Kinetic Modeling of
-Chloroprene Metabolism: I. In vitro Rates in Liver
and Lung Tissue Fractions from Mice, Rats, Hamsters, and Humans
Matthew W. Himmelstein,1 Steven C. Carpenter, and Paul M. Hinderliter
E.I. du Pont de Nemours and Company, Haskell Laboratory for Health and Environmental Sciences,
PO Box 50, 1090 Elkton Road, Newark, Delaware 19711
Received October 18, 2003; accepted January 26, 2004
Beta-chloroprene (2-chloro-1,3-butadiene, CD) is carcinogenic
by inhalation exposure to B6C3F1 mice and Fischer F344 rats but
not to Wistar rats or Syrian hamsters. The initial step in metab-
olism is oxidation, forming a stable epoxide (1-chloroethenyl)ox-
irane (1-CEO), a genotoxicant that might be involved in rodent
tumorigenicity. This study investigated the species-dependent in
vitro kinetics of CD oxidation and subsequent 1-CEO metabolism
by microsomal epoxide hydrolase and cytosolic glutathione S-
transferases in liver and lung, tissues that are prone to tumor
induction. Estimates for Vmax and Km for cytochrome P450-
dependent oxidation of CD in liver microsomes ranged from 0.068
to 0.29
mol/h/mg protein and 0.53 to 1.33
M, respectively.
Oxidation (Vmax/Km) of CD in liver was slightly faster in the
mouse and hamster than in rats or humans. In lung microsomes,
Vmax/Km was much greater for mice compared with the other
species. The Vmax and Km estimates for microsomal epoxide
hydrolase activity toward 1-CEO ranged from 0.11 to 3.66
mol/
h/mg protein and 20.9 to 187.6
M, respectively, across tissues and
species. Hydrolysis (Vmax/Km) of 1-CEO in liver and lung micro-
somes was faster for the human and hamster than for rat or
mouse. The Vmax/Km in liver was 3 to 11 times greater than in
lung. 1-CEO formation from CD was measured in liver micro-
somes and was estimated to be 2–5% of the total CD oxidation.
Glutathione S-transferase-mediated metabolism of 1-CEO in cy-
tosolic tissue fractions was described as a pseudo-second order
reaction; rates were 0.0016–0.0068/h/mg cytosolic protein in liver
and 0.00056–0.0022 h/mg in lung. The observed differences in
metabolism are relevant to understanding species differences in
sensitivity to CD-induced liver and lung tumorigenicity.
Key Words: 2-chloro-1,3-butadiene; microsomes; cytosol; liver;
lung; mouse; rat; hamster; human; in vitro kinetics.
Beta-chloroprene (2-chloro-1,3-butadiene, CD, CAS 126-
99-8) is a volatile colorless liquid used to manufacture poly-
chloroprene, a synthetic rubber (Lynch, M. A., 2001). Occu-
pational exposure occurs during monomer synthesis, shipping,
and polymerization processes, and inhalation is the only sig-
nificant route of exposure (Lynch, J., 2001). Cancer epidemi-
ology studies have been inconclusive (Acquavella and Leo-
nard, 2001; Colonna and Laydevant, 2001; Zaridze et al.,
2001).
The need to understand possible adverse health effects in
humans has led to extensive evaluation of CD in experimental
animals. Recent literature reviews have described the acute,
subchronic, and chronic toxicity (Melnick and Sills, 2001;
Valentine and Himmelstein, 2001). The most significant find-
ing was CD-induced tumorigenicity in F344/N rats and
B6C3F1 mice exposed to 80 ppm for 2 years (Melnick et al.,
1999, 1996; NTP 1998). Tumors in Fischer rats included the
lung, oral cavity, thyroid gland, kidney, and mammary gland.
Mouse tumors were observed in the lung, circulatory system,
Harderian gland, forestomach, kidney, mammary gland, skin,
mesentery, Zymbal gland, and liver. In contrast, no tumors
were observed in Syrian hamsters. A weak response in mam-
mary tissue was observed in female Wistar rats exposed to 10
or 50 ppm CD for up to 2 years (Trochimowicz et al., 1998).
In vitro genotoxicity studies revealed that CD is mutagenic,
primarily in bacterial reverse mutation assays with metabolic
activation, while other in vitro and in vivo assays for either
gene mutation or structural chromosomal damage were nega-
tive (Drevon and Kuroki, 1979; Foureman et al., 1994; NTP,
1998; Tice, 1988; Tice et al., 1988; Vogel, 1979). A recent
study showed that a known CD metabolite, racemic (1-chlo-
roethenyl)oxirane (1-CEO), was mutagenic in three Salmonella
typhimurium strains (Himmelstein et al., 2001b).
The in vitro biotransformation of CD has been studied to
identify metabolites that might contribute to toxicity or muta-
genicity (Bartsch et al., 1979). More recently, the microsomal
metabolism of CD to 1-CEO was confirmed as a cytochrome
P450-dependent reaction (Himmelstein et al., 2001a), and the
S- and R-enantiomers of 1-CEO were identified (Cottrell et al.,
2001). Enzyme-mediated hydrolysis of 1-CEO occurred by
microsomal epoxide hydrolase (Cottrell et al., 2001; Himmel-
stein et al., 2001a). Conjugation of 1-CEO with GSH has been
shown in liver cytosolic fractions (Himmelstein et al., 2001a),
consistent with earlier in vivo studies showing depletion of
1 To whom correspondence should be addressed. Fax: (302) 366-5003.
E-mail: matthew.w.himmelstein@usa.dupont.com.
Toxicological Sciences vol. 79 no. 1 © Society of Toxicology 2004; all rights
reserved.
TOXICOLOGICAL SCIENCES 79, 18–27 (2004)
DOI: 10.1093/toxsci/kfh092
Advance Access publication February 19, 2004
18

nonprotein sulfhydryls and the presence of thioethers in urine
of rodents exposed to CD (Jaeger et al., 1975; Plugge and
Jaeger, 1979; Summer and Greim, 1980).
The goal of this research was to quantify the in vitro rates of
CD-related biotransformation reactions in liver and lung tissue
fractions of the rodent strains tested in the cancer bioassays and
humans. Liver and lung were studied, because these tissues
were prone to tumor induction and represented tissues impor-
tant to xenobiotic metabolism. The simultaneous time course
of cytochrome P450-dependent oxidation of CD was compared
with rates of 1-CEO formation and hydrolysis, using a two-
compartment kinetic model to account for the volatility of both
analytes.
MATERIALS AND METHODS
Chemicals.
-Chloroprene (
99%) containing phenothiazine and N-ni-
trosodiphenylamine inhibitors was supplied by DuPont-Dow Elastomers LLC
(LaPlace, LA). The inhibitors were removed as previously described (Him-
melstein et al., 2001a). The synthesis of (1-chloroethenyl)oxirane (1-CEO,

98%) was previously described (Himmelstein et al., 2001a). Both the CD
and 1-CEO were stored under nitrogen headspace at -70°C and 20°C,
respectively. Other chemicals used were of the highest purity available.
Source of microsomes and cytosol. Rodent liver microsomes and cytosol
were purchased from two suppliers (In Vitro Technologies, Baltimore, MD or
XenoTech, Lenexa, KS). Both suppliers used differential centrifugation as the
method of preparation. Microsomes and cytosol were prepared from pooled
livers from male B6C3F1 mice, Fischer F344 rats, Wistar rats, or Golden
Syrian hamsters obtained from Charles River Laboratories (Raleigh, NC).
Rodent lung microsomes and cytosol were prepared in-house using animals
obtained from Charles River Laboratories. The fractions were prepared by
differential centrifugation as previously described (Csana´dy et al., 1992;
Guengerich, 1994). All fractions were stored at -70°C.
Human tissue fractions were also prepared by differential centrifugation. For
experiments involving hydrolysis of 1-CEO, pooled liver microsomes from 15
individuals were used (Lot #1032, In Vitro Technologies). Experiments in-
volving simultaneous CD and 1-CEO measurements used pooled liver micro-
somes from 10 individuals (Lot# 0010186, H1000, XenoTech). Human lung
microsomes were obtained from a pool of five individuals (Cat # HPM 501,
Lot 1.0, Human Biologics International, Scottsdale, AZ). Human liver cytosol
was obtained from a mixed pool of 15 individuals (Lot# HHC 280, Tissue
Transformation Technologies, Edison, NJ). Human lung cytosol was from a
single male (Lot # HG714100, Tissue Transformation Technologies). Human
donor demographic information and metabolic characterization is available but
not reported here for the sake of brevity.
Microsomal hydrolysis of 1-CEO. The time course of 1-CEO metabolism
was measured in liver and lung microsomes from each species by further
refinement of the method described in Himmelstein et al. (2001a). Vials (1 ml
liquid/10 ml vial) were prepared with 0.1 M phosphate buffer (pH 7.4), MgCl2,
(15 mM), EDTA (0.1 mM), glucose-6-phosphate (0.8 mM), and glucose-6-
phosphate dehydrogenase (1 U/ml). The vials were sealed with crimp top,
Teflon
-coated silicone septa (Gerstel US, Baltimore MD) with a ParafilmTM
sheet between the metal crimp top and septa and plastic tape around the outside
rim of the crimp top to insure a positive seal. Incubations were performed using
a programmable x-y-z robotic multi-purpose gas chromatograph (GC) sampler
(MPS2, Gerstel, Baltimore, MD). Sample vials were transferred to the MPS2
agitator-heater block and preheated for 11 min at 500 rpm. Known concentra-
tions of 1-CEO (prepared in Tedlar bags) were added by manually flushing the
vial headspace with approximately 15 ml of vapor (see Fig. legends for actual
concentrations used). The headspace concentration was immediately quantified
by robotic transfer from the vial to the GC inlet, using the MPS2 1.0 ml
gas-tight syringe. The optimal syringe temperature (38°C), sample injection
volume (200 l), and injection speed (800 l/s) were pre-determined experi-
mentally. The protein concentration (0.25–3 mg/ml) and duration of incubation
(0–60 min) were also optimized during preliminary experiments. Microsomal
protein for the definitive experiments (10–50 l to final concentration of 0.5
mg/ml) was manually added to the vial to start the enzymatic time course
reactions. Between injections, the headspace-sampling syringe was flushed
with helium (at 48.5°C for 3 min) to clear residual chemical. Prior to the next
sample (t  12 min), an injection of air was made to check for residual
chemical, which if present, was subtracted from the next headspace sample.
The air, sample injections, and syringe flushes were repeated (t  12, 24, 36,
48, and 60 min) to determine the time course of metabolism. Control incuba-
tions included phosphate buffer or heat-inactivated microsomes. The concen-
tration of 1-CEO in vial headspace was calculated using linear regression
analysis from gasbag standards. Headspace GC-mass spectrometry (GC/MS)
analysis by single ion monitoring (m/z 39/1-CEO) was conducted as described
previously (Himmelstein et al., 2001a). The limit of quantification of 1-CEO
(and CD in subsequent experiments described below) was 0.001 nmol/ml of
headspace (equivalent to 24 ppb).
Microsomal oxidation of 1-CEO. Vials were prepared with incubation
reagents as described above. Cofactor NADP was added to the vial 10 s
before the addition of microsomal protein. Time-course data for 1-CEO, with
and without NADP, were measured over a range of starting 1-CEO concen-
trations. In mouse liver microsomes, the only species to show significant
metabolism, additional incubations were conducted in the presence of 4-meth-
ylpyrazole (4-MP) or 1-aminobenzotriazole (ABT) as inhibitors of cytochrome
P450-dependent oxidation (Halpert et al., 1994). Stock solutions (100 mM) of
4-MP in phosphate buffer or ABT in acetonitrile were added to a final
concentration of 100 M; control incubation mixtures included equal amounts
of buffer or acetonitrile. Additional incubations were conducted to isolate the
potential oxidative metabolite of 1-CEO. Incubation mixtures containing
mouse liver microsomes were agitated in a Bucher shaker (Labconco, Lenexa,
KS) at 37°C with 1-CEO (10 mM) in 10 ml total volume in sealed 24 ml vials,
with or without NADP. After 30 min, the reactions were stopped by the
addition of cold ethyl acetate (10 ml) and NaCl (5 g/vial). Following centrif-
ugation (1700  g for 5 min) and extraction with ethyl acetate (10 ml, then 5
ml), the solvent was analyzed by full scan GC/MS. Instrument conditions were
similar to those used previously (Cottrell et al., 2001).
Cytosolic GSH conjugation of CD or 1-CEO. Preliminary experiments
showed no reactivity of CD with GSH in cytosol or heat-inactivated cytosol.
For 1-CEO, the incubation vials were prepared as described above for the
microsomal 1-CEO hydrolysis experiments, except that cytosolic protein from
each species (1–2 mg protein/ml) and GSH (10 mM) were used. Headspace
samples (400 l) were taken at 0, 12, 24, 36, 48, and 60 min, after the reactions
were started by the addition of cytosol and GSH. Control incubations in
phosphate buffer were conducted using 1-CEO alone, 1-CEO  GSH, and
1-CEO  GSH  heat-inactivated cytosol.
Microsomal oxidation of CD. The time course of CD disappearance and
1-CEO formation and hydrolysis was measured simultaneously in liver or lung
microsomes. For liver microsomal incubations from each species, vials were
prepared with the incubation reagents described above for microsomal 1-CEO
hydrolysis. After preincubation (37°C for 5 min), an equal volume of vial
headspace was removed from the vial and replaced with known concentrations
of CD vapor. The vial was equilibrated for approximately 10 min, and
reactions were started by the addition of microsomal protein (1 mg/ml) and
NADP (0.4 mg/ml of suspension). The protein concentration was optimized
during preliminary experiments (0.5–3.0 mg/ml). Control incubations were
performed without NADP or with NADP and heat-inactivated microsomes.
Incubation start times were staggered, and up to five vials per exposure
concentration were used to describe the time course of headspace 1-CEO
relative to the disappearance of CD from the headspace. The repeated head-
space samples (400 l, 4–6 injections per vial) were analyzed at 12-min
intervals for up to 1 h by GC/MS. For lung microsomes, the incubation
conditions were identical to those used for liver microsomes except for the
19
-CHLOROPRENE IN VITRO METABOLISM KINETICS