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Department of Comparative Biosciences and the Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, Wisconsin
(Received January 27, 2003; Accepted April 25, 2003)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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). BD
has also been detected in cigarette smoke, automobile exhaust, and urban air
(Brunneman et al., 1990).
Epidemiological studies have shown an increased incidence of hematopoietic
cancer among workers exposed to BD
(Santos-Burgoa et al., 1992;
Landrigan, 1993). In 2000, the
U.S. Department of Health and Human Services-National Toxicology Program
classified BD as a "known human carcinogen" (U.S. Department of
Health and Human Services, section 111-14). Long-term inhalation studies in
mice and rats resulted in multisite tumors in both species, although mice
exhibited a higher susceptibility to BD
(Owen et al., 1987;
Melnick et al., 1990).
BD initially is enzymatically converted to butadiene monoxide (BMO) via
cytochromes P450 (Duescher and Elfarra,
1994; Himmelstein et al.,
1994) and myeloperoxidase
(Duescher and Elfarra, 1992).
BMO can be either hydrolyzed to 3-butene-1,2-diol (BDD) by cytosolic or
microsomal epoxide hydrolase (Kemper and
Elfarra, 1996; Krause et al.,
1997), a major metabolic pathway in humans
(Bechtold et al., 1994), or
oxidized by cytochromes P450 to form diepoxybutane
(Csanady et al., 1992;
Krause and Elfarra, 1997). BD
toxicity is currently thought to be associated with the epoxide metabolites
BMO and diepoxybutane (Bechtold et al.,
1994; Xi et al.,
1997; Saranko et al.,
1998).
Although hydrolysis of BMO to BDD is considered a detoxification pathway of
BMO (Himmelstein et al.,
1994), BDD's role in BD toxicity is unclear. This is largely due
to limited research on the in vivo metabolism
(Kemper et al., 1998) and
toxicity of BDD. Previous studies in our laboratory showed that mice treated
with BDD (10–250 mg kg-1) excreted only 2 to 5% of the dose
as BDD (Kemper et al., 1998).
However, the fate of the remainder of the BDD dose was unclear. Earlier
studies in our laboratory (Kemper and
Elfarra, 1996) illustrated an in vitro metabolite of BDD. That
previous study showed the formation of 1-hydroxy-2-butanone (HBO) from BDD and
suggested the involvement of multiple steps involving alcohol dehydrogenase
(ADH; Kemper and Elfarra,
1996). However, it was not known whether HBO is an in vivo
metabolite of BDD in mice or rats.
Production of carboxylic acids from BDD in mice and rats is hypothesized to
occur via the metabolic pathway suggested by Kemper and Elfarra
(1996)
(Fig. 1, pathway 1), and from
the formation of unstable glucuronide/sulfate conjugates
(Fig. 1, pathway 2). In pathway
1, ADH-mediated oxidation of BDD can lead to the formation of 2-ketobutanal
which can be reduced by ADH in the presence of NADH to form HBO
(Kemper and Elfarra, 1996).
The 2-ketobutanal intermediate may also be oxidized by aldehyde dehydrogenase
to form 2-ketobutyric acid. Metabolic studies of 2-ketobutyric acid in rat
hepatocytes and homogenate indicate that 2-ketobutyric acid will readily
decarboxylate, resulting in the formation of propionic acid
(Steele et al., 1984;
Lapointe and Olson, 1985).
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The allylic hydroxy group of BDD may undergo glucuronide or sulfate
conjugation (Fig. 1, pathway
2). Solvolysis of these conjugates may then occur because of the expected
formation of a stable allylic carbonium ion in the transition state of the
reaction (Michejda and Koepke,
1994). However, the allylic carbonium ion may also undergo a
1,2-hydride shift to form 3-butenal, which could be oxidized by aldehyde
dehydrogenase to form vinylacetic acid. The proximity of the internal
methylene moiety of 3-butenal to the vinyl and aldehyde moieties makes these
internal hydrogens very acidic. As a result, 3-butenal could readily
tautomerize to form crotonaldehyde
(Elfarra et al., 1991;
Duescher and Elfarra, 1993).
Crotonaldehyde could then be oxidized to form crotonic acid via aldehyde
dehydrogenase. Therefore, crotonic acid, vinylacetic acid, 2-ketobutyric acid,
and propionic acid represent possible carboxylic acid metabolites of BDD.
These acids and HBO may be present in conjugated or free forms in the
urine.
The goal of this study was to investigate species differences in the
urinary excretion of BDD and its metabolites in male B6C3F1 mice and
Sprague-Dawley rats. Specifically, the metabolites investigated included
propionic acid, crotonic acid, vinylacetic acid, 2-ketobutyric acid, HBO, and
their respective glucuronide/sulfate conjugates. Male B6C3F1 mice and
Sprague-Dawley rats were used based on their known differences in the
metabolism of BD (Richardson et al.,
1999) and BMO (Sharer et al.,
1992; Krause and Elfarra,
1997). The use of multiple species can better indicate the
relative prevalence of specific BDD metabolic pathways.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Animals. Male B6C3F1 mice (24–35 g) were purchased from The Jackson Laboratory (Bar Harbor, ME), and male Sprague-Dawley rats (200–290 g) were purchased from Sasco (Omaha, NE). Mice and rats were maintained on a 12-h light/dark cycle and given water and food ad libitum. Mice were administered 1420 or 2840 µmol kg-1 BDD (equivalent to 125 or 250 mg kg-1, respectively) in saline i.p. and housed together (3/group) in polycarbonate metabolism cages (Nalgene; Nalge Nunc International, Rochester, NY). Rats were dosed with 710, 1420, 2272, or 2840 µmol kg-1 BDD (equivalent to 62.5, 125, 200, and 250 mg kg-1, respectively) i.p. and housed individually in metabolism cages. Control urine was collected from mice and rats before treatment. All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as recommended by the U.S. National Institutes of Health and the University of Wisconsin-Madison Animal Care Committee.
BDD and HBO Analysis. BDD was extracted from urine and derivatized
with BSTFA with 10% TMCS as described previously
(Kemper et al., 1998). Methods
for HBO extraction and derivatization were based on the methods previously
described by Kemper et al.
(1998) with slight
modifications. Briefly, an aliquot of urine (350 µl) and 100 µl of 10
µg/ml 2-butene-1,4-diol as internal standard were placed on the solid-phase
Extrelut packing material inside a glass column (1 g). After 5 min, two 3-ml
aliquots of methylene chloride were added to each column. The combined
methylene chloride extracts were concentrated to 100 to 200 µl under
N2 and then derivatized with BSTFA with 10% TMCS. For HBO
glucuronide and sulfate conjugates, ß-glucuronidase (5000 units) and
sulfatase (300 units) were added to the urine sample (350 µl). Aliquots of
the same urine with no added enzyme were run concurrently to determine the
increase in concentration of each carboxylic acid after
ß-glucuronidase/sulfatase treatment relative to urine without enzymatic
treatment. The solutions were incubated for 20 h at 37°C and then
extracted and derivatized as described above.
Carboxylic Acid Analysis. A method was developed to analyze for propionic acid, crotonic acid, vinylacetic acid, and 2-ketobutyric acid by GC/MS. Urine (900 µl) was combined with 100 µl of 200 µM isobutyric acid as internal standard. Urine was then acidified to pH 1.8 ± 0.05 with 1 mM HCl, and 650 to 700 mg of NaCl was added. The solution was then extracted with methylene chloride (three times, 2 ml). The extracts were concentrated to 2 ml under N2 and derivatized with ethereal diazomethane. The solutions were then further concentrated to 200 µl under N2 before analysis by GC/MS as described below. Some samples were treated with ß-glucuronidase (5000 units) and sulfatase (300 units) and incubated at 37°C for 20 h. Control samples without enzyme were also run to determine whether the acid concentration changed during the assay. The samples were extracted with methylene chloride, derivatized, and analyzed for free acids as described above.
GC/MS Conditions. Analyses of the carboxylic acids, BDD, and HBO were performed on a Hewlett Packard series 6890 gas chromatograph with a mass selective detector. The gas chromatograph was fitted with a DB-1 column (J&W Scientific, Folsom, CA). The injection port temperature was 250°C with a pressure of 1.55 psi. Injections (3 µl) were made in splitless mode at a column head pressure of 1.6 psi. For the carboxylic acid analysis, the initial oven temperature was set to 36°C. The temperature then increased at a rate of 70°C/min to 70°C, then 10°C/min to 90°C, 40°C/min to 130°C, and finally increased 70°C/min to 220°C where it was held for 1 min. Under these conditions, the retention times for propionic acid, isobutyric acid, vinylacetic acid, crotonic acid, and 2-ketobutyric acid were 2.75, 3.66, 3.95, 4.57, and 5.40 min, respectively (Fig. 2).
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Metabolites were initially identified based upon retention times and MS fragments obtained from authentic standards spiked into methylene chloride and methylated by diazomethane. Each acid was then later detected in urine using single ion mode. The ions used for detection of methyl propionate (methylated propionic acid) were at m/z 29 ([CH3CH2]+), 57 ([M-OCH3]+), and 88 ([M]+); for methyl crotonate, 41 ([CHCO]+ and [CH3CHCH]+), 69 ([M-OCH3]+), and 100 ([M]+); for methyl 2-ketobutyrate, 29 ([CH3CH2]+), 57 ([M-C(O)OCH3]+), and 116 ([M]+); for methyl isobutyrate, 41 ([CHCO]+), 43 ([CH2(CH3)CH3]+), 71 ([M-OCH3]+), and 87 ([M-CH3]+); and for methyl 3-butenoate, 41 ([CH2CHCH2]+), 59 ([C(O)OCH3]+), and 71 ([M-CH2CH]+). The concentration of each acid in urine was quantitated by calculating the peak area ratio of each acid to isobutyric acid. Values were corrected for any acids found in the urine prior to treatment. Standard curves (r > 0.99) of urine spiked with each acid along with isobutyric acid were prepared as indicated above. The limits of detection for vinylacetic acid and crotonic acid were 1 nmol ml-1; for 2-ketobutyric acid, 5 nmol ml-1; and for propionic acid, 25 nmol ml-1.
For BDD and HBO analysis the initial oven temperature was 70°C for 5
min. The temperature was then raised at a rate of 30°C/min to 250°C
and held for 5 min. The retention times of BDD, 2-butene-1,4-diol, and HBO
were 7.84, 8.63, and 6.80 min, respectively. BDD and HBO were originally
identified based upon retention times and MS spectra of authentic standards
silylated via BSTFA in ethyl acetate or methylene chloride, respectively. BDD,
2-butene-1,4-diol, and HBO were detected using single ion mode. Trimethylsilyl
(TMS) derivatives of BDD were detected using ions at m/z 103
([TMSOCH2]+), 129 ([M-CH2OTMS]+),
and 217 ([M-CH3]+); for 2-butene-1,4-diol, 129
([M-CH2OTMS]+), 142 ([M-6(CH3)]+),
and 217 ([M-CH3]+); and for HBO, 103
([M-CH2CH3 and 2(CH3)]+).
Concentrations of HBO and BDD were determined by calculating a ratio of HBO or
BDD to 2-butene-1,4-diol in urine before and after BDD treatment. Samples were
corrected for background concentrations found in the control urine. The
resulting ratio was compared to a ratio obtained from standard curves
(r 
0.99) of urine spiked with HBO or BDD along with
2-butene-1,4-diol prepared as indicated above. The limits of detection for BDD
and HBO were 0.1 and 10 µmol ml-1, respectively.
HPLC Analysis of Benzoic Acid and Hippuric Acid. Urine samples (50 µl) were added to deionized water (950 µl). The diluted samples were then deproteinized with 30 µl of iced concentrated HCl. Samples were centrifuged at 3000 rpm, and the supernatant was analyzed on a gradient-controlled HPLC system (Gilson Medical Electronics, Middleton, WI) equipped with a Brownlee Newguard guard column (Rainin Instruments, Woburn, MA) and a Beckman System Gold 166 variable wavelength detector (Beckman Coulter Inc., Fullerton, CA), using a Beckman Ultrasphere 5-µm ODS reverse-phase analytical column (4.6 mm x 25 cm) with UV detection at 229 nm. Samples (20 µl) were separated using 1% acetonitrile adjusted to pH 2.5 with trifluoroacetic acid as a mobile phase for pump A and 75% acetonitrile adjusted to pH 2.5 with trifluoroacetic acid as a mobile phase for pump B. The flow rate was 1 ml min-1. Initially 10% B was maintained for 5 min. The gradient then increased from 10% B to 60% B from 5 to 8 min, where it was then held for 3 min. The percentage of mobile phase B was then decreased from 60% to 10% B from 11 to 14 min and held for 6 min. Hippuric acid and benzoic acid eluted at 10.5 and 12.5 min, respectively. Concentrations of hippuric acid and benzoic acid were determined by comparing the peak area of each acid with standard curves prepared of known concentrations. Due to the presence of hippuric acid and benzoic acid in all urine samples, the standard curves were prepared in water to quantitate the total levels of hippuric acid and benzoic acid in each sample. The area response for both acids was linear from 2 to 2000 nmol ml-1 (r > 0.99). The limits of detection for both benzoic acid and hippuric acid were 2 nmol ml-1.
Statistical Analyses. All results were analyzed using Sigmastat software (SPSS Inc., Chicago, IL). A paired t test was used to determine significance between two groups. To determine significant difference between multiple groups of data, a one-way analysis of variance test was used.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Mice treated with 1420 or 2840 µmol kg-1 BDD excreted similar amounts of BDD whether the data were expressed as µmol kg-1 (Fig. 3) or as the percentage dose recovered as total BDD (3.4 and 1.7%, respectively). Compared to mice treated with 1420 and 2840 µmol kg-1 BDD, rats given an equivalent dose excreted a significantly higher total amount (µmol kg-1) of BDD (p < 0.05; Fig. 3). However, the amount of free BDD excreted by rats given 2840 µmol kg-1 BDD was not significantly different from the amount of free BDD excreted by mice given an equivalent dose.
Detection of HBO and Carboxylic Acid Metabolites of BDD. In both mice and rats treated with BDD, three carboxylic acid urinary metabolites of BDD were identified based upon comparisons of GC/MS retention time and mass spectra with known standards (propionic acid, crotonic acid, and 2-ketobutyric acid; Fig. 2). Vinylacetic acid was not detected in any sample. Propionic acid was present in urine collected before treatment. However, there was a 2- and 3-fold increase in the amounts of propionic acid excreted after mice were given 1420 or 2840 µmol kg-1 BDD, respectively. The amount of propionic acid present in rat urine also increased 2- and 6-fold after administration of 1420 and 2840 µmol kg-1 BDD, respectively (Fig. 2). Crotonic acid and 2-ketobutyric acid were only detected in urine samples after treatment with BDD and were not present in the control urine (Fig. 2). There were no significant differences in the amounts of propionic acid, crotonic acid, or 2-ketobutyric acid when comparing the levels of the three acids at the same dose of BDD within the same species (Table 1). There were no significant differences in the levels of each individual acid between doses within each species or between species given equivalent doses. Furthermore, there were no significant differences in the combined amounts of propionic acid, crotonic acid, 2-ketobutyric acid, and HBO between the different BDD doses within each species and between species given equivalent doses. The combined concentration for all acids for each treatment and species accounted for <1.0% of the dose. The finding that the amounts of carboxylic acids detected before and after enzymatic hydrolysis were similar suggests that only trace levels of glucuronide or sulfate conjugates of the carboxylic acids may have been formed.
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Both rats and mice excreted low levels of HBO as a result of BDD treatment (doses of 1420 or 2840 µmol kg-1; Table 1). HBO was not detected in the urine of mice given 1420 µmol kg-1 BDD (Table 1).
Ratio of Benzoic Acid to Hippuric Acid. Because of the
identification of the carboxylic acid metabolites and their known ability to
form coenzyme A (CoA) esters (Lapointe and
Olson, 1985; Brass et al.,
1986; Rognstad,
1991) and because CoA is necessary for conjugation of benzoic acid
with glycine to form hippuric acid (Gatley
and Sherratt, 1977), the effect of BDD treatment on benzoic acid
and hippuric acid levels was investigated. Rats treated with 1420 or 2272
µmol kg-1 BDD excreted a significantly higher ratio of benzoic
acid to hippuric acid in the urine 0 to 24 h after treatment relative to
control urine (Fig. 4). There
was no difference between the ratios of benzoic acid to hippuric acid in the
urine of rats treated with 710 µmol kg-1 relative to control
urine. Also, there were no significant differences in the ratios of benzoic
acid to hippuric acid in the urine before treatment in rats dosed with 710,
1420, or 2272 µmol kg-1 BDD. However, rats dosed with 1420 and
2272 µmol kg-1 BDD excreted a significantly higher ratio of
benzoic acid to hippuric acid in the urine 0 to 24 h after treatment when
comparing the urine 0 to 24 h after dosage in rats treated with 710 µmol
kg-1 (p < 0.05). There was no significant difference in
the ratio of benzoic acid to hippuric acid in the urine 0 to 24 h after
treatment of rats with 1420 or 2272 µmol kg-1 BDD. In mice
administered 1420 or 2840 µmol kg-1 BDD, there was no
significant difference between the ratio of benzoic acid to hippuric acid
before and after treatment.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Eder and Eder, 2000), and our
laboratory has detected crotonaldehyde in mouse liver microsomes incubated
with BD (Elfarra et al., 1991;
Sharer et al., 1992). Crotonic
acid formation may be low because the oxidation of crotonaldehyde to crotonic
acid is competing with other metabolic pathways, such as glutathione
conjugation (Gray and Barnsley,
1971) or nonspecific addition of crotonaldehyde to cellular
nucleophiles. The identification of crotonic acid is significant because
crotonaldehyde production from BDD could indicate that metabolism of BDD
represents an additional bioactivation pathway for BD.
2-Ketobutyric acid, propionic acid, and HBO are believed to be products of
the same metabolic pathway (Fig.
1, pathway 1). Their low levels indicate that there may be
competition for each step in the pathway. Our laboratory showed that the
primary and secondary hydroxyl group of BDD can be selectively oxidized by ADH
and P450s, respectively (Kemper and
Elfarra, 1996; Krause et al.,
2001). The oxidation of the secondary hydroxyl moiety of BDD
results in the formation of hydroxymethylvinylketone, a Michael acceptor, and
a very reactive compound. Oxidation of BDD to hydroxymethylvinylketone may be
competing with oxidation of the primary hydroxyl of BDD that results in the
formation of propionic acid, 2-ketobutyric acid, and HBO.
The formation of propionic acid from BDD may also elucidate the mechanism
of formation of CO2, a BD-derived metabolite. Richardson et al.
(1999) administered
14C-labeled BD to mice and rats and noted a significant amount of
radioactivity exhaled as 14C-labeled CO2, with rats
exhaling roughly 3 times the concentration (µCi kg-1) of
14C-labeled CO2 relative to mice receiving an equivalent
dose. However, the source of the CO2 was not clear. The
decarboxylation of 2-ketobutyric acid to propionic acid may likely be
contributing to the increase in exhaled CO2 observed in rats. This
notion is further supported by the higher rates of BMO hydrolysis observed in
rats as compared with mice (Krause et al.,
1997). The higher rates of BDD formation in rats could lead to a
greater production of 2-ketobutyric acid and thus be contributing to the
greater formation of CO2 in rats relative to mice.
Overall, the levels of the carboxylic acids recovered in the urine were
low, but this may be due to further metabolism of the carboxylic acids.
Multiple studies have indicated that conjugation with CoA is a major pathway
for the metabolism of carboxylic acids such as propionic acid and
2-ketobutyric acid (Lapointe and Olson,
1985; Brass et al.,
1986; Rognstad,
1991). CoA is a critical intermediate in the conjugation of
benzoic acid, a common component of rodent feed and a breakdown product of
aromatic amino acids, with glycine to produce hippuric acid
(Gatley and Sherratt, 1977).
However, unlike benzoic acid, these small carboxylic acids do not readily form
glycine conjugates because they do not have the appropriate functional groups
(arylacetic, aryloxyacetic, aromatic, and heteroaromatic carboxylic acids) for
glycine conjugation (Knights,
1998). Also, short-chain carboxylic acid CoA esters have been
shown to inhibit the enzymes and transporters necessary for the conjugation of
carboxylic acids with CoA (Sherratt,
1985; Matsuishi et al.,
1991). Therefore, hippuric acid formation may be inhibited by the
BDD-derived carboxylic acid-CoA esters depleting cellular CoA, inhibiting the
enzymes involved in the conjugation of benzoic acid with CoA, or some
combination of CoA depletion and enzyme inhibition. Evidence for this
hypothesis was obtained by investigating the effect of BDD treatment on the
conjugation of benzoic acid with glycine. The significant increase in the
ratio of benzoic acid to hippuric acid in rats treated with 1420 or 2272
µmol kg-1 BDD (Fig.
4) is likely the result of carboxylic acids forming CoA esters
that in turn inhibit benzoic acid conjugation.
The observed species differences in the inhibitory effect of BDD on benzoic
acid conjugation may be related to differences in the ability of rats and mice
to conjugate benzoic acid with glycine
(Seymour et al., 1987).
Because of their lower capacity to conjugate benzoic acid with glycine, rats
may be inherently more susceptible to chemically induced inhibition of
hippuric acid formation in comparison with mice. The formation of carboxylic
acids and the subsequent inhibition of hippuric acid formation may be of
toxicological significance. Carboxylic acids can interfere with glucose
synthesis and mitochondrial function
(Sherratt, 1985;
Brass, 1986). Also, elevated
levels of benzoic acid can interfere with urea synthesis and cause
ammonia-related toxicity (Maswoswe et al.,
1986; O'Connor et al.,
1987).
Mice and rats exhibited significant differences in the elimination of BDD after administration of equal doses (Fig. 3). Of particular interest, only rats exhibited significant differences in the ratio of benzoic acid to hippuric acid after administration of 1420 µmol kg-1 despite excreting significantly more of the dose as BDD compared with mice receiving the same dose. This suggests that the rat specific inhibition of hippuric acid formation is occurring despite rats eliminating a higher amount of the dose relative to mice. A similar trend was seen when comparing rats dosed with 2272 µmol kg-1 BDD and mice administered 2840 mg kg-1 BDD.
In summary, male B6C3F1 mice and Sprague-Dawley rats were dosed with BDD, a significant human in vitro metabolite of BD. Doses of 1420 and 2272 µmol kg-1 inhibited the conjugation of benzoic acid with glycine in the rat. Mice and rats also excreted the BDD metabolites, crotonic acid, propionic acid, 2-ketobutyric acid, and HBO. Although the amounts of the acids and HBO detected in the urine were low in terms of percentage of dose, their formation provides insight about pathways of BDD metabolism in vivo and the potential formation of the toxic metabolite, crotonaldehyde. Collectively, the results provide further characterization of BDD metabolism in rats and mice and illustrate species differences in BDD disposition. The potential role of BDD and its newly identified metabolites in BD toxicity warrants further investigation. Since BDD formation is expected to be a major pathway of BD metabolism in humans, the carboxylic acids identified in this present study may also be detected in the urine of humans exposed to BD.
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Acknowledgments |
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Footnotes |
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1 Abbreviations used are: BD, 1,3-butadiene; BMO, butadiene monoxide; BDD,
3-butene-1,2-diol; HBO, 1-hydroxy-2-butanone; ADH, alcohol dehydrogenase;
GC/MS, gas chromatography/mass spectrometry; HPLC, high-performance liquid
chromatography; BSTFA,
N,O-bis(trimethylsilyl)trifluoroacetamide; TMCS,
trimethylchlorosilane; HMVK, hydroxymethylvinylketone. 
Address correspondence to: Dr. Adnan A. Elfarra, 3154 School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706. E-mail: elfarraa{at}svm.vetmed.wisc.edu
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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palmitic acid and pyruvate metabolism in isolated rat hepatocytes.
Biochim Biophys Acta
888:
18–24.[Medline]
-Ketobutyrate
metabolism in perfused rat liver: regulation of -ketobutyrate
decarboxylation and effects of -ketobutyrate on pyruvate dehydrogenase.
Arch Biochem Biophys
242:
417–429.[CrossRef][Medline]
-ketobutyrate metabolism in rat tissues: effects of
dietary protein and fasting. J Nutr
114:
701–710.
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