Alcohol dehydrogenase- and rat liver cytosol-dependent bioactivation of 1-chloro-2-hydroxy-3-butene to 1-chloro-3-buten-2-one, a bifunctional alkylating agent

Article abstract of DOI:10.1021/tx300369b

1,3-Butadiene (BD) is an air pollutant whose toxicity and carcinogenicity have been considered primarily mediated by its reactive metabolites, 3,4-epoxy-1-butene and 1,2,3,4-diepoxybutane, formed in liver and extrahepatic tissues by cytochromes P450s. A possible alternative metabolic pathway in bone marrow and immune cells is the conversion of BD to the chlorinated allylic alcohol 1-chloro-2-hydroxy-3-butene (CHB) by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. In the present study, we investigated the in vitro bioactivation of CHB by alcohol dehydrogenases (ADH) under in vitro physiological conditions (pH 7.4, 37 °C). The results provide clear evidence for CHB being converted to 1-chloro-3-buten-2-one (CBO) by purified horse liver ADH and rat liver cytosol. CBO readily reacted with glutathione (GSH) under assay conditions to form three products: two CBO-mono-GSH conjugates [1-chloro-4-(S-glutathionyl)butan-2-one (3) and 1-(S-glutathionyl)-3-buten-2-one (4)] and one CBO-di-GSH conjugate [1,4-bis(S-glutathionyl)butan-2-one (5)]. CHB bioactivation and the ratios of the three GSH conjugates formed were dependent upon incubation time, GSH and CHB concentrations, and the presence of ADH or rat liver cytosol. The ADH enzymatic reaction followed Michaelis-Menten kinetics with a K_m at 3.5 mM and a k_cat at 0.033 s^-1. After CBO was incubated with freshly isolated mouse erythrocytes, globin dimers were detected using SDS-PAGE and silver staining, providing evidence that CBO can act as a protein cross-linking agent. Collectively, the results provide clear evidence for CHB bioactivation by ADH and rat liver cytosol to yield CBO. The bifunctional alkylating ability of CBO suggests that it may play a role in BD toxicity and/or carcinogenicity.

Full text of DOI:10.1021/tx300369b

Article
pubs.acs.org/crt
Alcohol Dehydrogenase- and Rat Liver Cytosol-Dependent
Bioactivation of 1‑Chloro-2-hydroxy-3-butene to 1‑Chloro-3-buten-2-
one, a Bifunctional Alkylating Agent
Adnan A. Elfarra*,† and Xin-Yu Zhang‡
†Department of Comparative Biosciences and the Molecular and Environmental Toxicology Center, University of
WisconsinMadison, Madison, Wisconsin 53706, United States
‡Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai
200444, People's Republic of China
ABSTRACT: 1,3-Butadiene (BD) is an air pollutant whose
toxicity and carcinogenicity have been considered primarily
mediated by its reactive metabolites, 3,4-epoxy-1-butene and
1,2,3,4-diepoxybutane, formed in liver and extrahepatic tissues by
cytochromes P450s. A possible alternative metabolic pathway in
bone marrow and immune cells is the conversion of BD to the
chlorinated allylic alcohol 1-chloro-2-hydroxy-3-butene (CHB) by
myeloperoxidase in the presence of hydrogen peroxide and
chloride ion. In the present study, we investigated the in vitro
bioactivation of CHB by alcohol dehydrogenases (ADH) under in
vitro physiological conditions (pH 7.4, 37 °C). The results provide
clear evidence for CHB being converted to 1-chloro-3-buten-2-
one (CBO) by purified horse liver ADH and rat liver cytosol. CBO readily reacted with glutathione (GSH) under assay
conditions to form three products: two CBO-mono-GSH conjugates [1-chloro-4-(S-glutathionyl)butan-2-one (3) and 1-(S-
glutathionyl)-3-buten-2-one (4)] and one CBO-di-GSH conjugate [1,4-bis(S-glutathionyl)butan-2-one (5)]. CHB bioactivation
and the ratios of the three GSH conjugates formed were dependent upon incubation time, GSH and CHB concentrations, and
the presence of ADH or rat liver cytosol. The ADH enzymatic reaction followed Michaelis−Menten kinetics with a Km at 3.5 mM
and a kcat at 0.033 s−1. After CBO was incubated with freshly isolated mouse erythrocytes, globin dimers were detected using
SDS-PAGE and silver staining, providing evidence that CBO can act as a protein cross-linking agent. Collectively, the results
provide clear evidence for CHB bioactivation by ADH and rat liver cytosol to yield CBO. The bifunctional alkylating ability of
CBO suggests that it may play a role in BD toxicity and/or carcinogenicity.
ity of BD,8,22 although it has been suggested that EBD might
INTRODUCTION
■
play a more important role in rats and humans.23,24
1,3-Butadiene (BD) is a major petroleum product used in the
Evidence for a possible alternative metabolic pathway
production of synthetic rubber and plastic. It is also found in
involving myeloperoxidase (MPO) has been obtained.25 BD
cigarette smoke, gasoline, and urban air.1 BD has been classified
has been demonstrated to be oxidized to 1-chloro-2-hydroxy-3-
as a human carcinogen by the U.S. Environmental Protection
Agency primarily because epidemiological studies showed
butene (CHB, 1) by isolated human MPO in the presence of
H2O2 and chloride ion (Scheme 1).25 The reaction probably
increased incidences of lymphohematopoietic cancers among
occurred through the initial formation of hypochlorous acid
workers occupationally exposed to BD.1
(HOCl) by MPO. EB was also detectable in these incubations,
The carcinogenicity of BD is widely believed to be mediated
by its primary and/or secondary cytochromes P450-dependent
metabolites (epoxides), namely, 3,4-epoxy-1-butene (EB),
1,2,3,4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol
(EBD).2 While EB, EBD, and DEB are all direct-acting
mutagens/carcinogens,3 DEB is the most potent mutagen
among these epoxides,3−5 possibly due to its higher reactivity
and cross-linking ability.6−8 DEB can induce in vitro/in vivo
DNA−DNA6,7,9−15 and in vitro DNA−protein cross-link-
ing.16−20 Interestingly, it almost exclusively causes DNA−
DNA cross-linking in cells.21 DEB is widely believed to be the
ultimate metabolite responsible for mutagenicity/carcinogenic-
but CHB was the major product detected at chloride
concentrations >50 mM.25 Although CHB is unlikely to be
produced in liver, lung, or kidney cells for lack of expression of
MPO and the extremely low intracellular H2O2 concentrations
(≤0.1−1 μM by estimation26), the MPO pathway is expected
to be effective in immune and bone marrow cells, such as
neutrophil granulocytes. Neutrophil granulocytes contain large
amounts of MPO and high concentrations of chloride ion (80−
111 mM)27 and can produce high concentrations of H2O2 (in
Received: September 2, 2012
Published: October 30, 2012
© 2012 American Chemical Society
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bromophenol blue, 2-mercaptoethanol, DTT, acetonitrile (HPLC
grade), purified horse liver ADH (catalog #A9589), nicotinamide
adenine dinucleotide (NAD), and GSH were obtained from Sigma-
Aldrich (St. Louis, MO). Rat liver cytosol was isolated from male
Sprague−Dawley rats as described previously.34,35 The whole blood of
male C57Bl mice was obtained from Harlan Bioproducts (Indian-
apolis, IN). CHB was synthesized as described previously.36
Scheme 1. Metabolic Pathway for the Formation of CHB (1)
and CBO (2) from BD and the Subsequent Reactions of
CBO with GSH To Form Three CBO−GSH Conjugates (3−
5)
Instruments and Methods. Mass spectra were obtained on an
MDS Sciex API 365 LC/MS/MS triple quadrupole electrospray
ionization (ESI) mass spectrometer. NMR spectra were recorded on a
Bruker Instruments DMX-400 Avance spectrometer (400 MHz).
HPLC separation was performed on a gradient-controlled HPLC
system (Gilson Inc., Middleton, WI) equipped with a Beckman System
Gold 166 variable wavelength detector (San Ramon, CA) using a
Beckman Ultrasphere 5 μm ODS reverse-phase analytical column (4.6
mm × 25 cm) with UV detection at 254 nm. A linear gradient program
was used, starting at 0 min from 0% pump B to 10% pump B over 7
min [pump A, 1% (v/v) acetonitrile with the pH being adjusted to 2.5
with TFA; pump B, 75% (v/v) acetonitrile at pH 2.5] at a flow rate of
1 mL/min and then at 7 min from 10 to 20% pump B over 1 min, at
12 min from 20 to 10% pump B over 1 min, and stopped at 17 min.
The retention times of the three CBO−GSH conjugates under these
conditions were 14.7 min for 3, 9.4 min for 4, and 7.1 min for 5. The
standard curves were linear with the ranges of quantities and
correlation coefficients as follows: 3, 0.6−484 nmol, 0.9980; 4,
1.33−106.4 nmol, 0.9968; and 5, 0.37−292.96 nmol, 0.9948,
respectively. For preparative separation, a Beckman Ultrasphere 5
μm ODS semipreparative column (250 mm × 10 mm) was employed.
The solvents in pump A and B were 1 and 15% acetonitrile at pH 2.5,
respectively. A 35 min isocratic program with 50% pump B was used
with UV detection at 220 nm.
Synthesis of CBO. CBO was prepared through oxidation of CHB
by Jones' reagent. The reaction was performed following a procedure
described in the literature.37 Jones' reagent was prepared by the
addition of concentrated H2SO4 (1.0 mL) to CrO3 (1.12 g) followed
by the careful dilution with water to a total volume of 8.33 mL. Jones'
reagent (1.1 mL) was added dropwise to a stirred solution of CHB
(0.106 g, 1.0 mmol) in acetone (4 mL) in an ice−water bath. After
complete addition of Jones' reagent, the ice−water bath was removed,
and the mixture was stirred at room temperature for 1 h. Methanol
(0.4 mL) was then added to remove excess of Jones' reagent.
Approximately 3 mL of water was added, and the mixture was
extracted three times with diethyl ether. The organic phase was washed
several times with saturated NaCl solution until it became colorless.
After it was dried with anhydrous MgSO4, the solvent was removed. A
pale yellow (74 mg, 71%) liquid was obtained. The obtained CBO,
whose purity was ∼90% as determined by GC-MS, was directly used in
the experiments without further purification. Pure CBO (>98%),
which was obtained by purification on a silica gel column with
dichloromethane as the eluent, was used as a reference for HPLC
analysis.
Reactions of CBO with GSH and Identification of Three
CBO−GSH Conjugates. These reactions were carried out to prepare
the standards for identification and quantification of CBO−GSH
conjugates produced in the enzymatic reactions. All reactions were run
in 50 mM, pH 7.4, Tris-HCl buffer at 37 °C. To prepare the di-GSH
conjugate 5, CBO (10 mg, 0.096 mmol) in Tris buffer (0.5 mL) was
added to a solution of GSH (88.2 mg, 0.287 mmol, CBO:GSH = 1:3)
in 0.5 mL of buffer. The mixture was incubated in a shaking water bath
for 30 min, and then, 40 μL of 35% perchloric acid was added to
terminate the reaction. Compound 5 (13 mg) was obtained through
fraction collection on preparative HPLC and then lyophilization. To
prepare the mono-GSH conjugates 3 and 4, a CBO:GSH molar ratio
of 2:1 was used. CBO (8.5 mg, 0.081 mmol) in 0.3 mL of buffer was
added to a solution of GSH (10.7 mg, 0.0348 mmol) in 0.3 mL of
buffer. After the reaction was allowed to proceed at 37 °C for 10 min,
the reaction mixture was subjected to HPLC separation as described
above. Compounds 3 (3.2 mg) and 4 (0.5 mg) were obtained through
fraction collection and lyophilization.
millimolar) for microbial killing when the cells are stimulated
by invading microorganisms or upon oxidative burst as part of
the inflammatory response. HOCl is believed to be the actual
agent to kill bacteria.28 When the chloride ion concentration is
higher than 50 mM, more than 90% of H2O2 is converted to
HOCl.27 Thus, there should be sufficiently high concentrations
of HOCl in neutrophil granulocytes to lead to the formation of
CHB when the cells are exposed to BD.
After its formation in vivo, CHB, a halogenated allylic
alcohol, is likely to undergo oxidation by alcohol dehydro-
genases (ADHs) and cytochromes P450s. Support for this
hypothesis is provided by our previous demonstration that 3-
butene-1,2-diol (BDD) can be metabolized by ADHs and
cytochromes P450 both in vitro and in vivo to yield the Michael
acceptor hydroxymethylvinyl ketone (HMVK).29−33 We
postulated that CHB could similarly be transformed to 1-
chloro-3-buten-2-one (CBO, 2) by ADHs (Scheme 1). On the
basis of its chemical structure, CBO is expected to act as a
bifunctional alkylating agent.
In the present study, we examined the in vitro transformation
of CHB to CBO by purified horse liver ADH and rat liver
cytosol. Glutathione (GSH) was included in the experiments as
a trapping agent. The results provide clear evidence for CHB
bioactivation to CBO by ADH and cytosolic ADH from rat
liver, and the generated CBO readily reacted with GSH to form
two CBO−mono-GSH conjugates (3 and 4) and one CBO−di-
GSH conjugate (5) (Scheme 1). Incubation of erythrocytes
with CBO resulted in the detection of globin dimers, providing
further evidence for the cross-linking ability of CBO.
EXPERIMENTAL PROCEDURES
Materials. BD (99.5%) was purchased from Matheson Gas (Joliet,
IL). Calcium hypochlorite, chromium oxide, perchloric acid, trifluoro-
acetic acid (TFA), Trizma base, glycerol, sodium dodecyl sulfate,
■
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1
Spectroscopic data of 5: ESI-MS: m/z 683.5. H NMR (400 MHz,
D2O): δ 4.63−4.55 (m, 2H, H-6 and H-16), 3.80−3.75 (m, 6H, H-8,
H-13, H-18, and H-23), 3.14−3.00 (m, 3H, H-5 and H-4a), 2.92−2.81
(m, 3H, H-15 and H-4b), 2.57−2.51 (m, 4H, H-11 and H-21), 2.24−
2.12 (m, 8H, H-1, H-3, H-12, and H-22).
one product peak at 7.1 min could be observed. The
protonated molecular ion peak of the product appeared at
m/z 683.5 (Figure 1A), which was consistent with a structure
Conversion of CHB to CBO by ADH. The enzymatic reaction
was carried out at in vitro physiological conditions (pH 7.4, 37 °C).
The reaction mixtures were prepared by adding ADH, NAD, and GSH
solutions (100 μL each, made in 50 mM, pH 7.4, Tris-HCl buffer) to
100 μL of Tris-HCl in glass test tubes (100 mm × 16 mm). The test
tubes were then placed in a shaking water bath at 37 °C for 3 min.
Then, CHB solution in Tris-HCl buffer (100 μL) was added, and the
reactions were allowed to proceed for 5−30 min. The total volumes of
the solutions were 500 μL, and the final concentrations of the
substances were as follows: ADH, 0.25 mg/tube; NAD, 1.5 mM; GSH,
1 or 5 mM; and CHB, 5 or 20 mM. Control incubations without
ADH, GSH, or CHB were also carried out. All incubations were
terminated by adding 20 μL of 35% perchloric acid to each test tube.
The reaction mixtures were cooled down on ice and then centrifuged
at 3000 rpm for 10 min to precipitate the protein. The supernatants
were analyzed by HPLC with UV detection at 220 nm.
To determine the kinetic constants of the enzymatic reaction, eight
CHB concentrations (0.25, 0.5, 1, 2.5, 5, 10, 15, and 20 mM) were
used. The mixtures were incubated for 15 min with the GSH
concentrations at 5 mM. Under these experimental conditions, only 5
was consistently detected, whereas 3 and 4 were trace products.
Therefore, only the amounts of 5 were determined and used in the
calculations.
Figure 1. ESI mass spectra of the three CBO−GSH conjugates. (A)
The CBO−di-GSH conjugate 1,4-bis(S-glutathionyl)butan-2-one (5).
(B) The CBO−mono-GSH conjugate 1-chloro-4-(S-glutathionyl)-
butan-2-one (3). (C) The CBO−mono-GSH conjugate 1-(S-
glutathionyl)-3-buten-2-one (4).
Conversion of CHB to CBO by Rat Liver Cytosol. Because rat
liver cytosol contains ADH, which has similar functions to horse liver
ADH, CHB (5 mM) incubations were also carried out with rat liver
cytosol (0.25 mg protein) and 5 mM GSH at pH 7.4 and 37 °C for 15,
30, and 60 min. The total volumes of the solutions were 500 μL. The
control experiments without CHB, GSH, or rat liver cytosol were also
performed.
that two molecules of GSH were added to one molecule of
CBO. The 1H NMR spectrum in D2O indicated the presence of
26 protons. Comparison of the NMR data with published
NMR spectroscopic data of GSH and 340 showed that the
spectrum was consistent with the expected structure 1,4-bis(S-
glutathionyl)butan-2-one (5). Therefore, 5 was the product
with the both ends (i.e., C-1 and C-4) of CBO reacting with
GSH; namely, it was the di-GSH conjugate of CBO (Scheme
2).
CBO-Induced Globin Cross-Linking Analyses by SDS-PAGE.
To obtain evidence for the ability of CBO to form protein cross-links
at physiological conditions, freshly isolated mouse erythrocytes were
incubated with CBO. After the removal of the plasma fraction from the
whole blood of male C57B1 mice, erythrocytes were washed three
times with an equal volume of saline and centrifuged at 3000 rpm for 5
min in between washes and then were resuspended in an equal volume
of phosphate-buffered saline (8.4 mM Na2HPO4, 1.6 mM KH2PO4,
and 154 mM NaCl, pH 7.4). Incubation with CBO was carried out in a
Dubnoff metabolic shaking water bath at 37 °C for 1 or 24 h as
previously described.38 The final concentrations of CBO were 0.2, 0.5,
1, 2, 5, and 10 mM. At the end of the incubations, erythrocytes were
lysed with equal volumes of cold doubly deionized water (ddH2O),
and globin was isolated using acidified acetone as described
previously.39 Globin (2.5 μg) dissolved in 2.5 μL of ddH2O was
added to the treatment buffer (500 mM, pH 7.4, Tris-HCl, 10%
glycerol, 10% SDS, 0.01 g/mL bromophenol blue, and 10% 2-
mercaptoethanol). After the addition of DTT (200 mM final
concentration) to reduce dimer formation between globin chains
due to disulfide bonds, the samples were kept at room temperature for
30 min before being loaded onto a 12.5% Tris-HCl Criterion Precast
gel and run at 200 V for 75 min. Gels were silver stained for
visualization of bands.
Scheme 2. Structure of the Di-GSH Conjugate of CBO (5)
When the reaction of CBO with GSH was performed with an
excess of CBO (the molar ratio of CBO:GSH was 2:1) at pH
7.4 and 37 °C for 10 min, two HPLC peaks were observed at
9.4 and 14.7 min in addition to the peaks for 5 and unreacted
GSH and CBO (their retention times were 7.1, 4.9, and 19.6
min, respectively). The ESI-MS of the compound eluted at 14.7
min showed a protonated molecular ion peak at m/z 412.6 with
an isotope peak at m/z 414.6 (∼1/3 of the intensity of the peak
at m/z 412.6) (Figure 1B), indicating that the compound
contains a chlorine atom. The molecular weight was consistent
with a structure that GSH was added to C-4 of CBO through
Michael addition. The protonated molecular ion peak of the
compound eluted at 9.4 min appeared at m/z 376.4 (Figure
1C), which was consistent with a structure that chlorine atom
in CBO was replaced by GSH. In addition, the isolated
RESULTS
■
Reactions of CBO with GSH under in Vitro Physio-
logical Conditions and Identification of Two CBO−
Mono-GSH Conjugates and One CBO−Di-GSH Conju-
gate. The reaction of CBO with GSH under in vitro
physiological conditions (pH 7.4, 37 °C) was performed first
with an excess of GSH (the molar ratio of CBO:GSH was 1:3).
After 30 min, the HPLC chromatogram of the reaction mixture
indicated that CBO had been completely consumed, and only
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compounds 3 and 4 both were readily converted to 5 upon
further incubation with GSH, providing evidence for these
compounds being the mono-GSH conjugates of CBO through
reactions on C-4 and C-1 of CBO, respectively. Thus, the two
products were identified as 1-chloro-4-(S-glutathionyl)butan-2-
one (3) and 1-(S-glutathionyl)-3-buten-2-one (4), respectively.
The reaction of CBO with GSH has been reported by Munter
et al. to yield two products: 5, as identified solely by mass
spectrometry, and 3, which was identified by both NMR and
mass spectrometry.40
Conversion of CHB to CBO by ADH. To examine if CHB
could be oxidized by ADH, CHB (5 mM) was incubated with
purified horse liver ADH at pH 7.4 and 37 °C. Because CBO is
a bifunctional alkylating agent, 5 mM GSH was included in the
incubations to serve as a trapping agent. To confirm that the
formation of CBO was attributed to the presence of ADH, a
control experiment without ADH was carried out. The three
CBO−GSH conjugates described above were used as the
standards for identification and quantification of the enzymatic
products.
GSH was included in the incubation. When higher CHB (20
mM) and lower GSH concentrations (1 mM) were used, ∼70%
of the CBO detected in the absence of GSH was consumed
when GSH was included. Collectively, the results clearly
indicated that CHB was converted to CBO by purified ADH,
and the formed CBO in turn reacted with GSH to yield the
three CBO−GSH conjugates 3−5.
Dependence of the Amounts and Ratios of the CBO−
GSH Conjugates Formed on the CHB and GSH
Concentrations. To examine how altering the CHB and
GSH concentrations could affect the amounts and ratios of the
CBO−GSH conjugates produced, two experiments with
different CHB and GSH concentrations (20:1 mM and 5:5
mM, respectively) were performed.
The total amounts of the three conjugates increased over
time in both experiments (Figure 3A), indicating that CBO was
The results showed that three products, including one major
and two minor ones, were formed. The major product was
identified as 5, and the two minor ones were 3 and 4. The
amount of 5 increased over time, and those of 3 and 4 changed
little with time (Figure 2A). In addition, a small amount of
Figure 3. Time-dependent formation of the CBO−GSH conjugates
when CHB was incubated with horse liver ADH. (A) Total CBO−
GSH conjugates formed at 20 mM CHB and 1 mM GSH or at 5 mM
CHB and 5 mM GSH. (B) Formation of the CBO−GSH conjugates
3−5 at 20 mM CHB and 1 mM GSH. All incubations were carried out
using 0.25 mg of ADH and 1.5 mM NAD. Values were corrected for
any non-ADH-dependent formation at all time points examined.
continuously produced. The total amounts of the conjugates
formed at 20 mM CHB were approximately 2−2.5-fold larger
than those observed at 5 mM CHB (Figure 3A).
Figure 2. (A) Time-dependent formation of the three CBO−GSH
conjugates 3−5 when CHB was incubated with horse liver ADH under
in vitro physiological conditions (pH 7.4, 37 °C). (B) Representative
graph showing the non-ADH-dependent formation of the CBO−GSH
conjugates (the total amounts) as compared to that formed in the
presence of ADH. All incubations contained 5 mM CHB, 1.5 mM
NAD, and 5 mM GSH. The ADH-containing incubations had 0.25 mg
of ADH.
The ratios of the three conjugates were dependent on the
concentrations of CHB and GSH. When the CHB concen-
tration was lower (5 mM) and the GSH concentration was
higher (5 mM), the di-GSH conjugate 5 was the over-
whelmingly predominant product (Figure 2A), apparently due
to a large excess of GSH over CBO formed. However, with
higher CHB concentration (20 mM) and lower GSH
concentration (1 mM), although generally 5 was still the
predominant product, the yields of the two mono-GSH
conjugates 3 and 4 greatly increased (Figure 3B). In particular,
the amount of 3, which increased by up to 11 times in
comparison with that in the experiment with 5 mM GSH, was
greater than that of 5 detected at the 5 min time point (Figure
3B).
CBO was also detected (data not shown). When ADH was
absent, only a trace amount of 3 was formed, probably due to
nonenzymatic oxidation of CHB by oxygen (Figure 2B). In the
control experiments without GSH, a significant increase in the
amount of CBO over time was observed (data not shown). A
comparison of the CBO amounts in the absence of GSH with
those in the presence of GSH revealed that ∼90% of the CBO
detected in the incubation without GSH was consumed when
Determination of Kinetic Constants of the Enzymatic
Reaction. The conversion of CHB to CBO by purified ADH
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followed the Michaelis−Menten kinetics (Figure 4). The Km
and kcat were determined as 3.5 mM and 0.033 s−1, respectively.
In the experiments, the GSH concentration was 5 mM, and the
incubation time was 15 min.
CBO for 24 h, globin isolated exhibited much more visible
darker dimer bands (Figure 6, lanes 2, 4, and 6) in comparison
Figure 6. SDS-PAGE and silver staining of globin samples isolated
after erythrocytes were incubated with (lanes 2, 4, and 6) and without
CBO (10 mM) (lanes 1, 3, and 5). The incubations were carried out at
37 °C for 24 h. Lanes 1 and 2 contained 5 μg of protein, lanes 3 and 4
contained 10 μg of protein, and lanes 5 and 6 contained 20 μg of
protein. Lane 7 contained the molecular weight marker. The gel image
represents the results obtained from a typical experiment.
Figure 4. ADH-dependent conversion of CHB to CBO, as measured
by the CBO−di-GSH conjugate 5, followed Michaelis−Menten
kinetics. The Km and kcat were determined to be 3.5 mM and 0.033
s−1, respectively. The GSH concentrations were 5 mM, and the
incubation time was 15 min.
with the corresponding control samples incubated with buffer
only (Figure 6, lanes 1, 3, and 5). Concentration-dependent
increases in dimer band intensity were also observed when the
CBO concentration was increased from 0.2 to 10 mM and the
incubation time was 1 h (data not shown). Collectively, the
results indicated that CBO was able to cause globin cross-
linking in erythrocytes.
Biotransformation of CHB to CBO by Rat Liver
Cytosol. Because liver cells contain abundant ADH, it was
expected that CHB could be converted to CBO by liver cytosol.
To confirm it, a mixture of 5 mM CHB and 5 mM GSH was
incubated with rat liver cytosol at 37 °C for 15, 30, and 60 min.
The results showed that all three CBO−GSH conjugates were
formed over time (Figure 5). Interestingly, the CBO−mono-
DISCUSSION
■
CHB, which was formed when BD was incubated with MPO in
the presence of H2O2 and chloride ion,25 is a structural
analogue of BDD. Similar to BDD, which was previously shown
to be oxidized to HMVK by purified horse liver ADH and
cytosolic ADH from mouse, rat, and human liver,29 and also by
human cytochromes P450s,30 the results presented in the
present study provide clear evidence for CHB oxidation by
isolated horse liver ADH and cytosolic ADH from rat liver to
yield CBO, which, in turn, was a structural analogue of HMVK.
Although CBO was expected to have reactivity toward
biological nucleophiles similar to that of HMVK due to their
similar structures, there are significant differences in reactivity
between the two compounds. Probably the principal difference
is that only one end of HMVK has reactivity, whereas both
ends of CBO are reactive. Thus, unlike HMVK, CBO is a
bifunctional alkylating agent, which was confirmed by the fact
that the di-GSH conjugate 5 was readily formed in the CBO−
GSH reactions. The bifunctional alkylating ability of CBO was
further demonstrated by the formation of dimer bands of globin
when CBO was incubated with mouse erythrocytes.
Figure 5. Time-dependent formation of the CBO−GSH conjugates
3−5 after CHB (5 mM) was incubated with rat liver cytosol (0.25 mg
protein). The reaction mixtures also contained 5 mM GSH and were
incubated at pH 7.4 and 37 °C.
GSH conjugate 3 was the predominant product in the
experiment, whereas the CBO−mono-GSH conjugate 4 and
the CBO−di-GSH conjugate 5 were minor products, consistent
with the increased formation of 3 at the early time points when
purified ADH was incubated with 20 mM CHB and 1 mM
GSH (Figure 3B).
CBO-Induced Cross-Linking of Hemoglobin. The
reaction of CBO with GSH produced predominantly the di-
GSH conjugate 5, indicating that CBO was a bifunctional
alkylating agent. Thus, CBO could probably induce cross-
linking of proteins. To obtain evidence for the protein cross-
linking ability of CBO at physiological conditions, mouse
erythrocytes were incubated with different concentrations of
CBO (0.2, 0.5, 1, 2, 5, and 10 mM) for 1 or 24 h, and then,
globin was isolated and subjected to SDS-PAGE and silver
staining. Indeed, CBO-induced globin dimer bands were
observed. When erythrocytes were incubated with 10 mM
Evidence for BDD oxidation to HMVK in mice and rats in
vivo41 was obtained by the detection of the urinary metabolite
4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (the mercaptu-
ric acid of HMVK−GSH conjugate)32 and detection of
HMVK−globin adducts in rats and mice given BDD.33 As a
reactive Michael acceptor, HMVK exhibited high reactivity
toward nucleophilic amino acids,42 hemoglobin,43 and nucleo-
sides and DNA.44 Thus, it is expected that CHB can also be
biotransformed to CBO in vivo, which can react with a variety
of biomacromolecules. Because of its high reactivity and cross-
linking ability, CBO may be highly mutagenic and could play a
significant role in the mutagenicity/carcinogenicity of BD.
Previous studies provide evidence for the presence of both a
high-affinity pyrazole-insensitive ADH and a low-affinity
pyrazole-sensitive ADH in human blood and bone marrow
cells.45,46 Human bone marrow has also been reported to
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express mRNA of cytochrome P450 1A1, 2A6/7, 2D6, 2E1, and
3A4.47 Cytochrome P450 2E1 was also detected by Western
blotting in human CD34+ bone marrow stem cells.48 In
addition, human blood monocytes and macrophages contain
multiple P450s (P450 1A1, 1A2, 1B1, 2B6/7, 2E1, 3A3/4, 3A7,
and 4B1) as determined by RT-PCR.49 Thus, it is reasonable to
speculate that CHB can undergo ADH- and cytochrome P450s-
mediated conversion to CBO in human bone marrow and
immune cells.
ABBREVIATIONS
■
ADH, alcohol dehydrogenase; BD, 1,3-butadiene; BDD, 3-
butene-1,2-diol; CBO, 1-chloro-3-buten-2-one; CHB, 1-chloro-
2-hydroxy-3-butene; ddH2O, doubly deionized water; DEB,
1,2,3,4-diepoxybutane; EB, 3,4-epoxy-1-butene; EBD, 3,4-
epoxy-1,2-butanediol; ESI, electrospray ionization; GSH,
glutathione; HMVK, hydroxymethylvinyl ketone; MPO,
myeloperoxidase; NAD, nicotinamide adenine dinucleotide;
TFA, trifluoroacetic acid
Chlorohydrins, such as ethylene chlorohydrin, propylene
chlorohydrin, and 3-chloro-1,2-propanediol, are considered
potential toxicants and carcinogens.50−56 In this regard,
exposure to chlorohydrins have been associated with leukemia
and pancreatic cancers54 and lymphatic and hematopoietic
cancers.55,56 Thus, mutagenicity and toxicity of CHB warrant
further investigations.
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: 608-262-6518. Fax: 608-262-3926. E-mail: elfarra@svm.
vetmed.wisc.edu.
Funding
This research was made possible by Grant R01 DK044295 from
the National Institutes of Health and Grant Number R01
ES06841 from the National Institute of Environmental Health
Sciences (NIEHS), NIH.
Notes
The authors declare no competing financial interest.
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