1,3-dichloropropene epoxides: Intermediates in bioactivation of the promutagen 1,3-dichloropropene

Article abstract of DOI:10.1021/tx980126y

1,3-Dichloropropene (1,3-D), a major soil fumigant nematicide, is genotoxic in many types of assays, leading to its classification as possibly carcinogenic in humans. This study tests in three steps the hypothesis that 1,3-D is a promutagen activated by epoxidation and further reaction of the 1,3-D-epoxides. Stereospecific epoxidation of 1,3-D (examined as the cis/trans mixture and as individual isomers) to the corresponding cis- and trans-1,3-D-epoxides is demonstrated here for the first time, both in vitro in a mouse liver microsome-NADPH system and in vivo in the liver of ip- treated mice, using GC/MS for product identification and quantitation. The cis epoxide is observed in higher yield than the trans epoxide, both in vitro and in vivo, and the cis isomer also reacts slower than the trans isomer with GSH alone or catalyzed by GSH S-transferase. cis- and trans-1,3-D-Epoxides are stable in acetone or chloroform but degrade completely in Me₂SO exclusively to 2-chloroacrolein (30 min at 40 °C). Epoxide decomposition is slower in pH 7.4 phosphate buffer (t(1/2) = 116 and 64 min for cis and trans, respectively, at 41 °C) with a > 99% yield of 3-chloro-2-hydroxypropanal (and its dimer) and <0.5% formation of 2-chloroacrolein (for which the t(1/2) is 248 rain at 41 °C). Mutagenicity assays in Salmonella typhimurium TA100 (standard plate incorporation) establish high potencies of 37, 17, and 150 revertants/nmol for cis- and trans-1,3-D-epoxides and 2-chloroacrolein, respectively. The mutagenicity of the epoxides is due either to their direct action or to a degradation product formed at physiological pH, i.e., 3- chloro-2-hydroxypropanal or its dehydrochlorination products. The candidate mutagens methylglyoxal and glycidaldehyde are not detected as breakdown products of 3-chloro-2-hydroxypropanal at pH 7.4 and also have low mutagenic activity in TA100. It is therefore proposed that the penultimate and ultimate mutagens of 1,3-D metabolism are the corresponding epoxides and their direct hydrolysis product 3-chloro-2-hydroxypropanal, respectively.

Full text of DOI:10.1021/tx980126y

Chem. Res. Toxicol. 1998, 11, 1137-1144
1137
1,3-Dich lor op r op en e Ep oxid es: In ter m ed ia tes in
Bioa ctiva tion of th e P r om u ta gen 1,3-Dich lor op r op en e
Manfred Schneider,† Gary B. Quistad, and J ohn E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science,
Policy and Management, University of California, Berkeley, California 94720-3112
Received J une 1, 1998
1,3-Dichloropropene (1,3-D), a major soil fumigant nematicide, is genotoxic in many types
of assays, leading to its classification as possibly carcinogenic in humans. This study tests in
three steps the hypothesis that 1,3-D is a promutagen activated by epoxidation and further
reaction of the 1,3-D-epoxides. Stereospecific epoxidation of 1,3-D (examined as the cis/trans
mixture and as individual isomers) to the corresponding cis- and trans-1,3-D-epoxides is
demonstrated here for the first time, both in vitro in a mouse liver microsome-NADPH system
and in vivo in the liver of ip-treated mice, using GC/MS for product identification and
quantitation. The cis epoxide is observed in higher yield than the trans epoxide, both in vitro
and in vivo, and the cis isomer also reacts slower than the trans isomer with GSH alone or
catalyzed by GSH S-transferase. cis- and trans-1,3-D-Epoxides are stable in acetone or
chloroform but degrade completely in Me2SO exclusively to 2-chloroacrolein (30 min at 40 °C).
Epoxide decomposition is slower in pH 7.4 phosphate buffer (t1/2 ) 116 and 64 min for cis and
trans, respectively, at 41 °C) with a >99% yield of 3-chloro-2-hydroxypropanal (and its dimer)
and <0.5% formation of 2-chloroacrolein (for which the t1/2 is 248 min at 41 °C). Mutagenicity
assays in Salmonella typhimurium TA100 (standard plate incorporation) establish high
potencies of 37, 17, and 150 revertants/nmol for cis- and trans-1,3-D-epoxides and 2-chloro-
acrolein, respectively. The mutagenicity of the epoxides is due either to their direct action or
to a degradation product formed at physiological pH, i.e., 3-chloro-2-hydroxypropanal or its
dehydrochlorination products. The candidate mutagens methylglyoxal and glycidaldehyde are
not detected as breakdown products of 3-chloro-2-hydroxypropanal at pH 7.4 and also have
low mutagenic activity in TA100. It is therefore proposed that the penultimate and ultimate
mutagens of 1,3-D metabolism are the corresponding epoxides and their direct hydrolysis
product 3-chloro-2-hydroxypropanal, respectively.
(6), induces DNA fragmentation in rat liver, gastric
mucosa, and kidney (7), and is genotoxic and cytotoxic
in cultured rodent and human cells, including metaboli-
cally deficient V79 Chinese hamster lung cells (8). There
is also a possible relationship between human exposure
to 1,3-D and the development of hematologic malignan-
cies (9).
In tr od u ction
1,3-Dichloropropene (1,3-D)1 is a preplant fumigant
used as a nematicide since 1943 (1) at rates of 75-200
lb/acre in the production of more than 40 different crops
(2). Its use in California was suspended in 1990 and
resumed in 1995 because alternative fumigants (methyl
bromide and metam sodium) were less effective or
projected to be phased out soon (3). In 1995, 1,3-D
ranked in the top five conventional pesticides with the
38-43 million pounds used in U.S. agricultural produc-
tion (4). The more active cis-1,3-D (>95% cis) is used in
The Netherlands as an improved formulation (5).
Three primary pathways are proposed for 1,3-D me-
tabolism in mammals. The first is cytochrome P450-
dependent epoxidation to cis- and trans-1,3-dichloropro-
pene epoxides (cis- and trans-1,3-D-epoxides) (Figure 1),
consistent with a common pathway for chloroalkenes (10,
11) but only recently demonstrated with 1,3-D (12). The
second is P450 oxidation to 3-chloroacrolein (13). The
third involves GSH conjugation at the chloromethyl
moiety leading to excretion of mercapturate metabolites
of 1,3-D in human urine (11, 14). The GSH/GSH S-
transferase (GST) conjugation pathway is the major
elimination route in mammals (2, and references therein).
1,3-D is classified as possibly carcinogenic in humans
on the basis of extensive genotoxicity studies by the
National Toxicology Program (2). It induces sister chro-
matid exchanges with and without metabolic activation
* To whom correspondence should be addressed. Telephone: (510)
642-5424. Fax: (510) 642-6497. E-mail: ectl@nature.berkeley.edu.
† Present address: Zeneca Ag Products, Western Research Center,
1200 S. 47th St., Richmond, CA 94804.
1,3-D-containing pesticides appeared to be direct-acting
mutagens in the TA100 and TA1535 strains of Salmo-
nella typhimurium (15), but this was due in TA100 to
polar impurities (16) subsequently identified as cis- and
trans-1,3-D-epoxides formed by autoxidation in equal
amounts (17). The 1,3-D-epoxide impurities must be
removed prior to studies of intrinsic genotoxicity and
1 Abbreviations: CI, chemical ionization; 1,3-D, 1,3-dichloropropene;
1,3-D-epoxides, 1,3-dichloropropene epoxides; GST, GSH S-transferase;
imp, solvent impurity; is, internal standard which is chlorobenzene;
MG, methylglyoxal; mEH, rat liver microsomal epoxide hydrolase;
RRT, relative retention time or kcompound/kis, where the capacity factor
k is (tR - tO)/tO and tO is the gas holdup time; S9 fraction, postmito-
chondrial supernatant fraction; sEH, rat liver soluble epoxide hydro-
lase; SIM, selected ion monitoring.
10.1021/tx980126y CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/02/1998

1138 Chem. Res. Toxicol., Vol. 11, No. 10, 1998
Schneider et al.
F igu r e 1. Metabolic epoxidation of 1,3-D isomers and hydrolysis of the epoxides to 3-chloro-2-hydroxypropanal proposed to be the
principal bioactivation sequence. The other dehydrochlorination, rearrangement, and dimerization reactions illustrated are less
important or not relevant under biological conditions. Major conjugation reactions of 1,3-D with GSH, mostly leading to detoxification,
are not shown. Reaction conditions were as follows: (a) cytochrome P450, (b) hydrolysis possibly by epoxide hydrolase, (c) reversible
dimerization (at high concentrations), (d) dehydrochlorination but not in pH 7.4 buffer, and (e) rearrangement and dehydrochlorination
in Me2SO but not in pH 7.4 buffer.
performed as follows: Hewlett-Packard 5890 gas chromatograph
coupled with a 5971A mass spectrometer in the chemical
ionization (CI) mode using methane; HP-1701 capillary column
(30 m × 0.32 mm i.d., 0.25 µm film thickness); helium carrier
gas at a constant rate of 33 cm/s; inlet at 250 °C and oven
program from 40 to 250 °C at 20 °C/min. CI/MS selected ion
monitoring (SIM) was based on the most abundant masses,
including the chlorine isotope ratios, i.e., [MH]+ and [M - Cl]+.
All quantitative analyses were in the linear range (r >0.99) of
the MS detector versus chlorobenzene as an internal standard
(is). These GC conditions gave tR values in minutes (and relative
retention times or RRT) of 3.71 (0.684) for cis-1,3-D, 4.08 (0.806)
for trans-1,3-D, 4.72 (1.000) for the internal standard, 5.23
(1.166) for cis-1,3-D-epoxide, and 5.63 (1.284) for trans-1,3-D-
epoxide.
metabolic activation and other toxicological investiga-
tions. Purified 1,3-D is not active in TA100 except on
P450 activation by rat liver postmitochondrial superna-
tant (S9 fraction) or microsomes with the putative
mutagenic metabolite(s) proposed to be 1,3-D-epoxide (17)
or 3-chloroacrolein (13). cis- and trans-1,3-D-Epoxides
do not require metabolic activation for mutagenic activity
in S. typhimurium TA100 (17) and TA1535 where the
cis isomer is twice as potent as the trans isomer (10).
The genotoxicity of cis- and trans-1,3-D-epoxides is also
evident from neoplastic transformations and increased
cell lethality in Syrian hamster cells (18) and the induc-
tion of squamous carcinomas in mouse skin (19).
This study focuses on the formation and toxicological
relevance of cis- and trans-1,3-D-epoxides from three
standpoints: direct evidence for stereoselective epoxida-
tion of 1,3-D in vitro and in vivo using the mouse as a
mammalian model, the decomposition of 1,3-D-epoxides
with special emphasis on possible formation of 2-chloro-
acrolein (a potent mutagen; 20) on rearrangement in Me2-
SO and 3-chloro-2-hydroxypropanal in aqueous medium,
and the mutagenic activity in S. typhimurium TA100.
Ch em ica ls. (A) 1,3-D a n d 1,3-D-Ep oxid es. 1,3-D from
Pfaltz & Bauer (Waterbury, CT) was 92% pure (1:1 cis:trans
determined by GC/MS). MS: m/z 111, 113, 115 [MH]+, 75, 77
[M - Cl]+. This sample contained 0.2% cis- and 0.2% trans-
1,3-D-epoxides (GC/MS). cis-1,3-D (99% pure) was from Supelco
(Bellefonte, PA) and trans-1,3-D (>96% pure) from TCI America
(Portland, OR). Oxidation of 1,3-D preparations with 3-chlo-
roperoxybenzoic acid (10) was used to prepare cis/trans-1,3-D-
1
epoxides (>96% pure by H NMR, 1:1 cis:trans by GC/MS and
1H NMR), cis-1,3-D-epoxide (80% cis, 6% trans; prepared from
80-90% cis-1,3-D), and trans-1,3-D-epoxide (97% pure by 1H
NMR). MS data were identical for cis- and trans-1,3-D-
epoxides: m/z (relative intensity) 127, 129, 131 (53, 36, 6 [MH]+),
91, 93 (100, 33 [M - Cl]+). 1H NMR (CDCl3): cis-1,3-D-epoxide,
δ 3.79 (2H, m), 3.33 (1H, m), 5.20 (1H, d); trans-1,3-D-epoxide,
δ 3.60 (2H, m), 3.42 (1H, m), 5.01 (1H, d). 13C NMR (acetone-
d6): cis-1,3-D-epoxide, δ 69.97 (ClCO), 55.83 (CCH2Cl), 42.21
(CH2Cl); trans-1,3-D-epoxide, δ 68.08 (ClCO), 59.48 (CCH2Cl),
Ma ter ia ls a n d Meth od s
Ca u tion : 1,3-D, 1,3-D-epoxides, 2-chloroacrolein, 3-chloro-
acrolein, and methylglyoxal are hazardous chemicals and must
be used under containment conditions.
Sp ectr oscop y a n d Ch r om a togr a p h y. NMR spectra were
determined with a Bruker AM300 spectrometer at 300 and 75
MHz for 1H and 13C, respectively. GC/MS measurements were

Bioactivation of 1,3-Dichloropropene
Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1139
42.59 (CH2Cl). The epoxides are stable without solvent for
months at 4 °C and in acetone or chloroform for several days at
25 °C.
(B) P r od u cts in p H 7.4 Bu ffer . A mixture of the 1,3-D-
epoxides (200 mg) and 100 mM phosphate buffer (pH 7.4, D2O,
500 µL) was kept for up to 7 days at 25 °C. At 3 days, the
epoxide had completely reacted and dissolved and no further
13C NMR spectral changes were observed.
(C) Rea ction Ra tes w ith GSH a n d GST. cis/trans-1,3-D-
Epoxides or 2-chloroacrolein (0.1 mM final concentration) added
in acetone (50 µL) to 100 mM phosphate buffer (pH 7.4, 1 mL)
with or without GSH (5 mM) and rat liver GST (200 µg of
protein; Sigma, St. Louis, MO) were incubated for 1, 30, 60, 90,
200, 325, and 425 min at 41 °C. They were analyzed by GC/
MS using 25 µL aliquots added to 5 mL of acetone containing 1
µg of internal standard. Rate constants were calculated as-
suming first-order reaction kinetics (r > 0.97).
(B) 2-Ch lor oa cr olein . 2-Chloroacrolein can be synthesized
by chlorination of acrolein and then dehydrochlorination of 2,3-
dichloropropanal with diethylaniline (21, 22). We find that
2-chloroacrolein is conveniently prepared by stirring a solution
of the 1,3-D-epoxides in Me2SO (5:1, v/v) for 60 min at 40 °C
(the reaction is complete within 30 min) and then distillation
(60 mm/50 °C) (60% yield, 92% purity by 1H NMR). MS: m/z
(relative intensity) 91, 93 (100, 33 [MH]+). 1H NMR (Me2SO-
d6): δ 9.42 (1H, s), 6.58 (1H, d), 6.40 (1H, d) (see also ref 23).
13C NMR (Me2SO): δ 186.78 (CHO), 140.10 (CCl), 134.81 (dCH2).
Me2SO and traces of dimethyl sulfide are the only impurities.
(D) In ter a ction w ith Ep oxid e Hyd r ola se. Rat liver
microsomal epoxide hydrolase (mEH) and soluble epoxide
hydrolase (sEH) were expressed in insect cells using recombi-
nant baculovirus (28) and assayed with selective substrates (50
µM): [3H]-cis-stilbene oxide for mEH in 100 mM Tris-HCl buffer
(pH 9, 100 µL) and [3H]-trans-stilbene oxide for sEH in 100 mM
phosphate buffer (pH 7.4, 100 µL) (29). Bovine serum albumin
(10 µg) was present in the buffer to control nonspecific reactions,
and enzyme levels were adjusted (by dilution of the crude
extract) to 2.1 (mEH) and 0.9 (sEH) nmol of diol formed min-1
mL-1, providing a linear relationship between the enzyme level
and diol yield in the standard 5 min assay. The potential
inhibitory effect of cis/trans-1,3-D-epoxides (1 mM final concen-
tration) was then examined as a function of preincubation time
(1-20 min) before [3H]stilbene oxide addition.
Mu ta gen icity of 1,3-D-Ep oxid es. S. typhimurium TA100,
which detects mutagens that cause base pair substitutions, was
used by the standard plate incorporation method, including all
required controls (30). cis- and trans-1,3-D-Epoxides were added
in acetone (100 µL) and 2-chloroacrolein in Me2SO (100 µL)
using a 2-fold dilution series. In some tests, washed microsomes
like those described above (0.64 mg of protein) and NADPH (1.5
mg) were added, each in 100 µL of 100 mM phosphate buffer
(pH 7.4). cis/trans-Diallate (40 nmol) (from Chem Service, West
Chester, PA) was used as a positive control in this activation
assay, giving 0 or 1000 revertants assayed directly or with
microsomes and NADPH, equivalent to about ∼15% conversion
of diallate to 2-chloroacrolein (20). The effect of GSH and/or
GST on the mutagenic activity of cis- and trans-1,3-D-epoxides
and 2-chloroacrolein was tested by addition of rat liver GST as
described above (10 µg in 100 µL of buffer) and varying levels
of GSH in buffer (100 µL). The spontaneous revertants (averag-
ing 180 per plate) were subtracted. Each assay dose involved
duplicate plates, and each experiment was carried out three
times. The mutagenicity values were determined from the
slopes of the linear dose-response range and are given in
revertants per nanomole of compound with standard deviations.
(C) 3-Ch lor oa cr olein . The procedure of Basu and Marnett
(24) was used to prepare an ether solution of 3-chloroacrolein
(20% cis, 80% trans as determined by GC/MS; same as the
reported ratio). MS data were identical for cis- and trans-3-
chloroacrolein (and 2-chloroacrolein): m/z (relative intensity) 91,
93 (100, 33 [MH]+). The RRTs compared with the internal
standard on the HP-1701 column are 0.592, 0.635, and 0.654
for 2-chloroacrolein and cis- and trans-3-chloroacrolein, respec-
tively; i.e., they can be easily separated for analysis.
(D) Meth ylglyoxa l. The 40% (w/w) aqueous solution of
methylglyoxal (MG) (Aldrich, Milwaukee, WI) was 92% pure
with ∼1:1 monohydrate and dihydrate (25) and 8% pyruvic acid
(1H NMR). 13C NMR (pH 7.4, 100 mM phosphate, trace of the
Me2SO standard): MG monohydrate, δ 210.61 (C-2), 91.55
(C-1), 23.59 (C-3); MG dihydrate, δ 93.98 (C-1), 83.64 (C-2), 26.22
(C-3).
Meta bolic Ep oxid a tion of 1,3-D. Male albino Swiss-
Webster mice (22-27 g) were obtained from Charles River
Laboratories (Wilmington, MA).
(A) In Vitr o. Mouse liver microsomes (2.6 mg of protein)
(26) were incubated with 1,3-D [0.1 mM cis or trans or a 0.2
mM cis/trans mixture; added in Me2SO (5 µL)], NADPH (0 or 1
mM), and GSH (0 or 5 mM) in 100 mM phosphate buffer (pH
7.4, 505 µL) for 15 min at 37 °C. The incubated mixtures were
spiked with 2 µg of internal standard (in 20 µL of ethyl acetate)
and immediately extracted with ethyl acetate (500 µL). Two
extractions led to the recovery of >95% of the internal standard,
and a third extraction did not produce additional 1,3-D-epoxides.
The extracts were combined and dried over sodium sulfate.
Aliquots (2 µL) were analyzed (GC/MS) for cis/trans-1,3-D and
cis/trans-1,3-D-epoxides.
(B) In Vivo. The 1,3-D-epoxides were analyzed in liver at
various times up to 150 min after ip treatment with 1,3-D (350
mg/kg for individual isomers or 700 mg/kg for cis/trans-1,3-D).
The carrier solvent was Me2SO, and 1,3-D solutions were held
for at least 4 h at 25 °C before the experiment to degrade any
epoxide impurities that might be present (see above). Some
mice were pretreated with metribuzin (300 mg/kg), a potent
GSH depletor (27), 90 min before 1,3-D administration. The
liver was homogenized in 100 mM sodium phosphate buffer (pH
7.4, 2 mL) containing ∼0.2 g of NaCl, and then ethyl acetate (2
mL) containing 2 µg of internal standard was added and the
mixture homogenized and centrifuged at 10000g for 30 min. The
organic layer was removed, and the pellet was extracted again
with ethyl acetate but without internal standard. Recoveries
for cis/trans-1,3-D and cis/trans-1,3-D-epoxides were 81-95%,
and a third extraction with ethyl acetate did not increase the
recoveries; hexane or dichloromethane for extraction gave poor
recoveries. The ethyl acetate extracts were analyzed as above.
Resu lts
Meta bolic Ep oxid a tion of 1,3-D. (A) Id en tifica -
tion of Ep oxid e Meta bolites. cis- and trans-1,3-D-
epoxides are identified as metabolites of the respective
1,3-D isomers in mice both in vitro (microsomes and
NADPH) and in vivo (liver) by GC/MS (Figure 2). The
structural assignments are based on (1) identical tR and
RRT values compared with standards, (2) detection of
both [MH]+ and [M - Cl]+, and (3) the expected isotope
ratios for two chlorines (m/z 127, 129, 131 in a 9:6:1 ratio
for [MH]+ and one chlorine m/z 91, 93 in a 3:1 ratio for
[M - Cl]+). 2-Chloroacrolein and cis- and trans-3-
chloroacrolein (easily separated from the other com-
pounds) were not detected as in vitro or in vivo metabo-
lites of cis- and trans-1,3-D.
Rea ction s of 1,3-D-Ep oxid es. (A) P r od u cts in Me2SO.
The conversion of the 1,3-D-epoxides in Me2SO-d6 to 2,3-
dichloropropanal and 2-chloroacrolein was monitored by 1H
NMR at 22 °C by integration of the epoxide proton at the
terminal carbon, which gives a well-isolated doublet for both
epoxide isomers. The rate constants were determined for
degradation of the epoxides to 2,3-dichloropropanal. 13C NMR
(Me2SO): δ 193.55 (C-1), 63.01 (C-2), 43.75 (C-3).
(B) Liver Micr osom es. Microsomal metabolism of
cis- and trans-1,3-D examined as individual isomers is
enhanced by addition of NADPH and further enhanced

1140 Chem. Res. Toxicol., Vol. 11, No. 10, 1998
Schneider et al.
F igu r e 3. Stereospecific epoxidation of cis-1,3-D in vitro in the
mouse microsome-NADPH system and trans-1,3-D in vivo in
mouse liver (4 min after treatment). The same stereospecificity
is observed for trans-1,3-D in vitro and cis-1,3-D in vivo.
Analyses were as described in the legend of Figure 2.
F igu r e 2. Detection of cis- and trans-1,3-D-epoxides as me-
tabolites of cis/trans-1,3-D (1:1 ratio) in vitro in the mouse liver
microsome-NADPH system and in vivo in mouse liver (150 min
after treatment). Analyses were by GC/MS.
Ta ble 1. Mou se Liver Micr osom a l Meta bolism of cis/
tr a n s-1,3-D a n d th e Effect of NADP H a n d GSH
recovery (%) with the indicated addition
compound
none
NADPH
NADPH and GSH
cis-1,3-D
parent
100a
<0.1
<0.1
89 ( 6
7 ( 1b
<0.1
26 ( 7
∼1
epoxide
acroleinsc
trans-1,3-D
parent
<0.1
100a
<0.1
<0.1
80 ( 5
2 ( 1b
<0.1
33 ( 5
<0.1
<0.1
epoxide
acroleinsc
a
Control normalized to 100%. Other values are mean ( SD (n
b
) 6). Molar amount relative to control. No cis/trans intercon-
version observed. c Includes 2-chloroacrolein and cis- and trans-
3-chloroacrolein.
by GSH with little isomeric specificity (Table 1). With
NADPH addition, the cis epoxide is recovered in 7% yield
versus 2% with the trans epoxide. The presence of GSH
(5 mM) greatly decreases the epoxide yield, but the cis
epoxide is still clearly evident, suggesting a higher rate
of formation or stability for the cis compared to the trans
epoxide (Table 1). Metabolism of cis/trans-1,3-D (1:1
ratio) leads to recovery of cis- and trans-1,3-D-epoxides
in an ∼4:1 ratio (Figure 2). The metabolism is stereo-
specific, converting cis-1,3-D to only cis-1,3-D-epoxide and
trans-1,3-D to only trans-1,3-D-epoxide (Figure 3).
(C) Liver in Vivo. Analysis of the liver 150 min after
treatment with cis/trans-1,3-D (1:1 ratio) reveals the
parent 1,3-D (with a cis:trans ratio of ∼1:2) and the
epoxide metabolites (with a cis:trans ratio of ∼4:1)
(Figure 2). As in the in vitro experiment, the metabolism
is stereospecific (Figure 3).
F igu r e 4. Levels of cis- and trans-1,3-D and their epoxides in
the liver of individual mice at various times after ip treatment
with cis/trans-1,3-D (1:1 ratio) at 700 mg/kg. Values with error
bars are for cis-1,3-D at 350 mg/kg administered 90 min after
metribuzin at 300 mg/kg (n ) 3).
and then their decay apparently follows first-order kinet-
ics with calculated half-lives of 36 and 50 min for cis-
and trans-1,3-D, respectively. The epoxide concentra-
tions are approximately 2 orders of magnitude lower than
those of the parent 1,3-D isomers. cis-1,3-D-Epoxide
reaches a maximum of ∼1 ppm at 30 min and is at a
The time-dependent concentration profiles of the par-
ent cis/trans-1,3-D or cis-1,3-D and their epoxide me-
tabolites are shown in Figure 4. The 1,3-D isomers reach
their maximum level in liver at ∼10 min after treatment,

Bioactivation of 1,3-Dichloropropene
Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1141
F igu r e 5. Increasing percentage of epoxide metabolites in liver
at various times after ip treatment with cis/trans-1,3-D
(1:1 ratio) at 700 mg/kg. Data for 1,3-D and epoxide levels are
from Figure 4.
F igu r e 7. 13C NMR spectrum showing 3-chloro-2-hydroxypro-
panal (signals a-c as a dimer and a′-c′ as a monomer) with a
trace amount of 2-chloroacrolein (signals d-f) formed on com-
plete hydrolysis of cis/trans-1,3-D-epoxides in pH 7.4 phosphate
buffer. Compounds not detected as hydrolysis products are 2,3-
dichloropropanal and methylglyoxal (see the text for NMR
chemical shifts).
Ta ble 2. Rea ction Ra tes of cis/tr a n s-1,3-D-Ep oxid es a n d
2-Ch lor oa cr olein in p H 7.4 Bu ffer Alon e or w ith GSH a n d
GST
a
rate constant k (×10-3 min-1
)
substrate
buffer
GSH
14 ( 1
25 ( 3
GSH and GST
cis-1,3-D-epoxide
trans-1,3-D-epoxide 10.8 ( 0.8
2-chloroacrolein
6.0 ( 0.6
920 ( 100
1380 ( 350
>9900b
2.8 ( 0.2 >9900b
a
Numbers must be multiplied by 10-3 to give the actual rate
b
constants. Minimal value based on the fastest analysis time.
F igu r e 6. Partial 1H NMR spectra showing 2,3-dichloropro-
panal at 155 min and 2-chloroacrolein at 280 min from rear-
rangement and dehydrochlorination of cis/trans-1,3-D-epoxides
in Me2SO-d6 at 22 °C.
corresponding to half-lives of 17 ( 2 and 32 ( 3 min,
respectively. Calculation from these rate constants of the
amount of trans-1,3-D-epoxide left after 155 min results
in an estimated residual of 3%, consistent with Figure
6.
level significantly higher than that of the trans isomer
10 min or more after treatment. The GSH depletor
metribuzin appears to elevate the amount of cis-1,3-D
at 30 and 90 min after treatment without significantly
altering the cis-1,3-D-epoxide levels (Figure 4). The
percentage of epoxide metabolites increases with time
after treatment, interestingly with a strong linear cor-
relation for each isomer; the slope is 9 times greater for
the cis than the trans epoxides (Figure 5).
(B) P r od u cts in p H 7.4 Bu ffer . 3-Chloro-2-hydroxy-
propanal hydrate and its dimer 2,5-dihydroxy-3,6-bis-
(chloromethyl)-1,4-dioxane in a ∼30:70 ratio are the only
major products after complete degradation of cis/trans-
1,3-D-epoxides in pH 7.4 buffer as is clearly evident by
13C NMR analysis (Figure 7). The 13C NMR chemical
shifts for the dimer are consistent with literature values
(31). 13C NMR (D2O buffer): 3-chloro-2-hydroxypropanal
(hydrate), δ 86.07 (C-1), 59.86 (C-2), 43.00 (C-3). The 1H
NMR signals of the monomer and dimer overlap suf-
ficiently that they are not individually assigned. 2-Chlo-
roacrolein is detectable in a trace amount (<0.5%) by 1H
NMR.
R ea ct ion s of 1,3-D-E p oxid es a n d 2-Ch lor oa c-
r olein . (A) P r od u cts in Me2SO. cis/trans-1,3-D-Ep-
oxides in Me2SO-d6 at 22 °C decompose completely to
2-chloroacrolein within 280 min as conveniently moni-
1
tored by H NMR (Figure 6). The Ha signal of cis-1,3-
D-epoxide disappears faster than that of the trans isomer.
2,3-Dichloropropanal is evident at 10 min (protons Hb and
Hc), is present in an amount equal to that of 2-chloro-
acrolein at 155 min, and is completely converted to
(C) Rea ction Ra tes w ith GSH a n d GST. GC/MS
analysis was used to determine the stability of the 1,3-
D-epoxide isomers and 2-chloroacrolein under biomimetic
conditions. The rate constant in pH 7.4 buffer at 41 °C
decreases in the order trans epoxide > cis epoxide >
2-chloroacrolein with about a 2-fold difference in each
case (Table 2). GSH increases the rate constants of the
epoxides about 2-fold, and GST further facilitates the
breakdown about 60-fold, with trans-1,3-D-epoxide al-
ways being more reactive than the cis isomer. GSH
greatly increases the reaction rate of 2-chloroacrolein
from a half-life of 248 ( 17 min in buffer to complete
2-chloroacrolein (proton Hd signal) after 280 min.
A
transitory but unidentified aldehyde (H?) is evident at
155 but not 280 min. There is no further change between
280 min and 7 days, indicating the stability of 2-chloro-
acrolein in Me2SO.
The first-order rate constants (min-1) for the rate-
limiting rearrangement step of cis- and trans-1,3-D-
epoxides at 22 °C are 0.041 ( 0.05 and 0.022 ( 0.002,

1142 Chem. Res. Toxicol., Vol. 11, No. 10, 1998
Schneider et al.
F igu r e 8. Inhibition of epoxide hydrolase activity by 1 mM
cis/trans-1,3-D-epoxides. mEH and sEH are rat liver microsomal
and soluble epoxide hydrolases, respectively. Data are mean and
SD values (n ) 3).
Ta ble 3. Mu ta gen icity in S. typh im u r iu m TA100
F igu r e 9. Mutagenicity of cis- and trans-1,3-D-epoxides and
2-chloroacrolein in S. typhimurium TA100.
revertants/nmola
compound
direct
<10-3
S9 or microsome-NADPH
1,3-D
cisb
0.02
1,3-D-epoxides
cis
trans
2-chloroacrolein
3-chloroacrolein
methylglyoxal
glycidaldehyde
37 ( 5
30 ( 3
14 ( 3
17 ( 3
150 ( 15c
<10-3 d
1e (3.6)f
0.1g (0.2)h
a
Standard plate incorporation procedure if not indicated oth-
erwise. Data given as mean ( SD are from this study using
b
microsomes. Calculated from ref 17 using S9. c Values of 113-
d
135 for direct and 49-74 with S9 from refs 32 and 33. Estimated
from ref 24. e From ref 34. f Preincubation test from ref 35.
g
h
Calculated from ref 36. Preincubation test from ref 36.
reaction within 30 s (the fastest analysis time) (i.e.,
>3500 times faster) (Table 2).
F igu r e 10. Effect of GSH and GST on the mutagenicity of cis-
and trans-1,3-D-epoxides and 2-chloroacrolein in S. typhimu-
rium TA100. All test compounds were present in 25 nmol
amounts. GSH at 400 molar equiv represents 5 mM in the top
agar. The following symbols are used: 9 and 0, 2-Cl-A (2-
chloroacrolein); [ and ], cis epoxide; b and O, trans epoxide;
dashed lines, with GST; and solid lines, no GST.
(D) In ter a ction w ith Ep oxid e Hyd r ola se. cis/
trans-1,3-D-Epoxides (1 mM) reduce the hydrolysis rate
of cis-stilbene oxide by mEH with progressively less effect
during a preincubation time of up to 20 min (Figure 8),
indicating that the mEH preparation almost completely
hydrolyzes the reacting 1,3-D-epoxide isomer(s) within
this time period. The sEH assays indicate that under
the test conditions it is less effective than mEH in
overcoming the inhibitory effect of the 1,3-D-epoxides
(Figure 8).
Discu ssion
Ep oxid a tion Is a Min or bu t Sign ifica n t P a th w a y
in 1,3-D Meta bolism . 1,3-D-Epoxides are unequivocally
established here for the first time as metabolites of 1,3-D
in a microsomal oxidase system [confirming the earlier
suggestion of Watson et al. (17) based on mutagenicity
studies] and additionally in vivo in the liver of 1,3-D-
treated mice (Figure 1). This P450-catalyzed oxidation
is stereospecific both in vitro and in vivo. cis- and trans-
1,3-D-Epoxides are at least 25 times more stable at pH
7.4 and 37 °C (t1/2 ) 288-300 min) than four similar
ethene and propene epoxides (10, 18, 31). The epoxide
stability is reflected in observing them here as 1,3-D
metabolites. Controversial putative or proposed metabo-
lites of 1,3-D are 2-chloroacrolein (Figure 1) (37) and
3-chloroacrolein (13), neither of which is observed in this
investigation.
Mu ta gen icity of 1,3-D-Ep oxid es. (A) Dir ect Activ-
ity. cis-1,3-D-Epoxide is 2-fold more potent than trans-
1,3-D-epoxide, and both isomers are >104 more potent
than cis-1,3-D (Table 3). The value of 17-37 revertants/
nmol for the 1,3-D-epoxide isomers is 11-25% of the
potency of 2-chloroacrolein (Figure 9). The S9 fraction
or microsomes with NADPH increase the potency of cis-
1,3-D, have little effect on that of the 1,3-D-epoxides, and
reduce the potency of 2-chloroacrolein. The reported
activities of 3-chloroacrolein, methylglyoxal, and glycid-
aldehyde are very low relative to that of cis- and trans-
1,3-D-epoxides (Table 3).
(B) Effect of GSH a n d GST (F igu r e 10). GSH at
2-400 molar equiv produces a dose-dependent decrease
in the mutagenicity for all compounds but even at the
highest level does not destroy but only ameliorates the
mutagenicity of cis-1,3-D-epoxide and 2-chloroacrolein.
GST appears to increase the effectiveness of GSH in
reducing the mutagenic potency of cis- and trans-1,3-D-
epoxides and of 2-chloroacrolein.
It has been shown that occupational exposure to 1,3-D
can result in a significant decrease in the blood level of
GSH and, additionally, GST activity (38). This and
several other reports (2) clearly show that the principal
pathway of 1,3-D metabolism is conjugation with GSH.
This reaction is strongly enhanced by GST (39). cis-1,3-D

Bioactivation of 1,3-Dichloropropene
Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1143
levels dissipate ∼2-fold faster than trans-1,3-D in mouse
liver in vivo (this study), a rate difference consistent with
findings using GSH and rat liver cytosol containing GST
(39). Mouse microsome-NADPH metabolism of cis- and
trans-1,3-D is greatly increased on adding GSH, and less
epoxide is formed, probably due mostly to 1,3-D conjuga-
tion by GSH. The epoxides themselves react very slowly
with GSH under physiological conditions (only 2-fold
faster than in buffer alone) but 60-fold faster on GST
addition in this study on reactivity. These findings and
the effect of GSH depletion with metribuzin indicate that
the GSH/GST pathway is much more important in
degrading the 1,3-D than the 1,3-D-epoxide isomers.
1,3-D-Ep oxid es Hyd r olyze to 3-Ch lor o-2-h yd r oxy-
p r op a n a l. 1,3-D-Epoxides undergo diverse hydrolysis,
dehydrochlorination, rearrangement, and dimerization
reactions, with those that are relevant under biological
conditions being of the greatest interest (Figure 1). We
find that the major reaction of the 1,3-D-epoxides at pH
7.4 and 25 °C is hydrolysis to 3-chloro-2-hydroxypropanal
(along with its dimer at high concentrations), and there
is essentially no formation (<0.5%) of 2-chloroacrolein (at
25 and 41 °C), confirming one earlier study at 20 °C (31)
but not another one at 37 °C (40). Other candidate
hydrolysis products not detected under these biomimetic
conditions are 2,3-dichloropropanal and methylglyoxal.
The degradation products of the 1,3-D-epoxides are
greatly influenced by the reaction solvent. The epoxides
are stable in acetone and chloroform (this investigation);
2,3-dichloropropanal is formed by partial rearrangement
in buffer containing 50% tetrahydrofuran (31), and
2-chloroacrolein is a degradation product of cis-1,3-D-
epoxide in Me2SO (17) and in fact is the sole end product
from degradation of cis- and trans-1,3-D-epoxides in this
solvent (this investigation). The solvent properties (par-
ticularly polarity and protic/aprotic characteristics) clearly
affect the dissipation of the 1,3-D-epoxides.
ruled out. Glycidaldehyde and methylglyoxal are known
mutagens, but they are not formed from the epoxides
under biomimetic conditions and have low mutagenic
activity relative to that of the epoxides. The prime
candidates are therefore the epoxides themselves or
3-chloro-2-hydroxypropanal. cis-1,3-D-Epoxide is 2-fold
more potent than its trans isomer in the TA100 mutage-
nicity assay, yet both hydrolyze completely to 3-chloro-
2-hydroxypropanal. This can be rationalized as both the
epoxides and the hydrolysis product have similar activi-
ties or the trans-1,3-D-epoxide is hydrolyzed more rapidly
and the hydrated aldehyde penetrates the bacteria more
slowly. The chirality of 3-chloro-2-hydroxypropanal may
also be an important aspect since the S-enantiomer is
an antifertility agent in many species and the R-enan-
tiomer [on further oxidation to the corresponding acid,
(R)-3-chlorolactate] is a renal toxicant (41). Structural
identification of four adducts formed on reaction of 1,3-
D-epoxides with deoxyguanosine in buffer (42)2 strongly
supports 3-chloro-2-hydroxypropanal as the ultimate
genotoxic agent because the adduct structures clearly
show that hydrolysis of the epoxides is occurring before
the aldehyde moiety of the resultant 3-chloro-2-hydroxy-
propanal is reacting at the guanine base.
Ack n ow led gm en t. This work was supported by
Grants P01 ES00049 and R01 ES04863 from the Na-
tional Institute of Environmental Health Sciences (NIE-
HS), National Institutes of Health (NIH), and its contents
are solely the responsibility of the authors and do not
necessarily represent the official views of the NIEHS,
NIH. The epoxide hydrolase inhibition assays were
performed in collaboration with C. H. Morrisseau and B.
D. Hammock at the University of California at Davis.
We thank B. N. Ames and C. Wehr (Department of
Molecular and Cell Biology, University of California at
Berkeley) for advice and assistance in the mutagenesis
assays.
1,3-D-Epoxides are potential substrates for epoxide
hydrolase which would presumably increase their rate
of conversion to 3-chloro-2-hydroxypropanal. Competi-
tion studies reported here indicate that mEH hydrolyzes
cis/trans-1,3-D-epoxide(s) but are less clear in defining
the role of sEH or the isomeric specificity.
Mu ta gen ic Activity of 1,3-D Th a t Ca n Be At-
tr ibu ted to 1,3-D-Ep oxid es or 3-Ch lor o-2-h yd r oxy-
p r op a n a l. The mutagenic activity of 1,3-D involves
oxidative bioactivation to either the epoxides (Figure 1)
or 3-chloroacrolein (by oxidation at the methylene group)
(13). The latter proposal is based on the increased
mutagenicity with cyanamide as an aldehyde dehydro-
genase inhibitor (13) but is not supported by the lack of
activity of 3-chloroacrolein in the TA100 assay (24) and
the failure to detect it as a metabolite in this study. It
is therefore proposed that epoxidation is the first step in
the mutagenic activation of 1,3-D, and if the formation
of an aldehyde is relevant (13), it must occur in the
subsequent fate of the 1,3-D-epoxides.
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TX980126Y