Stereochemical course of the biotransformation of isoprene monoepoxides and of the corresponding diols with liver microsomes from control and induced rats

Article abstract of DOI:10.1021/tx000061a

The stereochemical course of the biotransformation of isoprene by liver enzymes from control and induced rats has been determined. Between the two primarily formed metabolites, 2-methyl-2-vinyloxirane (2) and isopropenyloxirane (3), epoxide 2 is rapidly transformed into the corresponding vicinal racemic diol 4, predominantly through a nonenzymatic hydrolysis reaction. At variance, epoxide 3 is mainly biotransformed into the diol 5 by microsomal epoxide hydrolase (mEH) to give, before 50% conversion, selectively (R)-3-methyl-3-butene-1,2-diol, 5. The hydrolysis competes with the oxidation of the monoepoxide 3 to the corresponding diepoxides 6. Epoxidation of 3 catalyzed by P450 is characterized by a moderate stereoselectivity which, however, was strongly dependent on P450 induction. Treatment of rats with phenobarbital (PB) (an inducer of P450 2B1 and 3A) leads to threo-(2R,2'R)-(6 with a high selectivity, while with pyrazole (Pyr) (an inducer of P450 2E1), the formation of both erythro-(2S,2'R)- and threo-(2R,2'R)-6 is favored. The mEH-catalyzed hydrolysis of diepoxides 6 proceeds, although with a moderate turnover rate, with substrate and product diastereo- and enantio-selection by nucleophilic attack on the more substituted oxirane ring to give selectively (2R,3S)-3,4-epoxy-2-methyl-1,2-diol (7). Both diols 4 and 5 may be further oxidized on their double bond by P450. These reactions, which occur at a slow rate and are dependent on P450 induction with PB and Pyr, may be negligible in the overall isoprene biotransformation. On the other hand, the epoxydiol 7, which is formed by hydrolysis of diepoxides 6 but it is itself not hydrolyzable, may play an important role in the isoprene toxicity.

Full text of DOI:10.1021/tx000061a

Chem. Res. Toxicol. 2000, 13, 831-838
831
Ster eoch em ica l Cou r se of th e Biotr a n sfor m a tion of
Isop r en e Mon oep oxid es a n d of th e Cor r esp on d in g Diols
w ith Liver Micr osom es fr om Con tr ol a n d In d u ced Ra ts
Cinzia Chiappe,*,† Antonietta De Rubertis,† Viola Tinagli,† Giada Amato,‡ and
Pier Giovanni Gervasi*,‡
Dipartimento di Chimica Bioorganica e Biofarmacia, Universita` di Pisa, via Bonanno 33,
56126 Pisa, Italy, and Istituto di Mutagenesi e Differenziamento, CNR, Via Svezia 10,
56124 Pisa, Italy
Received March 13, 2000
The stereochemical course of the biotransformation of isoprene by liver enzymes from control
and induced rats has been determined. Between the two primarily formed metabolites,
2-methyl-2-vinyloxirane (2) and isopropenyloxirane (3), epoxide 2 is rapidly transformed into
the corresponding vicinal racemic diol 4, predominantly through a nonenzymatic hydrolysis
reaction. At variance, epoxide 3 is mainly biotransformed into the diol 5 by microsomal epoxide
hydrolase (mEH) to give, before 50% conversion, selectively (R)-3-methyl-3-butene-1,2-diol, 5.
The hydrolysis competes with the oxidation of the monoepoxide 3 to the corresponding
diepoxides 6. Epoxidation of 3 catalyzed by P450 is characterized by a moderate stereoselectivity
which, however, was strongly dependent on P450 induction. Treatment of rats with pheno-
barbital (PB) (an inducer of P450 2B1 and 3A) leads to threo-(2R,2′R)-6 with a high selectivity,
while with pyrazole (Pyr) (an inducer of P450 2E1), the formation of both erythro-(2S,2′R)-
and threo-(2R,2′R)-6 is favored. The mEH-catalyzed hydrolysis of diepoxides 6 proceeds,
although with a moderate turnover rate, with substrate and product diastereo- and enantio-
selection by nucleophilic attack on the more substituted oxirane ring to give selectively (2R,3S)-
3,4-epoxy-2-methyl-1,2-diol (7). Both diols 4 and 5 may be further oxidized on their double
bond by P450. These reactions, which occur at a slow rate and are dependent on P450 induction
with PB and Pyr, may be negligible in the overall isoprene biotransformation. On the other
hand, the epoxydiol 7, which is formed by hydrolysis of diepoxides 6 but it is itself not
hydrolyzable, may play an important role in the isoprene toxicity.
the isoprene carcinogenicity, but it is not known if an
important contribution to this toxicity is due to other
reactive metabolites such as epoxydiols 7 and 8, eventu-
ally formed by either further epoxidation of 4 and 5 or
diepoxide hydrolysis. That may be the case as for the
butadiene the 3,4-epoxy-1,2 butanediol was found to
exhibit a mutagenic activity (11) and to represent the
most abundant component of DNA adducts derived from
the butadiene epoxides (12).
In tr od u ction
Isoprene, the monomeric unit of natural rubber and
naturally occurring terpenes and steroids, is obtained
primarily as a byproduct of nafta cracking and is emitted
from both plants and animals at significant rates (1, 2).
Isoprene is of particular interest in atmospheric chem-
istry because it participates in the tropospheric reactions
that produce ozone; furthermore, its oxidation by the
hydroxyl radical reduces the oxidative capacity of the
atmosphere (3). The high-level diffusion in nature of this
chemical and its structural similarity to 1,3-butadiene,
a potent carcinogen, explain the relevance of the studies
focused on mammalian toxicology of isoprene and its
metabolites (4-6). Recent studies have indicated that
isoprene is carcinogenic to mice but much less if any to
rats (4, 5) and that among its metabolites only diepoxide
but not monoepoxides has been shown to be mutagenic
(7). Isoprene (1) is epoxidized by P450 to the isomeric
monoepoxides 2-methyl-2-vinyloxirane (2) and 2-isopro-
penyloxirane (3) (Scheme 1). Both epoxides are further
epoxidized to 2-methyl-2,2-bioxirane (6) in competition
with the hydrolysis to the corresponding vicinal diols 4
and 5 (4, 8-10). The diepoxide is thought to account for
The stereochemical aspects in the biotransformation
process of these compounds are also important in deter-
mining their toxicity. Studies carried out using liver
microsomes from uninduced rats and mice have shown
that the oxidation to 6 occurs with substrate enantio-
selectivity, product diastereoselectivity, and product
enantioselectivity (8). Furthermore, it has been shown
(8) that the microsomal epoxide hydrolase catalyzes the
in vivo hydrolysis of both monoepoxides 2 and 3. The
hydrolysis of racemic 2-isopropenyloxirane 3 with mouse
and rat liver microsomes occurs with substrate enantio-
selection; the (R)-enantiomer is preferentially hydrolyzed.
A much lower-level enantioselection was instead observed
in the hydrolysis of the more reactive 2-methyl-2-vinyl-
oxirane (2) which was attributed (8) to a competition
between the enzymatic hydrolysis and the spontaneous
process. It is, however, also noteworthy that, in the case
of 3, the presence of the double bond may favor the
* To whom correspondence should be addressed.
† Universita` di Pisa.
‡ CNR.
10.1021/tx000061a CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/03/2000

832 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Chiappe et al.
Sch em e 1
(()-2-Isopropenyloxirane (3) and a mixture of erythro- and threo-
2-methyl-2,2′-bioxirane (6) were prepared as previously reported
(8).
nonenzymatic oxirane ring opening, a racemic process
which can affect both the substrate and product enantio-
selectivity. The stereochemical behavior of the isoprene
biotransformation is therefore affected by the stereo- and
enantioselection of the oxidation processes as well as of
the hydrolysis reactions. With regard to oxidation pro-
cesses, it has been evidenced (13) that the epoxidation
of alkyl-substituted olefins, catalyzed by P450s, may
occur with a product stereoselectivity which strongly
depends on the specificity and composition of the P450
enzymes. Furthermore, induction may also favor the
mEH1 (microsomal epoxide hydrolase)-catalyzed hydroly-
sis of epoxides with respect to the nonenzymatic process.
In the oxidation of isoprene and its monoepoxides, the
prominent role of P450 2E1 has been demonstrated (9),
but the importance of this isoform in the stereochemical
course of these biotransformations has not been inves-
tigated.
Therefore, to gain further insight into the stereochem-
ical and mechanistic aspect in the in vivo metabolism of
isoprene, we investigated (i) the effect of the pretreatment
of rats with two P450 inducers, phenobarbital (PB) and
pyrazole (Pyr), on the stereochemistry of the oxidation
of racemic epoxides 2 and 3 to the isomeric diepoxides
erythro and threo-6; (ii) the biotransformation of diols 4
and 5, arising from the chemical and/or mEH-catalyzed
hydrolysis of the metabolites 2 and 3, to the correspond-
ing erythro and threo epoxydiols 7 and 8; (iii) the
mEH-catalyzed hydrolysis of diepoxides 6 to epoxydiols
7 and/or 8; and (iv) the mEH-catalyzed hydrolysis of both
7 and 8.
(()-2-Meth yl-3-bu ten e-1,2-d iol (4) a n d (()-3-m eth yl-3-
bu ten e-1,2-d iol (5) were prepared by acid-catalyzed hydrolysis
(1 N HClO4) from the corresponding oxiranes (2 and 3).
er yth r o- a n d th r eo-(()-3,4-Ep oxy-2-m eth yl-1,2-d iol (7).
To a solution of m-chloroperoxybenzoic acid (350 mg, 1.35 mmol)
in CH2Cl2 (25 mL), previously dried over MgCl2, was added diol
4 (69 mg, 0.67 mmol). The reaction mixture was allowed to stand
for 3 days at room temperature and 4 h at 4 °C. The solution
was filtered, and 80 mg (1.35 mmol) of KF, previously activated
by heating at 120 °C in vacuo (0.1 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 6:4 mixture of epoxy
diols erythro- and threo-7. 1H NMR (CDCl3): δ 1.21 (s, 3H), 1.25
(s, 3H), 2.70 (dd, J ) 5.3 and 4 Hz, 1H), 2.78 (dd, 1H), 2.79 (dd,
1H), 2.85 (dd, J ) 4 and 2.8 Hz, 1H), 3.50 (d, AB system, J )
11.5 Hz, 1H), 3.55 (d, AB system, J ) 11 Hz, 1H), 3.80 (d, AB
system, J ) 11.5 Hz, 1H), 3.65 (d, AB system, J ) 11 Hz, 1H).
13C NMR (CDCl3): δ 20.4, 22.0, 42.7, 44.6, 55.8, 56.5, 69.7, 70.5.
er yth r o- a n d th r eo-(()-3,4-Ep oxy-3-m eth yl-1,2-d iol (8).
A 6:4 mixture of erythro- and threo-8 was prepared from 5 like
7. 1H NMR (CDCl3): δ 1.37 (s, 3H), 1.39 (s, 3H), 2.66 (d, J ) 4.5
Hz, 1H), 2.68 (d, J ) 4.3 Hz, 1H), 2.94 (d, J ) 4.5 Hz, 1H), 2.99
(d, J ) 4.3 Hz, 1H), 3.78 (m, 6H). 13C NMR (CDCl3): δ 17.2,
18.3, 50.45, 51.5, 63.2, 63.5, 72.1, 73.6.
2(S)-3-Met h yl-3-b u t en e-1,2-d iol (5), 2(S)-2-Met h yl-3-
bu ten e-1,2-d iol, a n d 2(R)-2-Meth yl-3-bu ten e-1,2-d iol (4). A
3:1.3:0.7 mixture of diols 2(S)-3-methyl-3-butene-1,2-diol (5),
2(S)-2-methyl-3-butene-1,2-diol (4), and 2(R)-2-methyl-3-butene-
1,2-diol (4) was prepared from isoprene using AD-mix R,
according to the standard procedure reported by the Sharpless
group (14).
2(S)-3-Met h yl-1-[p -(t olu en esu lfon yl)oxy]-3-b u t en e-1,2-
diol (10), 2(S)-2-m eth yl-1-[p-(tolu en esu lfon yl)oxy]-3-bu ten e-
1,2-d iol, a n d 2(R)-2-m et h yl-1-[p -(t olu en esu lfon yl)oxy]-3-
bu ten e-1,2-d iol (9) were prepared from the mixture of diols,
arising from the asymmetric Sharpless hydroxylation, as previ-
ously described (8). Compound 9 and 10 were separated by
HLPC (Spherisorb S5 CN column, 96:4 hexane/2-propanol).
Ma ter ia ls a n d Meth od s
Ca u tion : The following chemicals are hazardous and should
be handled carefully: 2-isopropenyloxirane, 2-methyl-2-vinyl-
oxirane, and 2-methyl-2,2′-bioxirane.
Ma ter ia ls. PB and Pyr were obtained from common com-
mercial sources. Isoprene (1) and (()-2-methyl-2-vinyloxirane
(2) were purchased from Aldrich Chemical Co. (Milwaukee, WI).
1
Compound 9 (ee ) 33%, determined by GC). H NMR (CDCl3):
δ 1.27 (s, 3H), 2.45 (s, 3H), 3.88 (s, 2H), 5.15 (dd, J ) 0.8 and
10.5 Hz, 1H), 5.30 (dd, J ) 0.8 and 17.5 Hz, 1H), 5.80 (dd, J )
10.5 and 17.5 Hz, 1H), 7.35 (d, AB system, 2H), 7.80 (d, AB
system, 2H). 13C NMR (CDCl3): δ 21.6, 24.1, 75.7, 115.0, 127.9,
1
Abbreviations: mEH, microsomal epoxide hydrolase; PB,
phenobarbital; Py, pyrazole; TCPO, trichloropropene oxide; GC, gas
chromatography.

Isoprene Monoepoxide Biotransformation
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 833
129.9, 139.9, 145.0. Compound 10 (ee ) 95%, determined by
(consisting of 0.5 mM NADP+, 5 mM glucose 6-phosphate, and
0.5 unit/mL glucose-6-phosphate dehydrogenase), and TCPO (1
mM) to inhibit the mEH were initiated by the addition of 4 or
5 (30 mM). After 60 min, a saturating amount of NaCl was
added to precipitate the microsomal proteins and the reaction
mixtures were lyophilized. The solid phase was then dissolved
in ethyl acetate (2 mL), filtered, and analyzed by GC (NPGS
column, 120 °C) after addition of appropriate amounts of an
ethyl acetate stock solution of cycloheptanediol as an internal
standard.
1
GC). H NMR (CDCl3): δ 1.68 (s, 3H), 2.45 (s, 3H), 3.95 (dd, J
) 7.5 and 10.2 Hz, 1H), 4.10 (dd, J ) 3.4 and 10.2 Hz, 1H), 4.30
(dd, J ) 3.4 and 7.5 Hz, 1H), 4.95 (s, 1H), 5.05 (s, 1H), 7.35 (d,
AB system, 2H), 7.80 (d, AB system, 2H). 13C NMR (CDCl3): δ
18.5, 21.6, 72.34, 72.78, 113.58, 127.9, 129.9, 132.5, 141.9, 145.0.
2(S)-Isop r op en yloxir a n e (3). To a solution of 10 (50 mg)
in CH2Cl2 (2 mL) were added 50 mg of KOH and crown-18-ether
(5 mg). The mixture, stirred at room temperature for 18 h, was
filtered, and the solvent was evaporated to give (S)-3 (ee ) 95%,
determined by GC).
2(R)-Met h yl-2-vin yloxir a n e a n d 2(S)-Met h yl-2-vin yl-
oxir a n e (2). The 2:1 mixture of the two enantiomers of 2 was
prepared from the corresponding 2:1 mixture of 2(S)-2-methyl-
1-[p-(toluenesulfonyl)oxy]-3-butene-1,2-diol and 2(R)-2-methyl-
1-[p-(toluenesulfonyl)oxy]-3-butene-1,2-diol like 3. The crude
product was analyzed by GC.
(3) Deter m in a tion of th e P r od u ct En a n tioselectivity of
th e m EH-Ca ta lyzed Hyd r olysis of Mon oep oxid es 2 a n d 3.
Aliquots (10 µL) of an ethanolic stock solution of (()-2 or (()-3
were added to 1 mL of a microsomal preparation (control or PB-
induced) containing 2 or 4 mg of protein/mL in a such way to
obtain a substrate concentration of 5, 10, or 20 mM, and the
reaction mixtures were incubated at 37 °C. At prefixed times
(10, 25, 50, and 80 min), a saturating amount of NaCl was added
to precipitate the microsomal proteins, and the incubation
mixtures, after addition of a proper amount of cycloheptanol as
an internal standard, were analyzed directly by GC (Carbowax
column, 120 °C) to determine the diol yields.
(2S,2′R)- a n d (2R,2′R)-2-Meth yl-2,2′-bioxir a n e (6). Epoxi-
dation of 2(S)-isopropenyloxirane with m-chloroperbenzoic acid
in CH2Cl2 (2 h) gave a 6:4 mixture of (2S,2′R)- and (2R,2′R)-2-
methyl-2,2′-bioxirane (6). The crude product was analyzed by
GC.
Mixtu r e of Diep oxid es 6 En r ich ed in (2R,2′S)- a n d
(2R,2′R)-2-Meth yl-2,2′-bioxir a n e (6). Epoxidation of the 2:1
mixture of 2(R)-methyl-2-vinyloxirane and 2(S)-methyl-2-vinyl-
oxirane with m-chloroperbenzoic acid in CH2Cl2 gave a mixture
of the four epoxides enriched in the (2R,2′S)- and (2R,2′R)-
diastereoisomers. The crude product was analyzed by GC.
(2R,3R)- a n d (2R,3S)-3,4-Ep oxy-3-m eth yl-1,2-d iol (8). The
mixture of (2R,3R)- and (2R,3S)-8 was prepared from 2(S)-3-
methyl-3-butene-1,2-diol (5) like 8.
Mixtu r e of Ep oxyd iols 7 En r ich ed in (2R,3S)- a n d
(2R,3R)-3,4-Ep oxy-2-m eth yl-1,2-d iol (7). The mixture of ep-
oxydiols 7 enriched in the diastereoisomers (2R,3S)- and (2R,3R)-7
was prepared from 2(S)-2-methyl-3-butene-1,2-diol (4) like 7.
An im a ls a n d Micr osom a l P r ep a r a tion s. Male Sprague-
Dawley rats were purchased from Charles River. They were
treated for 3 days with PB ip (80 mg/kg daily) or Pyr (200 mg/
kg daily), and microsomes were obtained from the liver as
previously described (15). Microsomal protein concentrations
were assayed by using the method of Lowry et al. (16); the total
P450 concentration was measured according to the method of
Omura and Sato (17).
The enantiomer ratios and the absolute configurations of the
formed diols were determined, after extraction with ethyl
acetate (5 × 1 mL) 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, at 85 °C) by comparison of the
retention times with those of samples of 2(S)-3-methyl-3-butene-
1,2-diol [(S)-5], 2(S)-2-methyl-3-butene-1,2-diol [(S)-4], and 2(R)-
2-methyl-3-butene-1,2-diol [(R)-4] obtained as reported above.
(4) Deter m in a tion of th e P r od u ct En a n tioselectivity of
th e m EH-Ca ta lyzed Hyd r olysis of Diep oxid es 6. Aliquots
(10 µL) of an ethanolic stock solution of a 60:40 mixture of (()-
erythro-6 and (()-threo-6 were added to 1 mL of a microsomal
preparation (control or PB-induced) containing 2.5 or 5 mg of
protein/mL in a such way to obtain a substrate concentration
of 10, 25, or 50 mM, and the reaction mixtures were incubated
at 37 °C and pH 7.4. At prefixed times (30 min and 1, 2, 4, and
6 h), a saturating amount of NaCl was added to precipitate the
microsomal proteins, and the incubation mixtures, after addition
of a proper amount of cycloheptanediol as an internal standard,
were analyzed directly by GC (NPGS column, 120 °C) to
determine the epoxydiol yields.
En zym a tic In cu ba tion s. (1) Deter m in a tion of th e Cyto-
ch r om 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 in th e Oxid a tion of Mon oep oxid es 2 a n d
3. Incubation mixtures (2 mL) containing 100 mM potassium
phosphate buffer (pH 7.4), 4 mg of hepatic microsomal proteins,
a NADPH-generating system (consisting of 0.5 mM NADP+, 5
mM glucose 6-phosphate, and 0.5 unit/mL glucose-6-phosphate
dehydrogenase), and trichloropropene oxide (TCPO, 1 mM) to
inhibit the mEH were initiated by the addition of a proper
amount of 2 or 3. After 60 min, a saturating amount of NaCl
was added to precipitate the microsomal proteins. The reaction
products were extracted with ethyl acetate (2 × 5 mL) and
analyzed by GC (Carbowax column, 90 °C) after addition of
appropriate amounts of cycloheptanone as an internal standard.
The substrate concentrations ranged from 0.1 to 20 mM.
The enantiomeric composition of 6 was determined by GC
using a 30 m Chiraldex G-TA (ASTEC) column, with a helium
flow of 50 KPa, and with an evaporator and detector set at 200
°C, under the following conditions: 32 °C for 12 min, at a rate
of 10 °C/min, 55 °C for 4 min, at a rate of 10 °C/min, and 75 °C
for 22 min.
The enantiomer ratio and the absolute configurations of the
formed epoxydiols 7 and 8 at 20% conversion (6 h) were
determined, after dehydration and extraction with ethyl acetate,
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, at 90 °C) by comparison of the retention times
with those of samples of erythro-(2R,3S)-3,4-epoxy-2-methyl-1,2-
diol and threo-(2R,3R)-3,4-epoxy-2-methyl-1,2-diol obtained in
excess by epoxidation of a 2:1 mixture of (S)-2-methyl-3-butene-
1,2-diol and (R)-2-methyl-3-butene-1,2-diol, as reported above.
The erythro and threo isomers have been identified, on the same
column, on the basis of the ratio (around 6:4) between the two
pairs of enantiomers corresponding to the ratio determined by
NMR.
(5) Eva lu a tion of th e m EH-Ca ta lyzed Hyd r olysis of
E p oxyd iols 7 a n d 8. Aliquots (10 µL) of an ethanolic stock
solution of mixtures of (()-erythro-7 and (()-threo-7 or (()-
erythro-8 and (()-threo-8 were added to 2 mL of microsomal
preparation (control or PB-induced) containing 2.5 or 5 mg of
protein/mL in a such way to obtain a substrate concentration
of 50 mM, and the reaction mixtures were incubated at 37 °C
and pH 7.4. At prefixed times (at 30 min and 1, 2, 4, and 6 h),
a saturating amount of NaCl was added to precipitate the
microsomal proteins, and the incubation mixtures, after addition
of a proper amount of cycloheptanediol as an internal standard,
were analyzed directly by GC (NPGS column, 120 °C) to
determine the epoxydiol yields. The epoxydiols 7 or 8 were
always quantitatively recovered, showing that these compounds
were not substrates for the mEH.
The absolute configurations of the excess enantiomers of 6
were determined by comparison of the retention times of the
two enantiomers of each isomer with those of (2S,2′R)-, (2R,2′R)-,
and (2R,2′S)-6 obtained as reported above.
(2) Deter m in a tion of th e Cytoch r om e P 450 Activity in
th e Oxid a tion of Diols 4 a n d 5. Incubation mixtures (10 mL)
containing 100 mM potassium phosphate buffer (pH 7.4), 2 mg
of hepatic microsomal proteins, a NADPH-generating system

834 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Chiappe et al.
Ta ble 1. Ster eoch em ica l Cou r se for th e Oxid a tion of 3 by
Micr osom es fr om Con tr ol a n d Tr ea ted Ra tsa
(2R,2′S)-6 (2S,2′S)-6 (2S,2′R)-6 (2R,2′R)-6
microsomes
(a)
(b)
(c)
(d)
CTR
PB
Pyr
26
17
12
19
13
16
36
17
34
19
53
38
a
Each value represents the mean of at least two determinations.
Ta ble 2. Effect of P B a n d P yr P r etr ea tm en t of Ra ts on
th e Micr osom a l Oxid a tion of Mon oep oxid es 2 a n d 3a
microsomes
2 (nmol)
3 (nmol)
CTR
PB
Pyr
15 ( 4
26 ( 7
70 ( 15
275 ( 8
260 ( 19
475 ( 26
a
Values are means ( SD of three experiments performed with
different preparations of hepatic microsomes. The protein concen-
tration was 2 mg/mL, and the incubation time was 60 min.
oxirane (ee ) 33%) obtained from the corresponding diol
arising from the asymmetric Sharpless dihydroxylation
(14).
Only for epoxide 3 was it possible to obtain data related
to the stereochemical course of the oxidation of the
remaining double bonds (Table 1). Attempts to study the
substrate and product enantioselectivity of the epoxida-
tion of 2 failed due to the low biotransformation rate of
this substrate. Since conflicting results have been re-
ported about the oxidation rate of this epoxide by P450s
(4, 8), to obtain further information about the microsomal
biotransformation of 2 and 3, the reaction mixtures were
analyzed by GC using a Carbowax glass column. In this
case, all the stereochemical information was lost, but it
was possible to quantify the very low amounts of the
product.
F igu r e 1. Gas chromatographic enantiomer separation of
(2S,2′S)-6 (1), (2S,2′R)-6 (2), (2R,2′R)-6 (3), and (2R,2′S)-6 (4).
Analysis conditions are outlined in Materials and Methods.
Ra tes of th e Sp on ta n eou s Hyd r olysis of 2 a n d 3. Aliquots
(10 µL) of an ethanolic stock solution of (()-2 (0.5 or 1 M) or
(()-3 (1 or 2 M) were added to 1 mL of Tris-HCl buffer (pH 7.4),
and the reaction mixtures were thermostated at 37 °C. Samples
were withdrawn at 10 min intervals, neutralized with CaCO3,
and subjected to GC analysis (Carbowax column, 120 °C). The
reactions were carried out in duplicate. The diol concentration
versus time data were fitted to the integrated first-order rate
law (ln[diol] ) kt + ln[diol]0), and the rate constants k were
obtained with the usual least-squares procedure.
In agreement with the data previously reported by
Wistuba et al. (8), the epoxide 2 may also be oxidized by
the P450 system, although at a rate lower than that
obtained using epoxide 3 as the substrate (Table 2).
Furthermore, an increase in the oxidation rates of both
monoepoxides was observed in the Pyr-induced micro-
somes compared to the control microsomes according to
the high turnover toward these substrates shown by the
recombinant P450 2E1 among the P450 human isoforms
(9). The oxidation of 2 performed with control or induced
microsomes followed simple Michaelis-Menten kinetics,
which were linear up to 45 min and 1.5 mg of microsomal
protein. From the Lineweaver-Burk plots of 6 formation
data, we determined the apparent kinetic parameters
Vmax and KM illustrated in Table 3. Pyr microsomes
exhibited a Vmax that was higher (about 3-fold) than that
obtained with control microsomes, although also with a
higher KM. It is worth noting that the apparent KMs for
the oxidation of 2 shown in Table 3 are similar to the KM
observed for the oxidation of 3 by liver microsomes from
PB-induced rats (7).
Resu lts a n d Discu ssion
Ep oxid a tion of Mon oep oxid es 2 a n d 3 Ca ta lyzed
by Ra t Liver Micr osom es. The stereochemical course
of the double bond oxidation of the epoxides 2 and 3 in
the corresponding diepoxides 6 was investigated by
incubating 2 and 3 (20 mM) by using liver microsomes
obtained from untreated rats or rats pretreated with PB
and Pyr, classical inducers of 2B1/2, 3A, and 2E1,
respectively (15). At prefixed times (60 min), the reactions
were stopped by adding a saturating amount of NaCl,
and the incubation mixtures were extracted with ethyl
acetate and analyzed by GC on the chiral column. The
use of the chiral column allowed evaluation of not only
the diastereoisomeric ratio of the formed diepoxides 6
but also the enantiomeric ratio for each diastereoisomer
and the absolute configuration of the enantiomer in
excess (see Figure 1). The latter was evaluated by the
comparison of the retention time of the formed products
with those of samples with known configurations. A 6:4
mixture of (2S,2′R)- and (2R,2′R)-2-methyl-2,2′-bioxirane
(6)2 was obtained by epoxidation of the optically active
2(S)-isopropenyloxirane, prepared via a monotosyl de-
rivative from the corresponding diol, the latter synthe-
sized by asymmetric Sharpless dihydroxylation (14).
Similarly, a mixture of four diepoxides 6, enriched in the
isomers (2R,2′S)- and (2R,2′R)-2-methyl-2,2′-bioxirane
(6), was prepared by epoxidation of 2(S)-methyl-2-vinyl-
Related to the stereochemical behavior of the epoxi-
dation of 3, in accord with the results obtained by
2
The carbon atom C-2 of isopropenyloxirane becomes C-2′ in
2-methyl-2,2′-bioxirane because of the formal change of the carbon
atom numbering caused by the IUPAC rule. Furthermore, as
a
consequence of the formal change in the descriptor caused by the
priority rule of Chan, Ingold, and Prelog, the 2R (or 2S) stereochemistry
of the isopropenyloxirane becomes 2′S (or 2′R) in 2-methyl-2,2′-
bioxirane and the 2R (or 2S) stereochemistry of the 2-methyl-2-
vinyloxirane becomes 2S (or 2R) in 2-methyl-2,2′-bioxirane.

Isoprene Monoepoxide Biotransformation
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 835
Sch em e 2
Ta ble 3. Ap p a r en t Kin etic Con sta n ts for th e Oxid a tion of
Mon oep oxid e 2 by Micr osom es fr om Con tr ol a n d Tr ea ted
Ra tsa
both PB and Pyr, reverses the enantioface discrimination
on the (2S)-enantiomer, whereas the level becomes
extremely low on (2R)-3. Finally, the results reported in
Table 1 may be discussed in terms of product enantio-
selectivity, given by a versus c and by b versus d, arising
from the differentiation between the two enantiotopic
faces of the double bond of the two enantiomers. When
control microsomes are used, the enantiomeric pair
(2R,2′S)/(2S,2′R) was formed by epoxidation of the re face
of (2R)-3 and the si face of the (2S)-enantiomer with an
enantiomeric excess (ee) of 16% in favor of the (2S,2′R)-
enantiomer. This ratio decreases to practically zero with
PB-induced microsomes and increases again to 48% in
favor of the (2S,2′R)-enantiomer using Pyr-induced
microsomes. On the contrary, the enantiomeric pair
(2S,2′S)/(2R,2′R) was formed in a practically racemic
way when the incubations were carried out using control
microsomes, and it was characterized by enantiomeric
excesses of 60 and 40%, respectively, in favor of the
(2R,2′R)-enantiomer when the PB- or Pyr-induced micro-
somes were used.
Vmax
microsomes
KM (mM)
[nmol min-1 (mg of protein)-1
]
CTR
PB
Pyr
0.13 ( 0.06
0.28 ( 0.11
0.83 ( 0.32b
0.14 ( 0.03
0.20 ( 0.04
0.37 ( 0.06b
a
Values are means ( SD of three experiments performed
b
with different preparations of hepatic microsomes. Significantly
different from control microsomes as determined by a Student’s t
test (p < 0.05).
Wistuba et al. (8) using untreated rat liver microsomes,
the data reported in Table 1 show that the product
distribution, at least with control microsomes, is char-
acterized by a moderate selectivity. It must be mentioned
that the stereochemical results, arising from the competi-
tion among processes a-d (Scheme 2), may be rational-
ized considering three stereoselective processes: sub-
strate enantioselectivity, product diastereoselectivity, and
product enantioselectivity.
These results therefore seem to indicate that, as
previously observed (13) for the product enantioselectivity
in the P450 oxidation of prochiral olefins, more than one
isoform is involved in the epoxidation process and the
single enzymes catalyze the reaction with a different
stereoselectivity, substrate enantioselectivity, product
enantioselectivity, and product diastereoselectivity. The
substrate and product enantioselectivity imply a different
interaction of the two enantiomers of the substrate with
the active site of the distinct P450 enzymes in the
fundamental state (affecting the binding) and/or in the
activate complex (affecting kcat). On the other hand, the
product diastereoselectivity may arise, if the epoxidation
occurs through a concerted mechanism (13), exclusively
from a different face recognition which can be determined
by the site active architecture or by the ability of these
enzymes to use different iron species (peroxo, hydro-
peroxo, or oxenoid) as the active oxidant.
The substrate enantioselectivity, given by a and b
versus c and d, is extremely low (45:55) and in favor of
the (2S)-configured oxirane carbon. A moderate substrate
enantioselectivity in favor of the (2R)-enantiomer has
been reported by Wistuba (8). However, as it has been
recently shown (13), the stereoselectivity of the P450-
catalyzed double bond epoxidation may depend on sub-
strate concentration, and therefore, reactions carried out
under different conditions may be difficult to compare.
The main result is, however, the effect of the type of P450
induction on stereoselectivity. The substrate enantio-
selectivity increases indeed to 70:30, always in favor of
the (2S)-configured oxirane carbon, using PB- or Pyr-
induced microsomes. It is notheworthy that, while the
PB-induced microsomes do not modify the total amount
of products 6, the Pyr-induced microsomal preparations
give a larger amount of 6. This result is in agreement
with the fact that P450 2E1 is considered to be the
isozyme principally responsible for the biotransformation
of isoprene and the two isoprene monoepoxides (9). Also,
the product diastereoselectivity, given by a versus b and
c versus d, shows a behavior that is inducer-dependent.
The epoxidation of (2R)-3 with control microsomes occurs
preferentially at the re face, while that of (2S)-enantiomer
shows a selectivity toward the si face. The induction, with
Ep oxid a tion of Diols 4 a n d 5 Ca ta lyzed by Ra t
Liver Micr osom es. The ability of diols 4 and 5, arising
from the hydrolysis of epoxides 2 and 3, to be the
substrate of the P450s was checked by incubating 4 and
5 (30 mM) at 37 °C in 10 mM phosphate buffer (pH 7.4),
in the presence of a NADPH-regenerating system and
TCPO (1 mM), using liver microsomal preparations (10

836 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Chiappe et al.
Ta ble 4. En a n tiom er ic Ra tio a n d Absolu te Con figu r a tion
of Diols 4 a n d 5 F or m ed by Hyd r olysis of Ra cem ic
Mon oep oxid es 2 a n d 3 w ith Micr osom es fr om
P B-In d u ced Ra tsa
mL, 2 mg of protein/mL) obtained from untreated rats
or rats pretreated with PB and Pyr. At prefixed times,
the incubation mixtures were stopped and lyophilized.
The residue was suspended in ethyl acetate, and the
organic phase was analyzed by GC. Dehydration was
necessary to avoid the loss of the eventually formed
epoxydiols 8 and 9 as they are hardly recovered by ethyl
acetate extraction of their water solutions. Control
experiments ensured a recovery of the epoxydiols 7 or 8
of >85% using this procedure. No measurable amount
of epoxydiols 7 or 8 was, however, found when control
microsomes were used. On the contrary, when PB- or Pyr-
induced microsomes were used, very small amounts
[around 10 nmol in both cases, i.e., at a rate of 18 pmol
min-1 (mg of protein)-1] of the corresponding epoxydiols
were detected. In particular, it was found that only diol
5 was oxidized to epoxydiol 8 by using PB-induced
microsomes, whereas with Pyr-induced microsomes, only
diol 4 was transformed in the corresponding epoxydiol
7. Unfortunately, in this case, the amounts of formed
products were also too small to allow a stereochemical
study.
Hyd r olysis of Mon oep oxid es 2 a n d 3 a n d Di-
ep oxid es 6. The P450-catalyzed oxidation of mono-
epoxides 2 and 3 to diepoxides 6 competes with the
hydrolysis of the primarily formed monoepoxides to the
corresponding vicinal diols, a process which is a super-
position of a spontaneous and an enzymatic reaction. In
particular, although the spontaneous process is particu-
larly important for epoxide 2, it also cannot be neglected
in the case of epoxide 3. The rates of spontaneous
hydrolysis of 2 and 3 were measured in Tris-HCl buffer
(pH 7.4) at 37 °C, under conditions that were identical
to those employed for the enzymatic reactions. Samples
were withdrawn at intervals, neutralized by addition of
calcium carbonate, and analyzed directly by GC on a
Carbowax column, after addition of cyclohexanol as a
standard. The determinations of diols 4 and 5, at several
times during the course of triplicate runs for the hydroly-
sis reactions, were used to evaluate the kinetic constants
for the ring opening of epoxides 2 (at concentrations of
5 and 10 mM) and 3 (at concentrations of 10 and 20 mM).
In both cases, the diol formation obeyed a first-order rate
law with k values of (1.1 ( 0.1) × 10-3 and (3.1 ( 0.1) ×
10-4 s-1 for 2 and 3, respectively. These data show that
both epoxides underwent significant nonenzymatic hy-
drolysis. However, the rate of hydrolysis of 2 was about
4-fold as large as that of 3, as expected for a gem-
disubstituted epoxide as compared to a monosubstituted
one. The spontaneous hydrolysis is a racemic process able
to compete significantly also in the case of 3 with the
enzymatic reaction (diol 5 arising from nonenzymatic
hydrolysis represents ca. 35% of the total diol when the
incubations were carried out at a substrate concentration
of 20 mM and using a control microsomal preparation
containing 4 mg of protein/mL). To evaluate the stereo-
selectivity of the mEH-catalyzed hydrolysis, the incuba-
tions of 2 (5 mM) and 3 (10 mM) were carried out using
PB microsomes (in which mEH is induced) containing 4
mg of protein/mL. GC analysis on the chiral column of
diols 5 and 4, at 20 and 30% conversion, respectively,
arising from hydrolysis of 3 and 2, showed a 70:30 and a
40:60 ratio of the two peaks corresponding to the (R)- and
(S)-enantiomers of 5 and 4, respectively. After correction
for the racemic diol obtained nonenzymatically, the ee
of the enzymatically formed diol (R)-5 was 70% and that
diol
hydrolysis
(%)
enantiomeric
ratiob
absolute
configuration
substrate
2
3
30
20
4
5
40:60 (65:35)
70:30 (85:15)
S
R
a
Each value represents the mean of two determinations. The
incubations and analyses were performed as described in Materials
and Methods. b Values in parentheses are corrected for the amount
of racemic diol formed by nonenzymatic hydrolysis and give an
evaluation of the substrate selectivity of this reaction.
of (S)-4 30% (Table 4). It is noteworthy that, although a
substrate enantioselection was observed in the mEH
hydrolysis of 3 (8), the formation of a practically racemic
diol was reported to be independent of the incubation
time. Furthermore, when it is considered that with
monoalkyl- and gem-dialkyl-substituted oxiranes the
mEH-catalyzed ring opening occurs at the less hindered
oxirane carbon atom, the stereochemistry of the prefer-
entially formed diols is in agreement with the generally
observed mEH selectivity (18) and with the substrate
enantioselectivity previously reported for the hydrolysis
of 2 and 3 catalyzed by rat liver microsomes (8). In
particular using rabbit liver microsomes, it has been
shown that the hydrolysis of monosubstituted oxiranes
proceeds with a substrate selectivity that is strongly
dependent on substituent but always favoring the oxirane
ring opening of the (R)-enantiomer (19). The introduction
of a methyl group to give a gem-disubstituted oxirane
markedly increases the hydrolysis rate, reversing the
substrate enantioselection, and the transformation of the
(S)-enantiomer is generally favored (20).
Hyd r olysis of Diep oxid es 6 a n d Ep oxyd iols 7 a n d
8. To obtain a more complete picture of the biotransfor-
mation of isoprene and its metabolites, the mEH-
catalyzed hydrolysis of diepoxides 6 and epoxydiols 7 and
8 has been investigated. The experiments were carried
out by incubating the mixtures of the diastereoisomeric
diepoxides erythro-(()-6 and threo-(()-6, or of the dia-
steroisomeric epoxydiols erythro-(()-7 and threo-(()-7,
and erythro-(()-8 and threo-(()-8 (10-50 mM) with a
microsomal preparation from control or PB-induced rats
at 37 °C and in potassium phosphate buffer (pH 7.4)
containing 2.5 or 5 mg of protein/mL. The incubations
were stopped at several times (between 30 min and 6 h),
and the residual 6 or the formed epoxydiols 7 and 8 were
identified and quantified by GC, after addition of cyclo-
heptanediol as an internal standard. No biotransforma-
tion was observed in the cases of 7 and 8. The epoxydiols
were also quantitatively recovered after incubation for
6 h using PB-induced microsomes, showing that these
compounds were not substrates for the mEH. It is
noteworthy that, although lipophilic compounds are
generally the best substrates for mEH, epoxides bearing
a hydroxyl group may be hydrolyzed by this enzyme. The
four stereoisomers of 1,2-epoxyhexan-3-ol are not only
substrates for mEH but also biotransformed into the
corresponding triols with a high rate (13). The results
found in this work, related to the hydrolysis of epoxydiols
7 and 8, therefore suggest that the mEH-catalyzed
oxirane ring opening of epoxides bearing hydroxyl groups
is strongly dependent on the ability of the alkyl portion

Isoprene Monoepoxide Biotransformation
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 837
of the substituent to the oxirane ring to attenuate the
polar effect due to the OH group(s). This probably affects
the formation of the enzyme-substate complex which
arises from a delicate balance of entropic and enthalpic
factors (21).
Different from 7 and 8, diepoxides 6 were substrates
for mEH, although the biotransformation, which was not
under saturation conditions even at the high substrate
concentration (50 mM), occurred with a moderate rate.
The extent of product formation was indeed linear with
time, microsomal protein amount, and substrate concen-
tration, and a conversion higher than 20% has never been
obtained even after incubation for 6 h.
product (4), epoxide 2, is rapidly transformed into the
corresponding vicinal racemic diol 4, predominantly
through a nonenzymatic hydrolysis reaction. On the other
hand, epoxide 3, which is more stable toward the non-
enzymatic hydrolysis reaction, is biotransformed into the
diol 5 by microsomal epoxide hydrolase. The reaction
occurs with a fairly good substrate and product enantio-
selectivity favoring the hydrolysis of the (R)-enantiomer
to give, before 50% conversion, selectively (R)-3-methyl-
3-butene-1,2-diol, 5.
This hydrolysis reaction competes with the oxidation
of the monoepoxide 3 to the corresponding diepoxides 6.
When racemic compound 3 was used as a substrate, the
P450-catalyzed oxidation of the remaining double bond
is generally characterized by a moderate stereoselectivity
(substrate enantioselectivity, product diastereoselctivity,
and product enantioselectivity) which, however, strongly
depends on the composition of the P450 isoforms. Treat-
ment of rats with PB leads with a higher selectivity to
threo-(2R,2′R)-6, while with Pyr the formation of both
erythro-(2S,2′R)- and threo-(2R,2′R)-6 was favored.
The mEH-catalyzed hydrolysis of epoxides 6 proceeds,
although with a low turnover rate, with a substrate and
product diastereo- and enantioselection by nucleophilic
attack on the more substituted oxirane ring to give
selectively (2R,3S)-3,4-epoxy-2-methyl-1,2-diol (7).
Both diols 4 and 5 may be further oxidized by P450.
The epoxidation of the double bond, however, occurs
slowly, and the reaction is markedly dependent on P450
induction.
The reaction gave exclusively the epoxydiols 7 arising
from the selective opening of the more substituted
oxirane ring, and at 20% conversion, all four isomers of
7 were present in a 27:45:15:13 ratio.
At the present time, no data about the genotoxic
activities of the epoxydiols 7 and 8 are available, although
their activity is expected as seen for the butadiene
epoxydiol (11). Contribution to the overall toxicity of
isoprene in addition to other reactive intermediates and
in particular the diepoxide, a potent mutagen (7), may
be provided in particular by the epoxydiol 7. Indeed, only
this metabolite [namely, the (2R,3S)-3,4-epoxy-2-methyl-
1,2-diol] and not the epoxydiol 8 may be formed at a
consistent rate from the mEH-catalyzed hydrolysis of the
diepoxide, and it might represent an important toxic
metabolite of the isoprene biotransformation process as
in the case of butadiene (22). The formation of the
epoxydiols 7 and 8 would be possible through the P450-
dependent oxidation of the diols 5 and 6, but a significant
contribution of this pathway is not expected because of
the low rates observed in these enzymatic reactions.
Since the toxicity of many metabolites depends on their
stereochemistry, the results presented here, showing that
the stereochemical course of the isoprene biotrasforma-
tion is markedly affected by induction, suggest that
environmental or diet exposure to certain substances may
considerably modify the metabolic pathway of isoprene
and consequently the toxicity of this natural compound
largely produced by the petrochemical industry.
By co-injection of the reaction mixture product with
samples with a known configuration, obtained as re-
ported in Materials and Methods, it has been established
that the ratio between the diastereoisomeric pairs erythro
and threo was 70:30 and the reaction occurred with a
product selectivity favoring the formation of the enanti-
omer having the (R)-configuration at C-2.3 When the
regioselectivity generally observed in the mEH-catalyzed
oxirane ring opening of gem-dialkyl-substituted oxiranes
is considered (the selective attack occurs on the less
substituted carbon), the results presented here show that
of the two diastereoisomeric pairs of 6, the erythro
isomers are preferentially hydrolyzed with a moderate
selectivity in favor of the (2R,2′S)-enantiomer.
Con clu sion s
In conclusion, these results show that the stereochem-
ical course of the biotransformation of isoprene formu-
lates a composite picture markedly affected by the
composition of different P450 present in the control or
induced rat liver.
Ack n ow led gm en t. This work was supported by
grants from Consiglio Nazionale delle Ricerche (CNR,
Rome) and Universita` degli Studi di Pisa.
Between the two primarily formed metabolites, 2-meth-
yl-2-vinyloxirane (2) and isopropenyloxirane (3), the main
3
As a consequence of the formal change in the descriptor caused
Refer en ces
by the priority rule of Chan, Ingold, and Prelog, the 2R (or 2S)
stereochemistry of the 2-methyl-3-butene-1,2-diol becomes 2S (or 2R)
in 3,4-epoxy-2-methyl-1,2-diol and the 2R (or 2S) stereochemistry of
the 3-methyl-3-butene-1,2-diol becomes 2S (or 2R) in 3,4-epoxy-3-
methyl-1,2-diol.
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