Chemico-Biological Interactions 166 (2007) 93–103
Metabolism of 1,3-butadiene to toxicologically relevant
metabolites in single-exposed mice and rats
Johannes Georg Filser a,b,∗ , Christoph Hutzler b , Veronika Meischner a ,
Vimal Veereshwarayya a , György András Csanády a,b
a
Institute of Toxicology, GSF-National Research Center for Environment and Health, D-85764 Neuherberg, Germany
b Institut für Toxikologie und Umwelthygiene, Technische Universität München, München, Germany
Available online 17 April 2006
Abstract
1,3-Butadiene (BD) was carcinogenic in rodents. This effect is related to reactive metabolites such as 1,2-epoxy-3-butene (EB)
and especially 1,2:3,4-diepoxybutane (DEB). A third mutagenic epoxide, 3,4-epoxy-1,2-butanediol (EBD), can be formed from
DEB and from 3-butene-1,2-diol (B-diol), the hydrolysis product of EB. In BD exposed rodents, only blood concentrations of EB and
DEB have been published. Direct determinations of EBD and B-diol in blood are missing. In order to investigate the BD-dependent
blood burden by all of these metabolites, we exposed male B6C3F1 mice and male Sprague-Dawley rats in closed chambers over
6–8 h to constant atmospheric BD concentrations. BD and exhaled EB were measured in chamber atmospheres during the BD
exposures. EB blood concentrations were obtained as the product of the atmospheric EB concentration at steady state with the
EB blood-to-air partition coefficient. B-diol, EBD, and DEB were determined in blood collected immediately at the end of BD
exposures up to 1200 ppm (B-diol, EBD) and 1280 ppm (DEB). Analysis of BD was done by GC/FID, of EB, DEB, and B-diol by
GC/MS, and of EBD by LC/MS/MS. EB blood concentrations increased with BD concentrations amounting to 2.6 mol/l (rat) and
23.5 mol/l (mouse) at 2000 ppm BD and to 4.6 mol/l in rats exposed to 10000 ppm BD. DEB (detection limit 0.01 mol/l) was
found only in blood of mice rising to 3.2 mol/l at 1280 ppm BD. B-diol and EBD were quantitatively predominant in both species.
B-diol increased in both species with the BD exposure concentration reaching 60 mol/l at 1200 ppm BD. EBD reached maximum
concentrations of 9.5 mol/l at 150 ppm BD (rat) and of 42 mol/l at 300 ppm BD (mouse). At higher BD concentrations EBD
blood concentrations decreased again. This picture probably results from a competitive inhibition of the EBD producing CYP450
by BD, which occurs in both species.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: 1,3-Butadiene; 1,2-Epoxy-3-butene; 3-Butene-1,2-diol; 3,4-Epoxy-1,2-butanediol; 1,2:3,4-Diepoxybutane; Mouse; Rat; Inhalation
1. Introduction
1,3-Butadiene (BD) is a major industrial chemical,
the worldwide demand of which was almost 9 million
∗ Corresponding author at: Institute of Toxicology, GSF-National
Research Center for Environment and Health, Ingolstädter Landstrasse
1, D-85764 Neuherberg, Germany. Tel.: +49 89 3187 2977;
fax: +49 89 3187 3449.
E-mail address: johannes.filser@gsf.de (J.G. Filser).
metric tons in 2003 [1]. The gaseous olefin is primarily used in the production of synthetic rubbers and of
plastics as “shock-resistant polystyrene” (a two-phase
system made of polystyrene and polybutadiene), ABS or
MBS, copolymers of BD with acrylonitrile and styrene
and with methyl methacrylate and styrene, respectively.
BD is found ubiquitously in the environmental atmosphere, mainly because it is formed in combusting processes and during burning of organic material [2]. BD
concentrations in urban air generally range from less
0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2006.03.002
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J.G. Filser et al. / Chemico-Biological Interactions 166 (2007) 93–103
than 1 to 10 ppb [3]. The USEPA [4] reports for the
USA in 1994 average BD concentrations of 0.18 (rural
area), 0.45 (suburban area) and 0.62 ppb (urban area).
In rodents, inhaled BD caused dose-dependent increases
of tumors at various sites. Mice were far more sensitive
than rats [5–9]. In order to understand this species difference, metabolism of BD was investigated in several
laboratories. In both rodent species, BD is metabolized
extensively. A considerable number of metabolic intermediates has been detected in exhaled air, blood and
urine (summarized in [2,4]). These include the three
mutagenic epoxides 1,2-epoxy-3-butene (EB), its oxidation product 1,2:3,4-diepoxybutane (DEB), and 3,4epoxy-1,2-butanediol (EBD). The latter can be formed
by hydrolysis of DEB or by oxidation of 3-butene-1,2diol (B-diol), the hydrolytic product of EB (Fig. 1). All
four metabolites have been found in BD perfused livers of mice and all but EBD in BD perfused livers of
rats [10]. In vivo, however, only EB and DEB have
been measured in blood of both species when exposed
to BD ([11–14]: EB in both species and DEB in mice;
[13–16]: DEB in rats). Because of the relevance of these
metabolites for the species-specific toxicity and carcinogenicity of BD, it was the aim of the present study to
quantify the blood burden not only by EB and DEB
but especially by EBD and B-diol in male SpragueDawley rats and B6C3F1 mice single exposed up to 6 h to
constant atmospheric BD concentrations between 0 and
1200 ppm.
2. Material and methods
2.1. Chemicals
All commercial chemicals were purchased with
the highest purity available from Ridel-de-Haën,
Seelze, Germany, and Merck, Darmstadt, Germany.
Liquemin® N25000 (Heparin-Natrium) was obtained
from Hoffmann-La Roche, Grenzach-Wyhlen, Germany, and BD 99.5% from Linde, München, Germany. EB 98%, DEB 97%, 1,2-epoxybutane 99+%,
n-butylboronic acid 97% and 4-benzylpiperidine 99%
were from Sigma–Aldrich, Steinheim, Germany. Several compounds were obtained by custom synthesis: B-diol 99% and EBD 98% from EMKA-Chemie,
Neufahrn, Germany, EBD 98%, DEB-D6 98%, Bdiol-D8 98%, and EBD-D6 (3,4-epoxy-[1,1,2,3,4,42 H ]butane-1,2-diol) 98% from Synthon, Augsburg,
6
Germany. Handling of all chemicals during different sample preparations was carried out under the
hood.
2.2. Animals
Male Sprague-Dawley rats (200–300 g) and male
B6C3F1 mice (17–25 g) were purchased from Charles
River Wiga GmbH, Sulzfeld, Germany. All experimental procedures with animals were performed in
conformity with the “Guide for the care and use of
Fig. 1. Simplified scheme of the biotransformation of 1,3-butadiene (BD) showing the metabolites investigated.
J.G. Filser et al. / Chemico-Biological Interactions 166 (2007) 93–103
laboratory animals” (7th edition, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, National Academy
Press Washington, DC, 1996) under the surveillance
of the authorized representative for animal welfare of
GSF. Up to 1 week before use, two rats or five mice
were housed in the GSF-Institute of Toxicology in a
macrolon type III cage placed in an IVC top-flow system (Tecniplast, Buguggiate, Italy). This system provided the animals with filtered room air. A constant
12-h light/dark cycle was maintained in the chamber room. Animals had free access to standard chow
(Nr. 1324 from Altromin, Lage, Germany) and tap
water.
2.3. Exposure experiments
Animals were exposed to BD in closed all-glass
chambers under atmospheric pressure and room temperature, similarly as described earlier [17]. Two rats
or 5 mice were exposed together up to 8 h to constant
BD concentrations of 1, 5, 10, 30, 100, 300, 500, 1000,
2000, 3000, 6000, 10000 ppm (rats) and of 1, 5, 10, 30,
100, 300, 1000, 2000, 4000, 6000 ppm (mice) in closed
chambers of 6.5 l containing 35 g soda lime (Drägersorb
800; Drägerwerk, Lübeck, Germany) as CO2 adsorbent.
After establishing defined initial BD concentrations in
the chambers, the BD concentrations were maintained
in a pre-given range of ±10% by repeatedly re-injecting
gaseous BD to replenish the exposure-dependent losses
of BD in the gas-tight chambers. These losses resulted
only from BD uptake by the exposed animals as was
verified in chambers containing no animals but only
BD-gas and soda lime (35 g). Measurements of BD in
the chamber atmospheres were repeated in time periods between 8 and 23 min, depending on the exposure
concentration.
During the exposure experiments, the atmospheric
chamber concentration of exhaled EB was measured at
selected time points. With increasing time of BD exposure, EB increased in the chamber atmosphere until a
plateau concentration was reached. At such a steadystate condition, the air concentration of the endogenously
formed EB was in thermodynamic equilibrium with its
concentration in venous blood, and the rate of its endogenous formation equaled the rate of its metabolic elimination. Another possible way of EB elimination from
the chamber air, the reaction with soda lime (35 g), was
considered negligible. This reaction had been studied
in an exposure chamber containing EB vapor and fresh
or used (moistened) soda lime because soda lime could
hydrolyze the epoxide with different rates dependent on
95
its H2 O and CO2 content. The shortest elimination halflife was obtained with moistened soda lime. Because
it was longer than 2 days, the influence of soda lime
on the EB concentration in the chamber atmosphere
was not taken into account, and the EB concentration
in venous blood [mol/l] was calculated as the product
of the EB air concentration at plateau [mol/l] with the
blood-to-air partition coefficient of EB (83.4, measured
in rat blood [18]). For converting the EB air concentration from ppm to mol/l, the air concentration in ppm
was divided by 25.13, the molar volume of an ideal gas
at 25 ◦ C and 740 Torr. This procedure for determining
venous blood concentrations of an internally produced,
exhaled gas from its plateau concentrations in the atmosphere of a closed chamber principally yields higher
blood concentrations than actually exist in open systems,
where no re-inhalation occur. However, the difference
between the venous blood concentrations at steady state
in a closed and in an open system is less than 10% (investigated by physiological toxicokinetic modeling), provided that the blood-to-air partition coefficient is higher
than 50.
For direct measurements of DEB, B-diol, and EBD
in blood of BD exposed rodents, another closed exposure system was used. It consisted of a glass-sphere
(63 l), equipped with an 8 cm long neck (inner diameter 15 cm) closed by a round lid with 3 ports. Two ports
were closed by Teflon coated synthetic rubber septa;
the third one was connected to a passive oxygen supply system described in [19]. The sphere contained a
circular, foldable floor plate of solvent-inert polyvinylidene fluoride with a diameter of 40 cm. Exhaled CO2
was trapped by 80 g soda lime that was below the
floor plate. The BD concentrations in the chamber were
maintained constant (maximum coefficient of variation ±9%) by injecting BD gas repeatedly through one
of the septa into the chamber air in order to compensate for the loss of BD by metabolism. The target BD concentrations in the atmospheres were 0, 60,
100 (mice only), 150 (rats only), 300, 600, 900, and
1200 ppm BD for the determination of EBD and Bdiol with the exception that B-diol was not quantified
in rats exposed to 300 ppm BD. For the determination of DEB, exposure concentrations of BD were up
to 900 ppm in rats and 0, 67, 630, and 1270 ppm in
mice. For each BD concentration, two rats or 3–16
mice were exposed together for 6.0 h. Immediately thereafter, animals were sacrificed by cervical dislocation,
the ribcages were opened, and blood was taken from
the vena cava near to the heart using a disposable,
Liquemin® moistened syringe. This procedure lasted
less than 1 min.
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J.G. Filser et al. / Chemico-Biological Interactions 166 (2007) 93–103
For rats, the blood of two animals was pooled and
about 1 ml was used for B-diol, 1 ml for DEB, and 6 ml
for EBD analysis. For mice, blood of 12 animals (6 ml)
was pooled for one EBD determination and of 2 animals (1 ml) for one B-diol determination. Between 0.3
and 0.7 ml blood was obtained from each of three mice
simultaneously exposed to one BD concentration for the
individual determination of DEB. Blood was collected
in ice-cold 6 ml screw capped glass centrifuge tubes or
1.5 ml Eppendorf cups, each of which containing 10 l
of an ethanolic diethyl maleate solution (1 mol/l ethanol)
per ml of blood to be added. After immediate addition of
the internal standards (see below), centrifuge vials were
closed and the exact blood volumes were determined by
weighing. Thereafter, blood samples were either immediately analyzed or stored at −80 ◦ C until use. The analytes remained stable over the required time frame as had
been verified experimentally.
For safety reasons, all exposure experiments were
conducted under the hood.
2.4. Analysis of 1,3-butadiene
After intensive mixing the chamber air using a disposable syringe of 100 ml, air samples of 25 l or 10 ml
were taken repeatedly from the exposure chamber via
a Teflon® coated rubber septum using a gastight 25 l
syringe (RNS, series 1700, Hamilton, Bonaduz, Swizerland) or disposable syringe of 10 ml and injected on
column of a GC-8A gas chromatograph (Shimadzu,
Duisburg, Germany) equipped with a flame ionization
detector, either directly or via a gas sample loop (5 ml).
Separation was done at 155 ◦ C on a stainless steel
column (3.5 m × 1/8′′ × 2 mm) packed with Tenax TA
60–80 mesh (Chrompack, Frankfurt, Germany). Detector temperature was 200 ◦ C. Gas flows were: carrier
gas (nitrogen) 13.3 ml/min, hydrogen 60 ml/min, and
synthetic air 600 ml/min. Under these conditions, the
retention time of BD was 2.0 min. Chromatograms were
recorded and integrated by a C-R5A integrator (Shimadzu, Duisburg, Germany). Calibration curves were
constructed several times by generating BD gas concentrations ranging from 1 to 10000 ppm in atmospheres of
closed desiccators. Calibration curves were linear in the
whole range. Analysis of linear regression through the
origin revealed correlation coefficients of at least 0.997
between peak areas and atmospheric BD concentrations.
Each time before starting a BD exposure, a one-point
calibration was carried out in the concentration range
used in the actual experiment. The limit of detection
was not quantified because the smallest BD concentration of 1 ppm was more than 2 orders of magnitude
above the background noise when using the gas sample
loop.
2.5. Analysis of 1,2-epoxy-3-butene
EB concentrations in chamber air samples were quantified using a GC/MSD system from Agilent, Waldbronn, Germany (gas chromatograph HP 5890 Series
II Plus and mass selective detector HP 5972) equipped
with the Thermal Desorption Cold Trap Injector CP
4010 (Chrompack, Frankfurt, Germany). A chamber
air sample of 10 ml collected by means of a gas-tight
syringe (series 1010, Hamilton, Bonaduz, Switzerland)
was manually injected into the injector that contained a
CP Sil 5 CB capillary trap (length 30 cm, ID 0.53 mm;
Chrompack, Frankfurt, Germany). During the injection period (about 3 min) and up to 2 min thereafter,
the Cold Trap injector was cooled with liquid nitrogen to −100 ◦ C. The flow of the carrier gas helium
through the injector was 20 ml/min. Then the injector
was heated immediately to 200 ◦ C and analytes were
transferred to the separation column (PoraPlot U, length
25 m, ID 0.32 mm, equipped with a 2.5 m long particle trap; Chrompack, Frankfurt, Germany). Helium flow
was 2.4 ml/min. Column temperature was initially 50 ◦ C
for 1.0 min and then raised to 170 ◦ C with a rate of
30 ◦ C/min. The final temperature was held for 8 min
before cooling down the column again. The temperature of the GC/MSD interface was 280 ◦ C. The electron
ionization potential of the MSD was 70 eV. Retention
time of EB following transfer of the trapped material to the separation column decreased with increasing
water content of the air sample from 8.2 to 7.8 min. EB
was detected at BD exposure concentrations ≥100 ppm
in the scan mode (identified using the Wiley 138 MS
library) and at lower BD concentrations in the single
ion-monitoring (SIM) mode at m/z 69 [(M − 1)+ ] and
m/z 39 [(M - H2 COH)+ ]. Generally, EB was quantified
using m/z 69. Three calibration curves were constructed
over a concentration range from 0 ppb to 10 ppm using
14 different EB concentrations. Linear regression analysis through the origin revealed correlation coefficients
of at least 0.999 between peak areas and EB concentrations. Before each BD experiment for the determination
of EB, a three-point calibration curve through the origin
was constructed and an EB standard concentration, similar to the actual exhaled EB concentration, was prepared
in a desiccator and then repeatedly measured during the
exposure period. The detection limit for atmospheric
EB was 3 ppb for m/z 69 and 1 ppb for m/z 39 at an
injection volume of 10 ml and a signal-to-noise ratio
of 3:1.
J.G. Filser et al. / Chemico-Biological Interactions 166 (2007) 93–103
2.6. Analysis of 1,2:3,4-diepoxybutane
The detailed analytical procedure is given in [20]. The
internal standard DEB-D6 was immediately added to the
blood sample. After extraction with dichloromethane,
4 l were injected onto a pre-column (fused silica capillary, deactivated) protected HP-5MS capillary column
(30 m × 0.25 mm I.D.; film thickness 1.0 m; Agilent,
Waldbronn, Germany) in a gas chromatograph HP 6890
equipped with the mass selective detector HP 5973
(Agilent, Waldbronn, Germany). Analytes were detected
in positive chemical ionization mode using ammonia
as reagent gas. In the SIM mode, ions of m/z 104
[DEB + NH4 + ] and 110 [DEB-D6 + NH4 + ] were monitored for quantitative analysis. Racemic DEB was separated from the meso-form. The sum of both diastereomers was used to quantify DEB. The detection limit (3
times background noise) was 10 nmol DEB/l blood.
2.7. Analysis of 3-butene-1,2-diol
The detailed analytical procedure is given in [20].
Aliquots of blood were immediately spiked with the
internal standard (B-diol-D8) before erythrocytes were
removed by centrifugation. The supernatant was treated
with acetonic n-butylboronic acid (4 mg per ml acetone).
The obtained butylboronate of B-diol was extracted with
ethyl acetate; 4 l thereof were injected onto a HP-5MS
capillary column (30 m × 0.25 mm I.D.; film thickness
1.0 m) in a gas chromatograph HP 6890 equipped with
the mass selective detector HP 5973. Detection was in
the positive chemical ionization mode with methane as
reagent gas. In the SIM mode, the (M + H)+ ions of the
derivatized B-diol (m/z = 155) and B-diol-D8 (m/z = 161)
were chosen for identification. B-diol-D8 was also quantified using the ion of m/z 161. Because the ion of m/z 155
showed a disturbing peak in contrast to the less specific
ion of m/z 109, the latter was chosen for B-diol quantification. The detection limit (3 times background noise)
for B-diol was 20 nmol/l blood.
2.8. Analysis of 3,4-epoxy-1,2-butanediol
Aliquots of blood (6 ml) were immediately spiked
with the internal standard (EBD-D6) before erythrocytes were removed by centrifugation (5 min at 4000 g).
Potassium carbonate (3 g) was added to the mixture
of 3 ml supernatant, 27 ml isopropanol and 3 ml chloroform. After 5 min of vigorous shaking by hand,
phase separation was achieved by centrifugation (5 min,
4000 g). The organic (upper) layer, warmed to 45 ◦ C
by means of a metal block thermostat, was evapo-
97
rated under a stream of nitrogen. The residue containing EBD and EBD-D6 was derivatized for 1 h
at room temperature with 500 l of a methanolic 4benzylpiperidine solution (2 mg/ml). After the addition of 250 l acetonitrile, the samples were stored for
30 min on ice and then centrifuged (5 min, 4000 g). An
aliquot (5 l) of the supernatant containing the EBD
derivative 4-(4-benzylpiperidin-1-yl)-butane-1,2,3-triol
(C16 H25 NO3 , exact mass: 279.18 amu) and the corresponding derivative for EBD-D6 (C16 H19 D6 NO3 , exact
mass: 285.22 amu) was injected into an LC/MS/MS system, which consisted of an HP1100 LC, an autosampler
HP1100 equipped with a 100-well plate holder, and a
column oven (all from Agilent, Waldbronn, Germany),
an LC column Luna 150 × 2 mm I.D., 5 m, C18(2)
from Phenomenex, Aschaffenburg, Germany, and an
API 3000 triple quadrupole mass spectrometer with
a turbo ion spray interface from Applied Biosystems,
Darmstadt, Germany. Compounds were eluted within
7.5 min at 24 ◦ C with a flow of 250 l/min using an
isocratic mobile phase of 5 mmol/l aqueous ammonium
acetate (adjusted to pH = 4 with acetic acid):methanol
(60:40, v/v). The mass spectrometer was operated in
the multiple reaction monitoring mode and the turbo
ion spray source at a temperature of 350 ◦ C in the positive ion mode at an ion spray voltage of 4100 V. Nitrogen was used as curtain gas (setting 9), nebulizing gas
(setting 8) and collision gas (setting 4). The collision
energy was set at 38. The declustering potential and
focusing potential voltages were set at 40 and 150 V,
respectively. Unit resolution (at half peak height) was
used for both Q1 and Q3. Following HPLC separation, the peak area corresponding to the m/z 280 → 188
reaction ([M + H]+ - C3 H7 O3 ; dwell time 100 ms) for
the EDB-derivative was measured relative to that of
the m/z 286 → 190 reaction ([M + H]+ - C3 H3 D4 O3 ;
dwell time 100 ms) of the internal standard. The peak
areas of the m/z 280 → 202 and 280 → 91 reactions
(dwell time 100 ms for both) were used as qualifiers
for the EBD derivative and those of the m/z 286 → 205
and 286 → 91 reactions (dwell time 100 ms for both)
as qualifiers for the EBD-D6 derivative. For data processing, the software Analyst 1.3 (Applied Biosystems,
Darmstadt, Germany) was used. The detection limit
(3 times background noise) for EBD was 9.6 nmol/l
blood. Under analytical conditions, no separation of the
diastereomers was achieved. For every exposure experiment, a three-point calibration covering the expected
EBD concentration was performed. Analysis of linear
regression through the origin revealed correlation coefficients of at least 0.995 between peak areas and EBD
concentrations.
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J.G. Filser et al. / Chemico-Biological Interactions 166 (2007) 93–103
Fig. 2. Representative concentration-time courses of 1,3-butadiene
(BD) and 1,2-epoxy-3-butene (EB) measured in a closed chamber containing two rats exposed to an average BD concentration of 2000 ppm.
Arrows indicate the time points at which BD was administered into the
chamber air.
3. Results and discussion
A representative figure of the methodological procedure for maintaining BD exposure concentrations constant in the exposure chamber is given in Fig. 2. The
declines of the BD concentrations due to uptake by the
two exposed rats become obvious from the repeated mea-
surements. The arrows on the timeline represent the time
points at which defined amounts of BD gas were injected
into the chamber atmosphere in order to maintain the
BD concentration in the designed range. The figure
shows also the appearance of the exhaled metabolite EB.
Its concentration increased with time and reached after
about 2 h a plateau representing the equilibrium between
formation and metabolic elimination of EB.
Fig. 3 shows the EB concentration-time courses in
the air of the exposure chambers obtained with rats and
mice exposed to the different BD concentrations. In rats,
EB plateau concentrations were reached at all BD exposure concentrations. Up to 2000 ppm BD, the plateaus
increased with the BD concentrations, but at 6000 and
10000 ppm BD, almost the same EB plateau concentration of 1.3 ppm was obtained. This behavior results from
saturation of BD metabolism at BD concentrations above
2000 ppm [21]. In mice, the EB plateau concentrations
were much higher than in rats at comparable BD concentrations. Furthermore, at 2000 ppm BD no real EB
plateau was seen because after about 4 h of exposure,
the atmospheric EB concentration started to increase
again. This effect, which was even more pronounced at
the higher concentrations, had also been observed earlier
[22]. It was related to the breakdown of the glutathione
S-transferase mediated BD conjugation with cytosolic
Fig. 3. Concentration-time courses of 1,2-epoxy-3-butene (EB) measured in the air of closed chambers each containing either two rats (A1 and A2)
or five mice (B1 and B2) exposed to various 1,3-butadiene (BD) concentrations.
J.G. Filser et al. / Chemico-Biological Interactions 166 (2007) 93–103
Fig. 4. Calculated steady-state concentrations of 1,2-epoxy-3-butene
(EB) in venous blood of rats (△) and mice () versus the exposure
concentrations of 1,3-butadiene (BD). All EB concentrations reflect
single BD exposures over 2–8 h with the exception marked by the
asterisk. * : The EB concentration in blood of mice exposed to 2000 ppm
is reached after 3 h of BD exposure and will increase, if BD exposure
continues due to depletion of the EB conjugating glutathione.
glutathione (GSH) in livers of mice [18]. In this species,
continuous exposure to 2000 ppm BD for 7 and 15 h led
to a reduction of the non-protein thiol (NPSH) content
in the liver to 20% and less [23] and to about 4% [22],
respectively. At 1000 ppm BD (7 h of exposure [24]) and
1250 ppm BD (6 h of exposure [25]), the GSH decline
was only about 50%. In livers of equally BD exposed
rats, GSH depletion was much less expressed [22,23].
Even at a BD concentration of 8000 ppm, hepatic GSH
did not fall below 50% of the control level [25].
Fig. 4 shows the EB concentrations in venous blood
of rats and mice at steady-state conditions of single 8 h
exposures to BD between 1 and 10000 ppm in rats and
1 and 1000 ppm in mice, calculated from the plateau
concentrations in Fig. 3. Also plotted is the EB concentration in venous mouse blood that was reached after
3 h of exposure to 2000 ppm BD. Because EB in blood
of mice did not reach constant concentrations at higher
BD exposure concentrations, they were not included in
the figure. In mice, EB blood concentrations increased
almost linearly with the BD exposure concentration up to
1000 ppm BD. In blood of rats, the increase of EB deviates from linearity at much lower BD concentrations.
This flattening of the EB curve is due to the saturability of CYP450 mediated BD metabolism (see above). In
mice, a corresponding effect is counteracted at high BD
concentrations by the loss of glutathione S-transferase
mediated EB elimination due to depletion of glutathione.
Because of the species-specific saturation kinetics of BD
metabolism, the ratio of the EB blood concentrations
mouse-to-rat was not constant, even at BD concentrations far below 2000 ppm. It was between 2.0 and 2.6 at
BD concentration below 10 ppm, 3.8 at 100 ppm, 4.9 at
99
625, and 8.0 at 1250 ppm BD (Table 1). Similar ratios
were observed by comparing levels of the EB adduct
with the N-terminal valine in hemoglobin of mice and
rats following one- and four-week exposures (6 h/d, 5
d/w) to 10 and 100 ppm BD [26]: At 10 ppm BD the
adduct ratio mouse-to-rat was about 2 and at 100 ppm
BD about 4 [26].
Table 1 compares the EB blood concentrations
obtained in the present work with data published by other
authors at BD concentrations ≥62.5 ppm. Although different experimental methods were used in the three laboratories, there is pretty good agreement between the old
and new blood concentration data for EB.
Fig. 5 demonstrates the concentrations of DEB (mice
only), EBD, and B-diol in venous blood of mice and
rats, measured after 6 h of exposures to constant atmospheric BD concentrations and, for comparison, the EB
concentrations that are given in more detail in Fig. 4.
The DEB concentrations in blood of mice (means ±
S.D., n = 3), representing the sums of racemic and
Fig. 5. Metabolite concentrations in venous blood of rats (A) and
mice (B) at the end of 6 h exposures to various concentrations of
1,3-butadiene (BD). Symbols, each representing one measurement:
3-butene-1,2-diol (B-diol) pooled from 2 rats or 2 mice (n = 1–2);
△ 3,4-epoxy-1,2-butanediol (EBD) pooled from 2 rats or 12 mice
(n = 1–2); ♦ 1,2:3,4-diepoxybutane (DEB) in 1 mouse (n = 3). Lines:
1,2-epoxy-3-butene (EB) in blood (from Fig. 4).
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J.G. Filser et al. / Chemico-Biological Interactions 166 (2007) 93–103
Table 1
Concentrations of 1,2-epoxy-3-butene (EB) in venous blood of single 1,3-butadiene (BD) exposed male Sprague-Dawley rats and B6C3F1 mice
BD in air (ppm)
1
10
62.5
62.5
62.5
100
100
625
625
1250
1250
8000
8000
a
b
c
EB in venous blood (mol/l)
Rat
Mouse
0.003
0.028
0.12
0.07
0.036
0.19
0.1
1.6
0.94
2.0
1.3
4.3
4.03
0.006
0.073
0.46
0.6
0.295
0.73
0.38
7.9
3.7
16
8.6
Not done
Not done
EB ratio mouse to rat
Reference
2.0
2.6
3.8
8.6
8.2
3.8
3.8
4.9
3.9
8.0
6.6
This studya
This studya
This studya
[11]b
[13]c
This studya
[12]c
This studya
[11]b
This studya
[11]b
This studya
[16]c
Measured or calculated based on EB plateau concentrations in chamber air using the EB blood-to-air partition coefficient.
Mean values of the concentrations measured between 2 and 6 h of exposure.
After 4 h of exposure.
meso DEB (about 10% of the racemate), were 0.0,
0.30 ± 0.07, 2.2 ± 0.2, and 3.2 ± 0.6 mol/l at 0, 67, 630,
and 1270 ppm BD, respectively. They compare well with
values obtained under similar exposure conditions in the
same strain. Himmelstein et al. [11] determined DEB
concentrations of 0.65, 1.9, and 2.5 mol/l following 6 h
exposures to 62.5, 625, and 1250 ppm BD. ThorntonManning et al. [13] and Bechtold et al. [12] exposing
male mice for 4 h to 62.5 and 100 ppm BD, respectively,
reported DEB blood concentrations of 0.204 mol/l [13]
and of 0.33 mol/l [12]. Following single (6 h) and
repeated (6 h/d, 10 d) exposures of female B6C3F1 mice
to 62.5 ppm BD, DEB blood concentrations of 0.345 and
0.247 mol/l, respectively, were published [14].
In blood of male rats, no DEB could be detected at
all in the present study (detection limit 10 nmol/l), even
at the highest tested exposure concentration of 900 ppm
BD. Other authors, too, were unable to determine DEB
in rats exposed either to 100 ppm BD (4 h, DEB detection limit 10 nmol/l, [12]) or up to 1200 ppm BD (6 h,
DEB detection limit 130 nmol/l, [11]). After altering
the method described in [12] to reach a DEB detection limit of 1.6 nmol/l blood, DEB concentrations of
5 nmol/l [13] and 2.4 nmol/l [15] were measured following single exposures of male rats to 62.5 ppm BD.
Higher concentrations of DEB in blood were reported
for female Sprague-Dawley rats either once exposed over
6 h to 62.5 ppm BD (14 nmol DEB/l [14]) and 8000 ppm
BD (11 nmol DEB/l [16]) or repeatedly exposed over 10
d (6 h/d) to 62.5 or 8000 ppm BD (17 nmol/l at both concentrations [14,16]). In summary, the data from different
laboratories demonstrate for mice and rats a species difference in the DEB blood concentrations of more than
one order of magnitude, when exposed to around 65 ppm
BD. Also from the results of a hemoglobin binding
study in which the levels by N,N-(2,3-dihydroxy-1,4butadiyl)valine, the DEB-specific ring adduct to the Nterminal valine, were compared in both species following
a 2 week (6 h/d, 10 d) exposure to 3 and 62.5 ppm BD,
the authors concluded “that mice are much more efficient
at forming 1,2:3,4-diepoxybutane than rats, particularly
at low exposures” [27].
The present work is the first demonstrating EBD
and B-diol concentrations in blood of BD exposed
rodents (Fig. 5). EBD showed a maximum of 9.5 mol/l
at 150 ppm BD in rats and of 42 mol/l at 300 ppm
BD in mice. With further increasing BD concentrations, the EBD concentrations decreased in the rat
to become even smaller than the EB concentrations
at 900 ppm BD (EBD: 1.2 mol/l) and 1200 ppm BD
(EBD: 1.1 mol/l). In mice, the decrease of the EBD
concentration was much smaller. At BD concentrations of 900 and 1200 ppm mean blood concentrations of EBD were about 30 mol/l. Up to now, the
formation of EBD in BD exposed rodents was concluded indirectly from the determination of N-(2,3,4trihydroxybutyl)valine in hemoglobin [27,28] and of
N-7-(2,3,4-trihydroxybutyl)guanine in DNA [29,30].
Although these adducts are formed from EBD [31,32],
they might also result from DEB [33–35]). However, the
conclusion that N-(2,3,4-trihydroxybutyl)valine results
primarily from EBD is supported by comparing our
J.G. Filser et al. / Chemico-Biological Interactions 166 (2007) 93–103
data with the findings of Perez et al. [27] who measured the N-(2,3,4-trihydroxybutyl)valine levels in male
Wistar rats, repeatedly exposed to BD concentrations
of 50, 200, and 500 ppm (6 h/d, 5 d). The authors
described a similar dependency of the adduct levels
from the BD exposure concentration as obtained in the
present study for EBD concentrations in blood. The
adduct levels following exposures to 50 and 500 ppm
BD were almost identical but the level in the 200 ppm
exposed rats was twice as high. Also, the findings of
Koc et al. [29], who determined N-7 guanine adducts
of EB, DEB, and EBD in mice and rats exposed to
BD concentrations of 0, 20, 62.5, and 625 ppm BD,
do not disagree with our EBD data. The N7-(2,3,4trihydroxybutyl)guanine levels were the most abundant
exhibiting a nonlinear dose–response curve. The findings that N7-(2,3,4-trihydroxybutyl)guanine reached “a
plateau after 62.5 ppm” in rats but “continued to increase
in mice between 62.5 and 625 ppm BD” appear consistent with our results considering that the authors had no
information on the adduct levels at BD concentrations
between 62.5 and 625 ppm and above 625 ppm.
B-diol was first detected as a minor urinary BD
metabolite in mice and rats exposed up to 5 h to 800 ppm
13 C-BD [36]. Fig. 5 shows that B-diol concentrations
in blood of rats were higher than those of the epoxides at all BD concentrations above 60 ppm. In blood
of mice, B-diol concentrations surpassed those of EBD
only at 600 ppm. In both species, the B-diol concentrations increased continuously with the BD concentrations amounting to 60 mol/l at 1200 ppm. Below
60 ppm BD, the slopes of the curves yielding B-diol in
blood versus BD in air were less steep than at higher
concentrations.
The complex interaction pattern of the BD metabolites can be understood considering that they are not
only formed in a chain after re-uptake of their actual
parent compounds into the liver from its blood flow,
as Fig. 1 might suggest, but almost simultaneously in
the endoplasmic reticulum as immediate metabolites of
BD. This concurrent metabolite production was hypothesized [10,37] and strongly supported from experiments
with isolated, BD-perfused livers [10]. The immediate
formation of EB, B-diol, EBD, and DEB results from
the close spatial vicinity of CYP450 and microsomal
epoxide hydrolase both situated in the endoplasmic reticulum. In the mouse liver with the higher CYP450 activity
towards BD [38,39], the rates of EB and DEB formation
are higher than in that of the rat. However, the immediate
hydrolysis rates of EB to B-diol are probably higher in
the rat than in the mouse as evidenced from the ratios
B-diol to EB (compare Fig. 5). As a consequence, less
101
DEB is formed in the rat. Its release into the blood leaving the liver is so small that it could not be detected in
vivo by the present analytical method.
In both species, most of the produced DEB is assumed
to be immediately hydrolyzed to EBD before becoming systemically available. The EBD concentrations in
mouse blood are higher than in rat blood because mice
produce more DEB (due to their higher CYP450 activity to EB [40] and because more EB is available for
oxidation), which is immediately hydrolyzed. Additionally, B-diol is in part directly epoxidized to EBD as
shown in B-diol exposed rodents by EBD adducts to
hemoglobin and DNA of liver and lung [30]. However, only about 18% of the circulating B-diol was
detected in blood as EBD after intraperitoneal administration of B-diol (12 mg B-diol/kg body weight) to male
Sprague-Dawley rats (unpublished results). The group
of Elfarra investigating intensively the metabolism of Bdiol detected the oxidation product hydroxymethylvinyl
ketone [41]. From the urinary excretion of mercapturic
acids derived from B-diol, the formation of hydroxymethylvinyl ketone was suggested to be a prominent
route for B-diol metabolism in rats and mice [42].
Considering the continuous increase of the direct BD
oxidation product EB in blood of both species at BD concentrations up to 1200 ppm, the EBD maxima reached
at much lower BD concentrations (150–300 ppm rat and
about 300 ppm mouse), the steeper B-diol slopes at BD
concentrations above 60 ppm (both species), and the
nonlinear increase of DEB, it seems probable that BD
inhibits in both species the oxidation of EB to DEB and
of B-diol to EBD in a competitive manner by displacing
EB, B-diol and DEB from CYP450.
Mutagenic potencies of EB and EBD are comparable [43,44]. In rats, EB blood concentrations amount to
4 mol/l at 8000 ppm BD (Table 1; Fig. 4), the highest
BD concentration used in the carcinogenicity long-term
study with rats (0, 1000, 8000 ppm, 6 h/d, 5d/w, 2y; [9]).
Considering that EBD concentrations reach a maximum
of about 10 mol/l at a BD concentration of 150 ppm
(Fig. 5), it can be suggested that rats are at a similar
mutagenic risk at BD concentrations of about two hundred ppm BD as they had been in the long-term study at
8000 ppm BD.
Acknowledgements
The authors thank Mr. Christian Pütz for his excellent
technical assistance and Dr. Judith Baldwin for the quality assurance reviews. Financial support of the American
Chemistry Council is gratefully acknowledged.
102
J.G. Filser et al. / Chemico-Biological Interactions 166 (2007) 93–103
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