7R)-trans-10b. Also, in this case,
induction decreases the selectivity of the process. On the
contrary, the diepoxide cis-9 is formed in a practically
racemic way both when the incubations were carried out
using control or induced microsomes.
The stereochemistry of the products arising from the
oxidation of (1R,2S,4R)-trans-3b, the enantiomer that as
a consequence of the selective formation and/or reduced
elimination tends to accumulate in rat and mouse (11,
12), has been also investigated as described for the
epoxidation of the mixture of epoxides cis-2 and trans-3
by using liver microsomes obtained from untreated or
pretreated rats and the purified rat 2B1 and 2E1.
The data reported in Table 2 show that the epoxidation
of monoepoxide (1R,2S,4R)-3b occurs, independently of
inducer and species, with a practically complete product

62 Chem. Res. Toxicol., Vol. 16, No. 1, 2003
Chiappe et al.
Sch em e 5
Ta ble 2. Ster eoch em ica l Cou r se for th e Oxid a tion on th e
Exocyclic Dou ble Bon d of (1R,2S,4R)-3b by Micr osom es
fr om Ma le Con tr ol a n d P B- a n d P YR-Tr ea ted Ra ts or
w ith P u r ified P 450 (2B1 a n d 2E1)a
reaction, although this enzyme was capable, at low level,
to yield monoepoxides from VCH oxidation (12). To verify
if TCPO is able to inhibit the oxidation reactions, it was
incubated at concentration of 1 mM in a reconstituted
system with (1R,2S,4R)-3b, and the (1R,2S,4R,7R)-10b
was determined. In this condition no inhibition was
observed.
diepoxides
(1R,2S,4R,7S)-10b
(1R,2S,4R,7R)-10b
(nmolb)
(nmolb)
CTR
PB
PYR
2B1
2E1
4
5
87
212
traces
10.5
traces
Previously (12, 16, 19), stereoselectivity in the P450
oxidation of prochiral and chiral olefins has been re-
ported; it was also demonstrated that several isoforms
are involved in the epoxidation process with single
enzymes catalyzing the reaction with a different stereo-
selectivity: substrate enantioselectivity, product enan-
tioselectivity, and product diastereoselectivity. However,
a stereoselectivity as strong as that of monoepoxide cis-2
and trans-3 oxidation was not observed in other olefins
including VCH, but only in the epoxidation of polycyclic
aromatic hydrocarbons (20). It is possible that the
substrates cis-2 and trans-3, although are not molecules
of large size as the polycyclic aromatic hydrocarbons,
have sterical and chemical interactions with specific
amino acids in the active site of the P450 isoforms
catalytically implied.
n.d.
traces
n.d.
a
Each value represents the mean of at least two determinations.
The incubations with P450 2B1 and 2E1 were performed in a
reconstituted system as described in the Materials and Methods.
b
Average error: (5%. n.d.) not detectable. Traces: <2 nmol.
selectivity, to give (1R,2S, 4R,7R)-trans-10b diepoxide as
the sole product.
The stereoselectivity of the oxidation of (1R,2S,4R)-3b
was evaluated also by reconstituting the monooxygenase
reaction in the presence of DLPC, NADPH-cytochrome
P450 reductase, and purified rat liver 2B1 or 2E1 and
NADPH. The data, reported in Table 2, show that control
and PB-induced microsomes along with P4502B1, in a
reconstituted system, had practically a complete selectiv-
ity toward diepoxide (1R,2S,4R,7R)-10b. Of interest, it
is to note that P4502E1 was unable to perform this
Hyd r olysis of Diep oxid es cis-9 a n d tr a n s-10. The
enantio- and diastereoisomeric composition of the diep-
oxides cis-9 and trans-10 arising by the P450-catalyzed
oxidation of 1 depends on the stereoselectivity of the first
oxidation process, giving mainly the monoepoxides cis-2

Stereochemistry of 4-Vinylcyclohexene Biotransformation
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 63
Ta ble 3. Tim e a n d Ster eoch em ica l Cou r se of th e m EH Ca ta lyzed Hyd r olysis of th e Com m er cia l Mixtu r e of Diep oxid es 9
a n d 10a
residual diepoxides
(% of the starting material)b
time
(h)
hydrolysis
(%)
(1R,2S,4R,
7S)-10
(1R,2S,4R,
7R)-10
(1S,2R,4S,
7R)-10
(1S,2R,4S,
7S)-10
(1S,2R,4R,
7R)-9
(1S,2R,4R,
7S)-9
(1R,2S,4S,
7R)-9
(1R,2S,4S,
7S)-9
2
4
5
6
48
70
85
90
42
18
6
63
45
32
30
62
43
20
10
30
9
4
50
26
16
11
53
35
17
16
83
39
16
6
29
25
14
11
4
2
a
b
Each value represents the mean of at least three determinations. Average errors: (5%.
Sch em e 6
and trans-3, as well as on the stereoselectivity of the
conversion of the monoepoxides into the diepoxides.
Furthermore, it is affected by the stereoselectivity of at
least three other enzymatic processes, (i) the EH-
catalyzed hydrolysis of the monoepoxides to the corre-
sponding vicinal diols (12); (ii) the EH-catalyzed hydrol-
ysis of the formed diepoxides to epoxydiols; (iii) the
glutathione S-transferase transformation of mono- and
diepoxides to glutathione conjugates.
To evaluate the stereoselectivity of the mEH-catalyzed
hydrolysis of the diepoxides, the experiments were car-
ried out by incubating the commercial mixture of the
eight diepoxides cis-9 and trans-10 (20 mM) with a
microsomal preparation from control rats, containing 2-3
mg of protein/mL, at 37 °C and pH 7.4. The incubations
were stopped at different times, and the residual epoxides
were identified by GC, after addition of 2-cyclohexen-1-
one as an internal standard, on a chiral column. The
results are reported in Table 3.
substrate enantioselectivity. At 50% conversion, the ratio
between (1R,2S,4R,7R)-trans-10b and (1S,2R,4S,7S)-
trans-10a is around 70:30, a value which can be obtained
in the case of diepoxides (1S,2R,4S,7R)-trans-10a and
(1R,2S,4R,7S)-trans-10b at ca. 70% conversion. At vari-
ance, the hydrolysis of diepoxides cis-9 shows a lower
substrate enantioselectivity, which decreases on increas-
ing conversion.
In the mEH-catalyzed hydrolysis of diepoxides cis-9
and trans-10, the formal water attack can occur either
on the C1 or C2 carbon, with consequent opening of the
endocyclic epoxide ring or on the C8 carbon to give
exocyclic diols. In agreement with the previously observed
mEH-catalyzed hydrolysis of epoxy derivatives of cyclo-
hexenes (12, 21), the hydrolysis should take place exclu-
sively by diaxial opening of the oxirane ring, and the four
diaxial trans diol epoxides 12 should be the sole possible
product (Scheme 6). At variance, the mEH-catalyzed
hydrolysis of the exocyclic oxirane ring can give the
corresponding eight epoxy diols 11, Scheme 7. The 12
isomeric epoxydiols which may be formed either from
mEH-catalyzed hydrolysis of endocyclic and exocyclic
oxirane rings of diepoxides cis-9 and trans-10 may be
substrates for mEH and transformed into the corre-
It is noteworthy that all the diepoxides were substrates
for mEH. Furthermore, the stereochemical results re-
ported in Table 3 show that the hydrolysis of the mixture
of diepoxides cis-9 and trans-10 proceeds with a low
substrate diastereoselectivity. The hydrolysis of diep-
oxides trans-10 is, however, characterized by a fairly good
1
sponding tetrols. H and 13CNMR experiments carried

64 Chem. Res. Toxicol., Vol. 16, No. 1, 2003
Chiappe et al.
Sch em e 7
out on a sample arising by complete hydrolysis of the
commercial mixture of diepoxides cis-9 and trans-10 show
that the water attack occurs at both the oxirane carbons.
The comparison of the NMR spectra of the products
mixture arising by the mEH-catalyzed hydrolysis of
diepoxides cis-9 and trans-10 with the spectra of pure
11 and 12, prepared as reported in the Experimental
Section, shows indeed the presence of the signals related
to the carbons of the epoxy ring and to the carbons
bearing the hydroxy groups of both the diastereoisomeric
epoxydiols 12 besides other signals attributable to the
carbons of a sole diastereoisomer of the epoxydiols 11. It
is, however, noteworthy that the presence of a sole
diastereoisomer in this latter case may be due not only
to a high product diastereoselection but also a conse-
quence of a substrate diastereoselection, being probably
these epoxydiols substrate for mEH.
On the basis of the stereochemical data arising from
oxidation of monoepoxides cis-2 and trans-3 and hydroly-
sis of diepoxides cis-9 and trans-10, it must be remarked
that enantiomer (1R,2S,4R,7R)-trans-10b is the isomer
primarily formed by P450-catalyzed epoxidation of 1, and
it is also the isomer showing the lower propensity to
undergo mEH catalyzed hydrolysis. These two features
make this isomer a candidate for the mutagenic and
carcinogenic activity of 1. This indication, however, might
be considered affected by the stereoselective GSH-
catalyzed conjugation of VCH monoepoxides and diep-
oxides not taken in account in this study. Indeed, it is
well-known that VCH, VCH-1,2-epoxide, and VCH-diep-
oxides, when administered to rats or mice deplete the
hepatic GSH content, since these epoxides are substrates
of the glutathione S-transferases (22, 23). However, the
role of GSH conjugation on the overall metabolism and
disposition of VCH remains to be established.
In conclusion, the results arising from the present
investigation demonstrate that the stereochemical course
of the overall biotransformation of 4-vinylcyclohexene
formulate a composite picture markedly affected by the
composition of different P450 present in control or
induced rat liver.
The stereochemical composition of diepoxides cis-9 and
trans-10 depends on specificity and composition of the
P450 isoforms, but it is also affected by the substrate
enantioselectivity characterizing the mEH-catalyzed hy-
drolysis of these substrates. At variance with the oxida-
tion of endocyclic double bond of VCH, which is inducible
by phenobarbital and, although at low extent, by pyrazole
(12), in the oxidation of monoepoxides cis-2 and trans-3,
only phenobarbital is able to increase considerably
the yields of diepoxides, favoring the formation of
(1R,2S,4R,7R)-trans-10. Also the hydrolysis of diepoxides
trans-10 occurs with a good substrate enantioselectivity
favoring the hydrolysis of the diepoxide (1S,2R,4S,7S)-
trans-10 and (1R,2S,4R,7S)-trans-10 with respect par-
ticularly to the diepoxide (1R,2S,4R,7R)-trans-10. This
latter isomer is practically formed as the sole product by
P450-catalyzed epoxidation of (1R,2S,4R)-trans-3, the
major monoepoxide obtained from 4-vinylcyclohexene
oxidation (12). Diepoxide (1R,2S,4R,7R)-trans-10 is there-
fore in the rat the isomer primarily formed by P450-
catalyzed epoxidation of monoepoxides cis-2 and trans-
3, and it is also the compound showing the lower
propensity to undergo mEH-catalyzed hydrolysis; as a

Stereochemistry of 4-Vinylcyclohexene Biotransformation
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 65
of cytochrome P-450 enzymes after repeated exposure to 4-vinyl-
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elimination, it will have a longer half-life in vitro.
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The high biological reactivity and mutagenicity of diep-
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cellular macromolecules, DNA included (24). Thus, the
configuration of a specific diepoxide may be particularly
relevant for its reaction with various biomolecules and
to exert its own toxic potential.
Future studies will address this point; in addition the
stereochemical pattern of 4-vinylcyclohexene, whole me-
tabolism reported in this and previous papers (11, 12)
should be verified if it is maintained in humans.
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Rome) and Universita` di Pisa.
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Stereochemistry of the Biotransformation of 1-hexene and 2-methyl-
1-hexene with rat liver microsomes and purified P450s of rats
and humans. Chem. Res. Toxicol. 11, 1487-1493.
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