Oxidation of 3-butene-1,2-diol by alcohol dehydrogenase

Article abstract of DOI:10.1021/tx960090e

3-Butene-1,2-diol(BDD)is a metabolite of the carcinogenic petrochemical 1,3-butadiene. BDD is produced by cytochrome P450-mediated oxidation of 1,3- butadiene to butadiene monoxide, followed by enzymatic hydrolysis by epoxide hydrolase. The metabolic disposition of BDD is unknown. The current work characterizes BDD oxidation by purified horse liver alcohol dehydrogenase (ADH) and by cytosolic ADH from mouse, rat, and human liver. BDD is oxidized by purified horse liver ADH in a stereoselective manner, with (S)-BDD oxidized at approximately 7 times the rate of (R)-BDD. Attempts to detect and identify metabolites of BDD using purified horse liver ADH demonstrated formation of a single stable metabolite, 1-hydroxy-2-butanone (HBO). A second possible metabolite, 1-hydroxy-3-butene-2-one (HBONE), was tentatively identified by GC/MS, but HBONE formation could not be clearly attributed to BDD oxidation, possibly due to its rapid decomposition in the incubation mixture. Formation of HBO by ADH was dependent upon reaction time, protein concentration, substrate concentration, and the presence of NAD. Inclusion of GSH or 4-methylpyrazole in the incubation mixture resulted in inhibition of HBO formation. Based on these results and other lines of evidence, a mechanism is proposed for HBO formation involving generation of several potentially reactive intermediates which could contribute to toxicity of 1,3- butadiene in exposed individuals. Comparison of kinetics of BDD oxidation in rat, mouse, and human liver cytosol did not reveal significant differences in catalytic efficiency (V(max)/K(m)) between species. These results may contribute to a better understanding of 1,3-butadiene metabolism and toxicity.

Full text of DOI:10.1021/tx960090e

Chem. Res. Toxicol. 1996, 9, 1127-1134
1127
Oxid a tion of 3-Bu ten e-1,2-d iol by Alcoh ol Deh yd r ogen a se
Raymond A. Kemper and Adnan A. Elfarra*
Department of Comparative Biosciences and Center for Environmental Toxicology, School of
Veterinary Medicine, University of WisconsinsMadison, Madison, Wisconsin 53706
Received J une 4, 1996X
3
-Butene-1,2-diol (BDD) is a metabolite of the carcinogenic petrochemical 1,3-butadiene. BDD
is produced by cytochrome P450-mediated oxidation of 1,3-butadiene to butadiene monoxide,
followed by enzymatic hydrolysis by epoxide hydrolase. The metabolic disposition of BDD is
unknown. The current work characterizes BDD oxidation by purified horse liver alcohol
dehydrogenase (ADH) and by cytosolic ADH from mouse, rat, and human liver. BDD is oxidized
by purified horse liver ADH in a stereoselective manner, with (S)-BDD oxidized at ap-
proximately 7 times the rate of (R)-BDD. Attempts to detect and identify metabolites of BDD
using purified horse liver ADH demonstrated formation of a single stable metabolite, 1-hydroxy-
2
-butanone (HBO). A second possible metabolite, 1-hydroxy-3-butene-2-one (HBONE), was
tentatively identified by GC/MS, but HBONE formation could not be clearly attributed to BDD
oxidation, possibly due to its rapid decomposition in the incubation mixture. Formation of
HBO by ADH was dependent upon reaction time, protein concentration, substrate concentra-
tion, and the presence of NAD. Inclusion of GSH or 4-methylpyrazole in the incubation mixture
resulted in inhibition of HBO formation. Based on these results and other lines of evidence,
a mechanism is proposed for HBO formation involving generation of several potentially reactive
intermediates which could contribute to toxicity of 1,3-butadiene in exposed individuals.
Comparison of kinetics of BDD oxidation in rat, mouse, and human liver cytosol did not reveal
significant differences in catalytic efficiency (Vmax/K
m
) between species. These results may
contribute to a better understanding of 1,3-butadiene metabolism and toxicity.
In tr od u ction
reaction, which results in formation of 3-butene-1,2-diol
BDD), appears to be a major pathway for BMO metabo-
lism in humans (20, 21).
(
1
1
,3-Butadiene (BD) is a petrochemical used exten-
sively in the manufacture of synthetic rubber and ther-
moplastic resins. BD has also been found in gasoline,
automobile exhaust, and cigarette smoke (1). Long-term
inhalational exposure to BD produces tumors in multiple
organs in rodents, with mice being significantly more
susceptible to the carcinogenic effects of BD than rats
When BMO or BDD are administered to rodents, only
a small amount of BDD (<2%) appears in the urine.
However, a mercapturate metabolite (1,2-dihydroxy-4-
2
(
N-acetylcystein-S-yl)butane), which appears to be de-
rived from BDD, has been detected in the urine of
animals and workers exposed to BD (20, 21). These data
suggest that BDD may undergo extensive metabolism
prior to urinary excretion. Elucidation of the metabolic
fate of BDD may allow a better understanding of the
metabolism of BD and its mechanisms of toxicity and
carcinogenicity.
(
2-4). Furthermore, several epidemiological studies
suggest that BD is also a human carcinogen (5-7).
The carcinogenic and mutagenic effects of BD have
been largely attributed to its primary metabolite, buta-
diene monoxide (BMO), which is a direct-acting mutagen
(8, 9) and a rodent carcinogen (10). BMO is produced by
One likely route for metabolism of BDD is oxidative
dehydrogenation by cytosolic alcohol dehydrogenases
ADH). These enzymes are ubiquitous in animals and
oxidation of BD primarily by microsomal cytochrome
P450 enzymes (11-15). BMO can be metabolized further
by three separate enzyme systems: cytochromes P450,
glutathione S-transferase, and epoxide hydrolase. Oxi-
dation of BMO by cytochrome(s) P450 results in forma-
tion of butadiene diepoxide (16, 17), which is a more
potent mutagen and carcinogen in rodents than BMO (8,
(
are capable of catalyzing a variety of reactions including
oxidation of primary and secondary alcohols (22) and
carbonyl group reduction (23). Glycols and other diols
are substrates for ADH from a variety of mammalian
species including humans (24). In addition, there is good
evidence to suggest that horse liver ADH (HLADH)
catalyzes formation of R-hydroxyaldehydes from 1,2-diols
(25). ADH has also been shown to play a role in toxicity
of allyl alcohol (26) and ethylene glycol (27). Therefore,
ADH appears to be a good candidate for metabolism of
BDD and may increase the toxic potential of this com-
pound through the formation of reactive metabolites.
1
0). BMO can also be conjugated with GSH in the
presence of cytosolic glutathione S-transferase (18-20).
Finally, BMO can undergo enzymatic hydrolysis, medi-
ated by microsomal epoxide hydrolase (20, 21). The latter
*
To whom correspondence should be addressed at: Department of
Comparative Biosciences, School of Veterinary Medicine, University
of WisconsinsMadison, 2015 Linden Dr. W., Madison, WI 53706.
Phone: (608) 262-6518, Fax: (608) 263-3926, e-mail: elfarraa@
svm.vetmed.wisc.edu.
In the present paper, we report our findings on
oxidation of racemic, (R)-, and (S)-BDD by ADHs. The
results indicate that BDD can be oxidized by HLADH in
X
Abstract published in Advance ACS Abstracts, September 1, 1996.
Abbreviations: ADH, alcohol dehydrogenase; BD, 1,3-butadiene;
1
BDD, 3-butene-1,2-diol; BMO, butadiene monoxide; HBAL, 2-hydroxy-
3
2
-butenal; HBO, 1-hydroxy-2-butanone; HBONE, 1-hydroxy-3-butene-
-one; HLADH, horse liver alcohol dehydrogenase; 4-MP, 4-meth-
2
ylpyrazole.
R. D. Krause, J . E. Sharer, and A. A. Elfarra, Unpublished results.
S0893-228x(96)00090-2 CCC: $12.00 © 1996 American Chemical Society

1
128 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Kemper and Elfarra
a stereoselective manner and that this oxidation is also
catalyzed by ADH from rat, mouse, and human liver
cytosol. Furthermore, we have identified 1-hydroxy-2-
butanone (HBO) as a stable metabolite of BDD and
characterized its formation from BDD by HLADH in vitro
in the presence and absence of GSH and 4-methylpyra-
zole (4-MP), a classic ADH inhibitor (28).
time was 30 min. For inhibition experiments, 4-MP was added
to a final concentration of 50 µM. Reactions were terminated
by immersion in an ice water bath, and the contents were
transferred to 3 mL capacity solid phase Extrelut extraction
columns (EM Science, Gibbstown, NJ ), containing high pore
volume diatomaceous earth granules. Columns were tamped
lightly with a glass stirring rod prior to sample loading to repack
material loosened during shipping and handling. After 3 min,
samples were eluted with 12 mL (2 × 6 mL) of methylene
chloride. Extracts were concentrated to ∼100 µL by centrifuga-
tion under vacuum, and a 3 µL aliquot was removed for GC
analysis. GC analysis was carried out using a Hewlett Packard
Ma ter ia ls a n d Meth od s
Ha za r d ou s Ma ter ia ls. The toxicity of BDD has not been
investigated. However, its parent compound, BD, is an animal
carcinogen and probable human carcinogen. Thus, BDD should
be handled in a fume hood, and protective clothing should be
worn.
5
890 Series II gas chromatograph equipped with a flame
ionization detector (Hewlett Packard, Palo Alto, CA). Separa-
tion of reaction components was accomplished using a 12 m ×
0
.32 mm i.d. DB-1 capillary column (J and W Scientific, Folsom,
Ch em ica ls. (R)-, (S)-, and racemic 3-butene-1,2-diol (purity
CA). The injection port and detector temperatures were 200
and 250 °C, respectively. The column head pressure was 6 psi.
>99.5%) were obtained from Acros Organics (Pittsburgh, PA).
HLADH, HBO, 2-butanone, methyl vinyl ketone, 4-MP, GSH,
and NAD were obtained from Sigma Chemicals (St. Louis, MO).
GC-grade solvents were obtained from Fisher Chemicals (Cin-
cinnati, OH). 1-Butanol (HPLC grade) was obtained from
Aldrich Chemicals (Milwaukee, WI).
The column was maintained at 50 °C for 5 min and then
-1
increased to 200 °C at a rate of 30 °C min
temperature was maintained for 2 min.
. The final
Id en tifica tion of HBO by GC/MS. Combined extracts of
HLADH reactions were used for identification of reaction
products. Identification was accomplished using an HP 5790
mass selective detector interfaced with an HP 5890 Series II
gas chromatograph (Hewlett Packard, Palo Alto, CA). Separa-
tion of reaction components was accomplished using a 10 m ×
An im a ls, Hu m a n Liver Sa m p les, P r ep a r a tion of He-
p a tic Cytosol. Male B6C3F1 mice (20-25 g, J ackson Labo-
ratories, Bar Harbor, ME) and Male Sprague-Dawley rats (150-
2
50 g, Charles River Laboratories, Wilmington, MA) were
housed under standard laboratory conditions and allowed feed
and water ad libitum until sacrifice. Three human liver samples
were obtained from SRI International (Menlo Park, CA) and
maintained at -80 °C prior to subcellular fractionation. Sub-
jects H-19 and H-50 were both 36 year old, Caucasian, non-
smoking males. Subject H-92 was a 15 year old, Caucasian,
nonsmoking female. Mice and rats were sacrificed by cervical
dislocation and decapitation, respectively. Livers were excised,
weighed, and homogenized in 3 volumes of 0.1 M potassium
phosphate buffer (pH 7.4). Cytosol was prepared from whole
liver homogenate by differential centrifugation as described
previously (11) and stored at -80 °C until use. Protein
concentrations were determined by the method of Lowry et al.
0
.25 mm i.d. DB-1 capillary column (J and W Scientific, Folsom,
CA). The temperature program used was the same as described
above, except that the initial column temperature was 40 °C,
the column head pressure was 7.5 psi, and the detector tem-
perature was 280 °C. The mass spectrometer was programmed
for scanning mode with a scan range of 10-400 amu.
Qu a n tita tion of HBO by GC. For quantitation of HBO,
1
-butanol (10 µL of a 1:1000 dilution in water) was added to
reaction mixtures just prior to extraction as an internal
standard. The concentration of HBO in HLADH reaction
mixtures was determined by comparison of the peak area ratio
(HBO/1-butanol) with standard curves prepared from known
concentrations of HBO in reaction buffer and extracted as
described above. The detector response for HBO was linear
(29).
Deter m in a tion of BDD Deh yd r ogen a se Activity. Kinet-
-1
between 1.5 and 100 nmol mL , and the limit of detection was
ics of BDD oxidation were determined spectrophotometrically,
by measuring the rate of reduction of NAD to NADH. Reactions
were carried out at 37 °C in disposable 1.5 mL semimicrocu-
vettes (Fisher Scientific, Cincinnati, OH) and contained 0.05 M
Tris-HCl (pH 7.4), 0.0125 mg of HLADH or ∼0.2 mg of rat,
mouse, or human liver cytosolic protein, 1.35 mM NAD, and
∼
1 nmol. Recovery of HBO under these conditions was ∼70%.
Recovery of HBO from HLADH reactions was accounted for by
preparing standards in buffer and processing them exactly like
the enzyme reactions.
Resu lts
2
.5-200 mM BDD ((R)-, (S)-, or racemic mixture), in a final
St er eoselect ive Oxid a t ion of BDD b y H LADH .
(R)-, (S)-, and racemic BDD were tested for their ability
to serve as substrates for purified HLADH. Representa-
tive velocity versus substrate concentration plots are
shown in Figure 1. Oxidation was rapid with all three
substrates, though high concentrations were required to
achieve saturation of the enzyme. Oxidation of (S)-BDD
was significantly more rapid than oxidation of the (R)-
enantiomer over the concentration range examined. The
rate of oxidation of the racemic mixture was similar to
that of (S)-BDD. Kinetic parameters derived from non-
linear regression of these data to the Michaelis-Menten
volume of 1.0 mL. The reaction time was 3 min for HLADH
experiments, or 10 min for cytosol experiments. These times
were chosen based on the duration of the linear phase of the
reaction. For cytosolic experiments, only racemic BDD was used
as a substrate. Rates of BDD oxidation were corrected for
background NADH production measured in the absence of BDD.
For inhibition experiments, 4-MP was added to a final concen-
tration of 0.125 (HLADH) or 0.5 µM (mammalian cytosol).
Ca lcu la tion of Mich a elis-Men ten P a r a m eter s for Oxi-
d a tion of BDD by HLADH. Initial estimates of K
m
and Vmax
were obtained from double reciprocal plots and used as a
starting point for nonlinear curve fitting. The velocity vs
substrate concentration data were fitted to the Michaelis-
Menten equation using the nonlinear curve fitting feature of
the Sigmaplot software package (J andel Scientific, San Raphael,
CA). This fitting routine employs the Marquardt-Levenberg
algorithm to determine the parameters which provide the
optimal fit of the data to the equation.
equation are presented in Table 1. While the K
m
values
for all three substrates were comparable, the Vmax for
oxidation of the (S)-enantiomer is almost 7-fold higher
than that of the (R)-enantiomer. Consequently, the
m
catalytic efficiency (Vmax/K ) is much greater for (S)-BDD
Detection a n d Qu a n tita tion of P r od u cts of BDD Oxid a -
tion by GC. For initial detection of products of BDD oxidation
by HLADH, reactions were carried out in 0.05 M Tris-HCl (pH
compared to (R)-BDD. The catalytic efficiency of the
racemic mixture is similar to that of the (S)-enantiomer,
suggesting that the kinetics of oxidation of the racemic
mixture are dominated by the (S)-enantiomer.
GC An a lysis of HLADH-Med ia ted Meta bolism of
BDD a n d Id en t ifica t ion of HBO b y GC/MS. The
7
1
1
.4) at 37 °C. Reaction mixtures contained 0.25 mg of HLADH,
.35 mM NAD, and 150 mM racemic BDD in a total volume of
mL. Control reactions in which enzyme, cofactor, or substrate
was omitted were carried out in parallel. The initial reaction

Oxidation of 3-Butene-1,2-diol
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1129
F igu r e 2. Detection of HBO by GC. Reactions were carried
out as described under Materials and Methods and contained
0
.25 mg of HLADH, 150 mM racemic BDD, and either 1.35 mM
+
NAD (top panel) or an equal volume of buffer (bottom panel).
GC conditions are as described under Materials and Methods.
F igu r e 1. Stereoselective oxidation of BDD by HLADH.
Representative velocity versus substrate concentration plots are
shown. (A) (R)-BDD, (B) (S)-BDD, (C) racemic BDD. Reactions
rate of BDD oxidation measured spectrophotometrically
(
∼0.014% of Vmax with racemic BDD), suggesting that
were carried out for 3 min at 37 °C in 50 mM Tris buffer (pH
+
7
.4) and contained 1.35 mM NAD , 125 µg of protein, and
very little of the original oxidation product is converted
to HBO (Figure 4). HBO formation exhibited first order
kinetics at substrate concentrations up to 200 mM
substrate in a final volume of 1.0 mL. Data points represent
observed velocity. Lines indicate computer generated fit of data
points to the Michaelis-Menten equation.
(Figure 4C). The reaction was linear with respect to
Ta ble 1. Mich a elis-Men ten P a r a m eter s for Oxid a tion of
reaction time up to at least 2 h and increased in a linear
fashion with increasing enzyme concentration between
BDD by HLADH
-
1
Vmax (nmol min-1 mg-1
0.125 and 1.0 mg mL
(Figure 4A,B).
substrate
Km (mM)
)
Vmax/Km
Formation of HBO by HLADH was subject to both
enzymatic and chemical inhibition (Figure 5). Inclusion
of 50 µM 4-MP in the reaction mixture decreased HBO
formation by approximately 85% relative to control
reactions, as would be expected for an ADH-mediated
reaction. Likewise, addition of GSH (5 mM) to HLADH
reactions also significantly inhibited formation of HBO
(∼60%). Addition of GSH after termination of the
(
(
(
R)-BDD
S)-BDD
R,S)-BDD 34.0 ( 1.3
28.1 ( 5.9a
33.8 ( 5.6
306 ( 27
2124 ( 191
2496 ( 126
11.1 ( 1.5
63.6 ( 5.8
73.5 ( 3.4
a
Mean (SD for 3-5 separate determinations.
expected reaction products of ADH-mediated oxidation
of BDD are 2-hydroxy-3-butenal (HBAL) and 1-hydroxy-
3
-butene-2-one (HBONE). Efforts were made to detect
these products in extracts of HLADH reactions by GC
analysis. A single unique peak was detected in extracts
of reaction mixtures (Figure 2). This peak, which had a
retention time of 6.4 min under the GC conditions used,
4
enzymatic reaction with ZnSO , followed by an additional
30 min incubation at 37 °C, did not result in decreased
formation of HBO (data not shown). Furthermore,
incubation of commercial HBO (100 µM) with GSH (5
mM) at 37 °C for 60 min did not decrease HBO concen-
trations relative to controls (data not shown), suggesting
that conjugation of electrophilic precursor(s) of HBO with
GSH is responsible for the observed inhibition by GSH.
Because HBONE could be a precursor of HBO, experi-
ments were carried out to determine if HLADH was
capable of directly reducing the double bond of BDD or
methyl vinyl ketone (150 mM), a structural analog of
HBONE, in the presence of NADH (1.35 mM). Neither
of the products expected from these reactions (1,2-
butanediol or 2-butanone) was detected by GC. Further-
more, HBO was not detected in incubations containing
NADH and BDD, which is contaminated with HBONE
(data not shown).
+
was dependent on the presence of enzyme, NAD , and
BDD and was attenuated in the presence of 4-MP (see
below), suggesting that it represented an ADH-dependent
metabolite of BDD. Organic extracts of HLADH-BDD
reactions were pooled, concentrated, and subjected to GC/
MS analysis. The reaction product was identified as
1
-hydroxy-2-butanone (HBO), based on comparison of its
GC retention time and its electron impact mass spectrum
to those of commercial HBO (Figure 3). These data
demonstrate that HBO is a product of ADH-mediated
metabolism of BDD.
Ch a r a cter iza tion of En zym a tic HBO F or m a tion
by HLADH. For a given substrate concentration, the
rate of formation of HBO was very low compared to the

1
130 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Kemper and Elfarra
F igu r e 4. Characterization of HBO formation by HLADH.
Reactions were carried out as described under Materials and
Methods. (A) Time dependence. Reactions contained 0.25 mg of
F igu r e 3. Identification of HBO by GC/MS. Reactions were
carried out and extracted as described under Materials and
+
+
Methods and contained 0.25 mg of HLADH, 1.35 mM NAD ,
HLADH, 1.35 mM NAD , and 150 mM racemic BDD. (B)
and 150 mM racemic BDD. Extracts were pooled for GC/MS
analysis. (A) Pooled reaction extracts. (B) Authentic HBO.
Dependence on protein concentration. Reactions contained 1.35
+
mM NAD and 150 mM racemic BDD. Reaction time was 30
+
+
Fragments m/ z 88, 57, and 29 correspond to M , M - CH
2
-
min. (C) Dependence on substrate concentration. Reactions
+
+
OH, and M - C
2
H
3
O
2
respectively.
contained 0.25 mg of HLADH and 1.35 mM NAD . The reaction
time was 30 min.
Oxid a tion of BDD by Mou se, Ra t, a n d Hu m a n
Liver Cytosol. Oxidation of BDD by cytosolic ADH from
mouse, rat, and human liver was evaluated using racemic
BDD as the substrate. Representative velocity versus
BDD concentration plots for the three species are shown
in Figure 6, and the corresponding apparent kinetic
parameters are presented in Table 2. The apparent K
m
values observed for oxidation of BDD by rat liver cytosol
were comparable to those for purified HLADH, while the
lower values observed with mouse and human cytosol
suggest a somewhat greater affinity for the substrate.
The apparent Vmax for rat liver cytosol was found to be
about twice that in mice, which resulted in comparable
catalytic efficiencies for both species. The variability for
the three human liver cytosol samples was remarkably
low. The catalytic efficiency in humans is comparable
to that in both of the other two species examined. Thus,
these data failed to reveal any marked species differences
in hepatic ADH-mediated oxidation of BDD in mice, rats,
and humans.
F igu r e 5. Inhibition of HBO formation by 4-MP and GSH.
Reactions were carried out as described under Materials and
To confirm the role of ADH in oxidation of BDD,
reactions were carried out in the presence or absence of
+
Methods and contained 0.25 mg of HLADH, 1.35 mM NAD ,
and 150 mM racemic BDD. The reaction time was 30 min. 4-MP
(50 µM) or GSH (5 mM) was added as indicated. Control
reactions contained an equal volume of buffer.
4
-MP, an inhibitor of ADH (28). The effect of 4-MP on
oxidation of BDD by HLADH and rat liver cytosol is
illustrated in Figure 7. The data in panel A show that
4
-MP (0.125 µM) effectively inhibits HLADH-mediated
liver cytosol. Similar inhibition by 4-MP was observed
in mouse and human liver (data not shown). The data
shown in Figure 7B demonstrate the effect of 0.5 µM
oxidation of BDD. The data in panel B suggest that ADH
is the enzyme system involved in BDD oxidation in rat

Oxidation of 3-Butene-1,2-diol
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1131
F igu r e 6. Oxidation of racemic BDD by mouse, rat, and human
liver cytosol. Representative velocity versus substrate concen-
tration plots are shown. Reactions were carried out as described
under Materials and Methods and contained 0.20 mg of cytosolic
F igu r e 7. Inhibition of BDD oxidation by 4-MP. Representative
velocity versus substrate plots are shown. (A) HLADH. Reac-
+
tions contained 0.125 mg of HLADH, 1.35 mM NAD , and
+
protein, 0.675 mM NAD , and racemic BDD in a final volume
substrate in a final volume of 1.0 mL. The concentration of 4-MP
of 1.0 mL. (A) Mouse; (B) rat; (C) human (sample H-19). Values
shown have been adjusted for background NADH formation.
Data points represent observed velocity data. Lines indicate
computer generated fit of observed data to the Michaelis-
Menten equation.
was 0.125 µM. (B) Rat liver cytosol. Reactions contained 0.5 mg
+
of cytosolic protein, 0.675 mM NAD , and substrate in a final
volume of 1.0 mL. The concentration of 4-MP was 0.5 µM.
were quite high (28-34 mM), suggesting that metabolism
of BDD resulting from exposure to BD will be first order
in vivo. This is consistent with values observed for
oxidation of ethylene glycol and 1,2-propanediol by
HLADH (24).
A marked stereoselectivity was evident in HLADH-
mediated oxidation of BDD. While similar K
m
values
were observed for the (R)- and (S)-enantiomers of BDD,
the maximal reaction rate (Vmax) of (S)-BDD was almost
Ta ble 2. Ap p a r en t Kin etic P a r a m eter s for Oxid a tion of
BDD by Mou se, Ra t, a n d Hu m a n Liver Cytosol
species
Km (mM)
20.1 ( 2.2a
32.7 ( 4.7
Vmax (nmol min- mg-1
1
)
Vmax/Km
mouse
rat
human
H-19
10.4 ( 1.2
21.4 ( 1.0
0.53 ( 0.11
0.66 ( 0.09
b
18.3
19.6
22.5
11.6
10.6
9.1
0.63
0.54
0.40
H-50
H-92
7
-fold greater than Vmax for (R)-BDD. This in turn results
a
Mean (SD for three separate determinations. b Values for
in much higher catalytic efficiency (Vmax/K ) for oxidation
m
human samples represent average of two separate determinations.
of (S)-BDD compared to (R)-BDD. The catalytic efficiency
for oxidation of racemic BDD was comparable to that of
the (S)-enantiomer. These data are consistent with the
observed stereoselectivity of ADH toward secondary
alcohols (30, 31), 1,2-diols, and 2-amino alcohols (25).
The major products expected from oxidation of BDD
were HBAL and HBONE. The latter compound could
arise from direct oxidation of the C-2 hydroxyl group, or
from rearrangement of HBAL. We were unable to detect
HBAL by GC or GC/MS in organic extracts of ADH
reaction mixtures. Probable reasons for this include the
relatively high water solubility of short chain hydroxy-
carbonyl compounds, which would complicate organic
extraction, and, more likely, the instability of 2-hydroxy-
aldehydes in aqueous media, where these compounds are
quite prone to hydration, polymerization and rearrange-
ment (25, 32, 33). A second compound (retention time
5.6 min, in Figure 2) found in reaction mixture extracts
had an MS fragmentation pattern consistent with the
4
-MP, though at higher inhibitor concentrations (>5 µM)
complete inhibition of the cytosolic reaction is observed
data not shown).
(
Discu ssion
The present work demonstrates that BDD, a metabo-
lite of the industrial chemical BD, is a substrate for ADH
from horse, rat, mouse, and human liver. Furthermore,
formation of HBO, a stable metabolite of BDD, was
demonstrated and characterized. Since enzymatic hy-
drolysis of BMO to BDD appears to be a significant
metabolic pathway, particularly in primates and humans
(20, 21), the fate of this compound may be important in
overall toxicity and/or carcinogenicity of BD.
HLADH rapidly and stereoselectively oxidized BDD.
However, the Michaelis constants for these oxidations

1
132 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Kemper and Elfarra
+
2
structure of HBONE (major fragments: 55 [M - CH -
+
+
3 3
OH], 31 [M - C H O], 86 [M ], data not shown) but was
subsequently found to be a contaminant in the com-
mercial BDD (∼0.1-0.5%, depending on lot, as deter-
mined by GC analysis). While enzymatic incubations
suggested that 30-35% of the compound present could
not be accounted for by contamination of BDD, its
concentration in ADH reaction extracts could not be
clearly associated with enzymatic oxidation of BDD in
4
-MP inhibition experiments (data not shown). Attempts
to remove this compound from BDD by fractional or
vacuum distillation, or by column chromatography, were
unsuccessful. Preincubation of BDD in buffer alone led
to a rapid decrease in the area of this peak, suggesting
that HBONE readily decomposes in aqueous solutions.
HBO was the only metabolite of BDD conclusively
identified in our experiments. This was somewhat
surprising, since formation of HBO from BDD does not
involve any change in the overall oxidation state of the
molecule. Spontaneous rearrangement of BDD to HBO
was ruled out as a mechanism, since formation of HBO
was strictly dependent on the presence of BDD, NAD,
and enzyme and was inhibited by 4-MP. Another pos-
sible explanation for HBO formation was that oxidation
of BDD merely was acting to supply NADH for an ADH-
catalyzed reduction of some contaminant in the reaction
mixture, such as HBONE. However, substitution of
NADH for NAD as cofactor in the reaction did not result
in formation of HBO (data not shown). Furthermore,
HLADH was incapable of reducing methy vinyl ketone
to 2-butanone, suggesting that direct reduction of the
double bond of BDD or HBONE does not occur in the
presence of HLADH.
F igu r e 8. Proposed scheme for ADH-mediated formation of
HBO from BDD. Species in brackets are hypothetical.
The data are consistent with a mechanism of HBO
formation in which ADH-mediated oxidation of BDD is
followed by rearrangement and subsequent reduction of
the initial product. A possible pathway for this mecha-
nism is illustrated in Figure 8. In this scheme, BDD is
assumed to be oxidized to HBAL. This assumption is
supported by the findings of Matos et al. (25), who
demonstrated that HLADH and yeast aldehyde dehy-
drogenase, coimmobilized on a polyacrylamide gel, oxi-
dized BDD to 2-hydroxy-3-butenoic acid (vinylglycolic
acid). Though quantitative data were not reported, these
results suggest that HBAL was an intermediate in this
reaction. Rearrangement of HBAL could proceed by the
shift of the relatively acidic R-proton from C-2 to C-4. A
similar shift has been proposed for the rearrangement
of 3-butenal to crotonaldehyde (34, 13). The resulting
enol could then rapidly tautomerize to form the 1,2-
dicarbonyl compound 2-ketobutanal (ethylglyoxal). Re-
duction of the aldehyde moiety of the latter compound
by HLADH would generate HBO. Reduction of glyoxals
by ADH has been demonstrated for methyl- and phe-
formation. As mentioned previously, R-hydroxyaldehydes
are highly susceptible to hydration, polymerization, and
rearrangement and are also known react readily with
GSH (36) and other nucleophiles (37). Thus, the amount
of ethylglyoxal available for reduction by ADH would
likely be quite low, resulting in the first order kinetics
of HBO formation observed in our experiments.
Given the well documented species differences ob-
served for in vivo and in vitro metabolism of BD (38-
40) and BMO (41-43), it was of interest to examine
possible species differences in oxidation of BDD by
cytosolic ADH. Racemic BDD was found to be a substrate
for ADH in cytosolic preparations from rat, mouse, and
human liver. Further evidence for the involvement of
ADH in oxidation of BDD by liver cytosol was obtained
by inclusion of 4-MP, an inhibitor of ADH. While both
the Km and Vmax for oxidation of BDD were significantly
lower in mouse liver cytosol than in rat liver cytosol, the
catalytic efficiencies for these two species were compa-
rable. Oxidation of BDD by cytosol from three individual
human liver samples yielded kinetic parameters which
more closely resembled the mouse parameters rather
than rat, but again, the catalytic efficiencies were com-
parable for all three species examined. Thus, these
experiments did not reveal any striking species differ-
ences associated with hepatic ADH-mediated oxidation
of BDD, in agreement with the well documented similar-
ity in structure and catalytic activity among mammalian
ADHs (44, 24). The lack of species differences observed
in the present study is in contrast with the marked
species differences seen in microsomal metabolism of BD
(11, 12) and BMO (12, 17) between rat, mouse, and
human tissue preparations. The data presented here
suggest that differences in hepatic ADH-mediated oxida-
nylglyoxal, both of which are good substrates (K
mM and 0.3 mM, respectively) for ADH (35).
m
) 0.6
The rate of HBO formation is extremely low compared
to the rate of BDD oxidation, as measured by generation
of NADH. The most likely cause for this result is the
instability of the hypothesized intermediates involved.
HBAL, HBONE, and the other postulated intermediates
depicted in Figure 8 are expected to be unstable and/or
reactive compared to BDD and HBO. The inhibitory
effect of GSH on HBO formation observed in our experi-
ments, along with the observation that HBO does not
react directly with GSH, is consistent with the involve-
ment of reactive electrophilic intermediates in HBO

Oxidation of 3-Butene-1,2-diol
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1133
tion of BDD probably do not contribute significantly to
observed species differences in carcinogenicity and toxic-
ity of BD. The possible contributions from ADH in other
tissues remain to be examined.
crotonaldehyde by mouse liver microsomes and chloroperoxidase.
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The data from the experiments reported here suggest
that enzymatic dehydrogenation of BDD to potentially
reactive carbonyl intermediates by hepatic ADH may be
a mechanism for metabolic elimination of BDD. The
biological significance of this metabolic pathway, which
will probably depend on the availability of alternative
routes of BDD metabolism and elimination, remains to
be established. Given the apparent importance of BDD
as a metabolite of BD in humans (20), further investiga-
tion of its metabolic fate is needed.
(
(
(
(
Ack n ow led gm en t. This study was supported by
Grant ES 06841 from the National Institutes of Health.
The authors would like to thank Dr. Harrell E. Hurst of
the University of Louisville Therapeutics and Toxicology
Laboratory for providing GC/MS facilities for this study.
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