Characterization of Urinary Metabolites from Sprague-Dawley Rats

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Chem. Res. Toxicol. 1996, 9, 764-773

Characterization of Urinary Metabolites from Sprague-Dawley Rats and B6C3F1 Mice Exposed to [1,2,3,4-13C]Butadiene Sara K. Nauhaus,†,‡ Timothy R. Fennell,† Bahman Asgharian,† James A. Bond,† and Susan C. J. Sumner*,† Chemical Industry Institute of Toxicology, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, North Carolina 27709, and Department of Chemistry, Duke University, Box 90348, Durham, North Carolina 27708 Received November 20, 1995X

1,3-Butadiene (BD) is used in the production of synthetic rubber and other resins. Carcinogenic effects have been observed in laboratory animals exposed to BD, with mice being more sensitive than rats. Metabolic oxidation of butadiene to epoxides is believed to be a crucial step in the initiation of tumors by BD. However, limited information is available that describes the in vivo metabolism of BD. Male Sprague-Dawley rats and B6C3F1 mice were exposed to 800 ppm [1,2, 3,4-13C]butadiene for 5 h, and urine was collected during and for 20 h following exposure. Urinary metabolites were characterized using 1- and 2-dimensional methods of NMR spectroscopy. Three metabolites previously detected in vivo, N-acetyl-S-(2-hydroxy-3-butenyl)L-cysteine, N-acetyl-S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine, and N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine, were present in both rat and mouse urine, accounting for 87% and 73% of the total metabolites excreted, respectively. A fourth metabolite, previously detected in vitro, 3-butene-1,2-diol, was also present in both rat and mouse urine and comprised 5% and 3% of the total metabolites excreted, respectively. An additional metabolite detected only in mouse urine that is derived from glutathione conjugation with epoxybutene was identified as S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine (4%). N-Acetyl-S-(1-hydroxy-3-butenyl)-L-cysteine (4%), detected in mouse urine, is a thiohemiacetal product of 3-butenal. Additionally, mice excreted N-acetyl-S-(3-hydroxypropyl)-L-cysteine (5%) and N-acetyl-S-(2-carboxyethyl)-L-cysteine (5%), which could be derived from further metabolism of N-acetyl-S-(3,4-dihydroxybutyl)L-cysteine or from glutathione conjugation with acrolein. Mice excreted N-acetyl-S-(1(hydroxymethyl)-3,4-dihydroxypropyl)-L-cysteine (5%), which could be derived from glutathione conjugation with diepoxybutane (BDE), while rats excreted 1,3-dihydroxypropanone (5%), which may be derived from hydrolysis of BDE. These studies indicate that reactive aldehydes are produced as metabolites of BD in vivo, in addition to the reactive monoepoxide and diepoxide of BD. The greater toxicity of BD in mice compared with rats may be attributed to the greater ability of rats to detoxify BDE via hydrolysis, and/or to the production of reactive aldehydes.

Introduction 1,3-Butadiene (BD)1 is a colorless, noncorrosive gas that has major use in the manufacture of styrenebutadiene and polybutadiene rubbers and thermoplastic resins (1). BD has also been detected in cigarette smoke, automobile exhaust, gasoline vapor, and urban air (2, 3). BD is one of 189 pollutants listed in the 1990 Clean Air Act Amendments (4). Mice and rats exposed to BD exhibit toxic and carcinogenic effects, with mice being the more sensitive species to BD-induced carcinogenicity (1). In rats, tumor sites include the mammary gland, Zymbal gland, thyroid, uterus, testis, and pancreas. The sites in mice include the mammary gland, ovary, heart, lung, forestomach, bone marrow, and liver. Characterization of the geno* Address correspondence to: Dr. Susan C. J. Sumner, Chemical Industry Institute of Toxicology, 6 Davis Dr., Research Triangle Park, NC 27709. Phone: 919-558-1343; Fax: 919-558-1300. † Chemical Industry Institute of Toxicoloy. ‡ Duke University. X Abstract published in Advance ACS Abstracts, May 1, 1996. 1 Abbreviations: BD, 1,3-butadiene; BMO, 3,4-epoxy-1-butene; BDE, 1,2,3,4-diepoxybutane; CCC2DQ, carbon-carbon correlation spectroscopy; HET2DJ, two-dimensional heteronuclear J-resolved spectroscopy.

S0893-228x(95)00196-2 CCC: $12.00

toxic properties of BD using a variety of in vitro and in vivo mutagenicity assays suggests that the initiation of cancer by BD requires in vivo metabolic activation to DNA-reactive metabolites, epoxybutene (BMO) and diepoxybutane (BDE) (5). BD is a genotoxicant in vivo in B6C3F1 mice but not in Sprague-Dawley rats (6, 7), suggesting that species differences in the metabolism of BD to the DNA-reactive epoxides may contribute to the greater sensitivity of mice to BD-induced carcinogenicity (8). This conclusion is supported by in vivo studies that demonstrate that mice have higher blood and tissue levels of BMO and BDE compared with rats exposed to BD by inhalation (8-10). The metabolism of BD has been studied in vitro in subcellular fractions of human, mouse, and rat lung and liver (11, 12), and in vivo in mice, rats, and monkeys (8, 9, 13, 14). Metabolic oxidation of BD to BMO is mediated principally by cytochrome P450 2E1 (11). Csana´dy et al. showed that mouse liver and lung microsomes metabolize BD to BMO at a significantly faster rate than microsomes from either rat or human tissues (11). The rates of BMO oxidation to BDE in liver microsomes are significantly greater in mice compared with rats or humans (15). BDE was detected in the blood of mice exposed to BD (62.5© 1996 American Chemical Society

Characterization of Butadiene Urinary Metabolites

1250 ppm) but not rats exposed to the same concentrations of BD (9). However, Thornton-Manning recently detected low levels of BDE (≈50-fold less than mice) in rats exposed to BD (62.5 ppm) by inhalation (10). BD can also undergo oxidation to BMO by peroxidases, including human myeloperoxidase (16), and in bone marrow cell lysates in vitro (17). The oxidation of BD can also lead to the production of 3-butenal and crotonaldehyde in vitro (16, 18-20). However, the formation of these aldehydes in vivo following adminstration of BD has not been demonstrated. S-(2-Hydroxy-3-buten-1-yl)glutathione and S-(1-hydroxy3-buten-2-yl)glutathione have been detected in the bile of rats administered BMO by ip injection (21) and are derived from glutathione conjugation with BMO at the 1 or 2 carbon. The N-acetylcysteine derivatives of these compounds have been detected in the urine of rats and mice administered BMO by ip injection (22). N-AcetylS-(3,4-dihydroxybutyl)-L-cysteine has been detected in urine from rats and mice and is believed to be formed by oxidation of 3-butene-1,2-diol (the hydrolysis product of BMO) to 1-hydroxy-2-oxo-3-butene, followed by conjugation with glutathione and subsequent reduction of the keto conjugate back to its hydroxyl form (23, 24). These metabolites constitute 66% of total radioactivity in urine collected from rats and 78% of total radioactivity in urine collected from mice administered 8000 ppm [14C]BD (23). Urinary metabolites derived from BDE have not been observed, although glutathione conjugation with BDE in liver cytosolic fractions from rats, mice, and humans has been described in vitro (25). Additional BD metabolites have been detected in urine from animals administered 8000 ppm [14C]BD using HPLC with radioactivity detection (23). However, these metabolites (34% of total excreted in rats, 22% of total excreted in mice) have not been characterized. Characterization of urinary metabolites using these methods is often limited by poor HPLC resolution and the proper selection of synthetic standards for identification and quantitation. Quantitation of urinary metabolites has been complicated by unassigned metabolites and the presence of structural isomers of BD-derived metabolites. Rats mainly form N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (52%), while mice form more of the mixture of mercapturic acids of BMO (80%) following administration of 11.7 ppm BD (24). S-(2-Hydroxy-3-buten-1-yl)glutathione and S-(1-hydroxy-3-buten-2-yl)glutathione account for approximately 7% of the administered dose in the bile of rats administered BMO (14.3 or 143 µmol/kg ip) (21). There have been no studies conducted to characterize and quantitate all urinary metabolites of BD and to compare species differences in flux through metabolic pathways. To investigate further the mechanisms involved in BD metabolism between rats and mice, a straightforward method for characterization and quantitation of all metabolites is necessary. Methods of nuclear magnetic resonance (NMR) spectroscopy have been used to characterize and quantitate metabolites in the urine of mice and rats following administration of carbon-13-labeled toxicants (26-28). The use of NMR spectroscopy to characterize metabolites overcomes methodological challenges associated with more conventional techniques by enabling identification and quantitation of metabolites directly in urine. In the present study described in this paper, these methods were employed to examine metabolites in urine from rats and mice exposed by inhalation

Chem. Res. Toxicol., Vol. 9, No. 4, 1996 765

to 800 ppm [1,2,3,4-13C]BD. Two metabolites were characterized in urine from rats (10% of total metabolites), and 6 metabolites were characterized in urine from mice (26% of total). Two metabolites known to be derived from metabolism of acrolein and/or acrylic acid (29-31) were detected in mouse urine. Species differences were observed in the formation of the regioisomers produced from glutathione conjugation with BMO. This study represents the first in vivo identification of metabolites derived from BDE, and implicates reactive aldehydes as intermediates in the formation of some metabolites.

Materials and Methods Chemicals. Carbon-13-labeled (97%) butadiene ([1,2,3,4-13C]BD, CAS Registry No. 106-99-0)2 was obtained from Merck and Co. (St. Louis, MO), with a 97% isotopic enrichment and a 99% chemical purity. The purity was established by the vendor using 1H NMR and GC/MS. The GC/MS gave a m/z value of 58, consistent with 4 labeled carbons. The 1H NMR contained three doublets centered at 6.38 (C-2 H), 5.26 (C-1 Htrans), and 5.14 (C-1 Hcis) ppm with one bond JC-H of 160 Hz. 1,3-Dihydroxypropanone (dimer, 98%, CAS Registry No. 62147-49-3) and 1,4dioxane (HPLC grade, CAS Registry No. 123-91-1) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Animals. Male Sprague Dawley rats (250-270 g, 8 weeks) and male B6C3F1 mice (25-27 g, 8 weeks) were purchased from Charles River Laboratories (Raleigh, NC). They were housed according to recommendations listed in the guide for care and use of laboratory animals (DHEW Publication No. NIH 86-23). They were supplied food (NIH 07 diet) and deionized water ad libitum, except during butadiene exposure, and maintained on a 12-h light-dark cycle at a temperature of 22 ( 2 °C and relative humidity of 55 ( 5%. Exposure System. Animals (3 per group) were exposed to [1,2,3,4-13C]BD using a modified Cannon nose-only exposure system (32). [1,2,3,4-13C]BD was supplied in a sealed glass tube. The tube was placed in an acetone ice bath (-80 °C), condensing the [1,2,3,4-13C]BD. A T-connector with a septum in one port and a bag delivery line in the second port was connected to the exit of the sealed tube. A stainless-steel tube was forced through the septum, breaking the seal and passing into the glass. Air at 2 L/min flowed for 22.5 min through the stainless steel tube and carried the [1,2,3,4-13C]BD to a 45-L bag, reaching 9913 ppm [1,2,3,4-13C]BD. A peristaltic pump running at 0.115 L/min carried the BD from the bag to the nose-only units (NOU). The BD was diluted with 0.965 L/min air to bring the total flow to 1.080 L/min before entering the two NOU, which operated on a push-pull basis. Flow rate through each NOU was proportional to the minute ventilation of each species. Of the total flow rate, 0.900 L/min went into the unit that housed the rats, and the other 0.180 L/min entered the unit that hosted the mice. The animals were exposed for 5 h. The average exposure concentration was 814 ( 222 ppm for rats and 811 ( 235 ppm for mice, as determined by sampling 24 times at a port during the exposure. Sample Collection. Immediately following exposure, animals were placed in glass metabolism chambers for collection of urine for 20 h. Urine was collected from the nose-only tubes and combined with that collected from the metabolism cages. For a control group, male rats and mice (3 animals per group) were exposed to air alone in the Cannon nose-only tower, as described above. At the end of the exposure, the animals were placed in metabolism cages, and control urine was collected for 20 h. All urine samples were centrifuged at 2000g for 20 min to remove particulate material and analyzed immediately or stored at -80 °C. Sample Preparation. Samples were prepared for NMR analysis by concentrating 1 mL of urine (under N2 gas) to 250 µL and adding 200 µL of D2O (mice) or by adding 3.5 mL of 2

CAS Registry Nos. were supplied by the author.

766 Chem. Res. Toxicol., Vol. 9, No. 4, 1996 methanol to 3.5 mL of urine, centrifuging, reducing the volume of the supernatant with N2 gas to 300 µL, and adding 175 µL of D2O (rat). One rat urine sample and one mouse urine sample were prepared for quantitation by adding dioxane as an internal standard. NMR Spectroscopy. NMR spectra were acquired with a 5-mm dual proton-multinuclear probe on a Varian VXR-300 spectrometer (Palo Alto, CA). Carbon-13 NMR spectra were acquired in the double precision mode with an acquisition time of 0.9 s, 30K data points, a relaxation delay of 10 s, and a 45° pulse width. All carbon spectra were acquired with approximately 8000 transients and are referenced to urea at 162.5 ppm. Two-dimensional carbon-carbon connectivity with quadrature in both domains (CCC2DQ) spectra were acquired using the CCC2DQ program from the Varian pulse sequence library. Relaxation delays ranging between 5 and 10 s and τ values corresponding to coupling constants of 40 and 60 Hz were used to acquire data over the entire spectral window. Broad-band decoupling was employed throughout the pulse sequence, with phase-insensitive or phase-sensitive modes, 2048 complex points in t2, and 32 complex points in t1. Two-dimensional heteronuclear J-resolved spectra, with gated decoupling, were acquired using the HET2DJ program from the Varian pulse sequence library. NMR spectra were acquired with 2048 complex points in t2 and 64 complex points in t1. Calculated values of shift for carbons of biochemically feasible metabolites were obtained using incremental substituent effects for alkanes (33), 13C-NMR Module, a Chemintosh application for calculating 13C-shift values (Softshell International, Grand Junction, CO), or C13NMR files available through STN International (Columbus, OH). Metabolite Quantitation. Quantitation of metabolites was carried out by adding dioxane as an internal standard to a urine sample from one mouse and one rat. Metabolite concentrations were calculated by comparing the intensities of metabolite carbon signals to that of dioxane (26, 28). The total amount of BD metabolites present was 5 µmol in the mouse urine sample and 13 µmol in the rat urine sample. In a previous study, the amount recovered in urine after exposure to 1000 ppm [14C]butadiene for 6 h is 7 ( 0.4 µmol in mouse and 21 ( 4 µmol in rat (13). Assuming the total micromoles of BD excreted in urine to be linear relative to dose between 800 and 1000 ppm, the expected amount of butadiene metabolites recovered in urine is 6 µmol for the mouse and 17 µmol for the rat, consistent with 5 and 13 µmol detected in this study. Metabolites of acrylonitrile and acrylamide have also been quantitated using these methods (26, 28), providing results consistent with those observed in studies using radiolabeled chemicals. For rats (n ) 3) and mice (n ) 3) urinary metabolites are presented as a percentage of the total excreted 13C-label. Relative quantitation of metabolites was performed by recording the intensity for one carbon signal for each metabolite. The intensity was multiplied by 2 for carbons that give rise to doublet patterns and by 4 for carbons that give rise to a doublet of doublets pattern. The sum of the intensities for one carbon in each metabolite was adjusted to 100, and each metabolite was recorded as the percentage of total metabolites in the 24 h urine. Statistics. An arcsin transformation was applied to the data, and p values were calculated using Student’s t-test. The 95% confidence intervals were calculated on the arcsin scale and transformed back to the original scale.

Results Structural Assignments. The 1H-decoupled 13C spectrum of control mouse urine (Figure 1A) has an intense peak for urea, referenced to 162.5 ppm (28). Other peaks present in the spectrum are due to endogenous compounds such as creatinine and hippurate (34). Signals not present in spectra of control samples were detected in urine from mice administered 800 ppm BD (Figure 1B,C). Metabolites derived from [1,2,3,4-13C]BD

Nauhaus et al.

Figure 1. 13C NMR spectra of control mouse urine (A) and urine collected for 25 h following exposure to 800 ppm [1,2,3,413C]BD (B, C). Signals for metabolites are labeled according to metabolite number (see Table 1 and Scheme 1) and a letter designating the carbon derived from 3,4-epoxy-1-butene, a, b, c, and d representing the 4, 3, 2, and 1 carbons, respectively.

are recognized by coupling patterns that are produced by spin-spin interactions between the labeled carbons. A 13C nucleus with one adjacent 13C nucleus gives rise to two equally intense resonances, while a 13C nucleus with two adjacent 13C nuclei gives rise to four equally intense resonances. The chemical shift values (for the center of the multiplet patterns) and carbon-carbon coupling constants for the metabolites of BD are listed in Table 1. Each carbon atom was assigned a number corresponding to the metabolite and a letter designating its derivation from 3,4-epoxy-1-butene (a, b, c, and d representing the 4, 3, 2, and 1 carbons, respectively). Carbon-carbon coupling constants measured from the 1-D spectrum may be used to determine signals that arise from adjacent carbons in each metabolite. However, similar metabolite structures can give rise to similar coupling constants and patterns. An unambiguous determination of carbon connectivity can be obtained using 2-D NMR methods for correlating carbon signals. An expanded region of a CCC2DQ spectrum of a concentrated sample of mouse urine is shown in Figure 2. Contours with the same double quantum frequency (i.e., aligned horizontally along the F1 axis) characterize the connectivity of carbons with different chemical shifts (F2 axis). For example, contours arise between some signals associated with carbons from metabolites 1,1′, 2,2′, and 3 in the 13C spectrum (F2 axis), indicating that these signals arise from adjacent carbons in each metabolite. The heteronuclear 2-D J-resolved spectrum (not shown) permits identification of the number of hydrogen atoms attached to each carbon atom. The number of hydrogens



Table 1. 13C NMR Chemical Shifts (ppm), Carbon-Carbon Coupling Constants (Hz) and Multiplicities, Carbon-Carbon Correlations, and Proton Multiplicities for Resonances of Metabolites in Rat and Mouse Urine Produced after Administration of [1,2,3,4-13C]Butadienea compd no.b

chemical shift (ppm)c

7d 3d 8d 3c 7c 8c 1a 1′a 4b 2b 9b 2′b 6b 6a 2a 2′a 7b 5ak 5*ak 3a 10a,c 10a,c 3b 4a 6c 1b, 1′b 5b 5*b 1d 5d 5*d 2d 2′d 4d 4c 2c 2′c 5c 5*c 1c 1′c 8b 10b

27.4 27.8 29.2 32.2 33.8 37.5 38.2 38.4 40.5 50.1 50.3 50.5 53.6 62.9 63.2 63.2 64.8 64.9 64.9 65.2 67.3 67.5 70.5 70.6 l 71.3 72.8 72.8 116.2 116.7 116.7 117.9 118.0 118.8 133.2 135.2 135.4 136.6 136.4 138.3 138.1 180.7 211.8

C-C coupling constants (Hz) and multiplicityd 37 37, 3 37 37, 37 37, 37 37, 54 38, 3 38 36, 41 38, 44 39, 43 38, 44 30, 49 30 38 38 37 41 41 41, 3 41 41 36, 41 36

d d d d, d d, d d, d d d d, d d, d d, d d, d d, d d d d d d d d d d d, d d

38, 46 41, 46 41, 46 69, 3 69, 3 69, 3 69, 3 69, 3 69, 3 42, 69 44, 69 44, 69 46, 69 46, 69 46, 69 46, 69 54 41, 41

d, d d, d d, d d d d d d d d, d d, d d, d d, d d, d d, d d, d d d, d

carbon connectivity (CCC2DQ)e

proton multiplicity (HET2DJ)f

7c 3c 8c 3b, 3d 7b, 7d 8b, 8d 1b

CH2 CH2 CH2 CH2 CH2 CH2 CH2 ndi CH2 CH CH CH nd CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH CH

4a, 4c 2a, 2c 2′a, 2′c 6a, 6c 6b 2b 2′b 7c 5b 5*b 3b 10b 10b 3a, 3c 4b 6b 1a, 1c 5a, 5c 5*a, 5*c 1c 5c 5*c 2c 2′c 4c 4b, 4d 2b, 2d 2′b, 2′d 5b, 5d 5*b, 5*d 1b, 1d 8c 10a, 10c

CH CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH CH CH CH CH CH CH C C

calcd valuesg

13CDatabase or Chemintoshh

31 28 30 35 33 34 40 40 41 52j 52j 52j 58 63 67 67 60 68 68 69 65 65 73 73 78 74 73 78 nci nc nc nc nc nc nc nc nc nc nc nc nc nc nc

27 25 26 35 34 37 38 38 42 55 55 55 44 63 67 67 62 68 65 70 68 68 73 76 73 76 59 72 115 115 117 119 119 115 137 134 134 134 135 137 137 177 202

a Metabolite structures: 1,1′, RSCH CH(OH)CHdCH and/or R′CH CH(OH)CHdCH ; 2,2′, RSCH(CH OH)CHdCH ; 3, RSCH CH 2 2 2 2 2 2 2 2 CH(OH)CH2OH; 4, RSCH(OH)CH2CHdCH2; 5, NHRCH(CH2OH)CHdCH2; 5*, CH2(OH)CH(OH)CHdCH2; 6, RSCH(CH2OH)CH(OH)CH2OH; 7, RSCH2CH2CH2OH; 8, RSCH2CH2CO2H; 9, R′SCH(CH2OH)CHdCH2; 10, HOCH2C(O)CH2OH [R ) N-acetylcysteine, R′ ) cysteine]. Molecular structures 1-10 are proposed from the NMR data and values of shift calculated for mechanistically feasible metabolites. b The carbon atom is assigned a number corresponding to the metabolite and a letter designating its corresponding carbon in butadiene monoepoxide (a, b, c, or d corresponding to the 4, 3, 2, or 1 carbons of 3,4-epoxy-1-butene, respectively). c Chemical shifts are listed for the center of the multiplet patterns. Carbon signal 6d was not resolved due to a poor signal-to-noise ratio and overlap with signals at 65 ppm. Other signals for metabolite 9 overlap the signals of metabolite 2,2′. d Carbon multiplicities were obtained by inspection of the 1-D spectrum; d ) doublet, d, d ) doublet of doublets. e Carbon connectivities were obtained through CCC2DQ spectroscopy and interpretation of 1-D carbon coupling constants. f Proton multiplicities were obtained through HET2DJ spectroscopy. g Calculated values were obtained through addition of incremental effects for alkanes. h Values calculated using SPECIFINO or C13NMR files , or Chemintosh. i nd, not determined; nc, not calculated. j Value corrected for two terminal groups attached to the CH carbon. k Structures 5 and 5* are two possible structures for the same NMR shifts. l Connectivity established via CCC2DQ.

attached to a carbon at a chemical shift position equals the number of contour peaks minus one. For example, a doublet having 3 contours in the F1 dimension defines a CH2 carbon. The assignment of metabolite structures was carried out using the data obtained on proton multiplicity, carbon connectivity, and calculated values of shift. Since the NMR data are obtained for the portion of each metabolite derived from the carbon-13 labels of BD, the remainder of each metabolite structure was assigned by consideration of substituent effects on chemical shift, or comparison with chemical shifts reported for previously assigned metabolites, or calculated values of shift for mechanisti-

cally feasible metabolites. The assigned metabolites and the proposed pathways of formation are shown in Scheme 1. Metabolites in Mouse Urine. Carbon-carbon connectivities for metabolite 1 (Figure 2, Table 1) are established between signals at 71 ppm (1b) and 38 ppm (1a). Correlations are also present between signals at 71 and 138 ppm (1c) and between 138 and 116 ppm (1d). Contours in the HET2DJ spectrum arise from CH2 carbons for signals at 38 and 116 ppm and CH carbons for the peaks at 71 and 138 ppm. The chemical shift positions of 116 and 138 ppm are characteristic of a CdC group. These data suggest an XCH2CHYCHdCH2 struc-

768 Chem. Res. Toxicol., Vol. 9, No. 4, 1996

Nauhaus et al.

Scheme 1. Proposed Metabolism of BD in Rats and Micea

a Processes that should involve several transformations are indicated by broken arrows. Compounds in brackets are suspected intermediates that were not detected in this study. RS ) N-acetylcysteine and Cys ) cysteine.

Figure 2. Expanded region of a CCC2DQ spectrum of mouse urine collected for 25 h following inhalation administration of 800 ppm [1,2,3,4-13C]BD. The urine was concentrated as described under Materials and Methods. Signals for metabolites are labeled according to metabolite number (see Table 1 and Scheme 1) and a letter designating the carbon derived from 3,4epoxy-1-butene, a, b, c, and d representing the 4, 3, 2, and 1 carbons, respectively.

ture. The functional groups X and Y are determined using incremental shift values for substitution on an ethane. The R and β effects for a CdC substituent are 20 and 6, respectively. If X is an RS group (R ) 20, β ) 7) and Y is an OH group (R ) 41, β ) 8), the shift for the ethane (6) CH2 is 40 (20 + 8 + 6 + 6), and the shift for the ethane CH is 74 (20 + 41 + 7 + 6). The values

calculated from substituent effects on an ethane group are near those observed for metabolite 1 (38, 71 ppm) in urine (Table 1). N-Acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine, which is consistent with this proposed structure, has been previously detected in the urine of mice administered BD or BMO (22, 35). Signals with identical coupling constants but 9-fold less intensity than those assigned to metabolite 1 also arise near 38 and 71 ppm. These signals (metabolite 1′) are assigned to either the stereoisomer of metabolite 1 or the cysteine derivative that precedes metabolite 1. The presence of a diastereomer would be due to chiral centers in the BD-derived portion of the molecule (CH group) and in the N-acetylcysteine portion of the molecule (CHR). The presence of small quantities of cysteine derivatives in comparison to the N-acetylcysteine derivatives has been reported in the urine of mice administered 25 ppm [13C]ethylene oxide.3 Metabolite 2 (designated 2,2′) has two sets of similar multiplet patterns (with a ratio of 5:4) with similar chemical shifts and coupling constants (Table 1). CCC2DQ correlations are present between the signal at 50 ppm (2,2′b) and signals at 63 (2,2′a) and 135 (2,2′c) ppm. Correlations are also present for the signals at 135 (2,2′c) and 118 ppm (2,2′d). The peaks at 63 and 118 ppm are assigned to CH2 carbons, and peaks at 135 and 50 ppm are assigned to CH carbons via HET2DJ spectroscopy. Chemical shifts of 118 and 135 ppm are characteristic of a carbon-carbon double bond. Together, these data indicate an XCH(CH2Y)CHdCH2 structure where diastereomers can account for the similar multiplet patterns (28). Calculated shifts using incremental values for X ) RS and Y ) OH (RSCH(CH2OH)CHdCH2) provide values 3

S. C. J. Sumner and T. R. Fennell. Unpublished results.


consistent with those observed in urine. N-Acetyl-S-(1(hydroxymethyl)-2-propenyl)-L-cysteine has previously been described as a urinary metabolite of BMO in the mouse (22), indicating that RS is N-acetylcysteine. For metabolite 3, carbon signals are correlated between 32 (3c) and 28 (3d) ppm and between 32 and 70 (3b) ppm. Correlation also arises between the signals at 65 (3a) and 70 ppm. The HET2DJ spectrum results in assignment of CH2 carbons for 3a, 3c, and 3d and a CH carbon for 3b. The resulting structure is XCH2CH2CHYCH2Z. Calculated values of shift (Table 1) are consistent with X ) RS, Y ) Z ) OH (RSCH2CH2CHOHCH2OH). N-AcetylS-(3,4-dihydroxybutyl)-L-cysteine has been previously described (23, 24) in rat and mouse urine. Metabolite 4 has correlations between the carbon signal at 41 ppm (4b) and signals at 70 (4a) and 133 ppm (4c). A signal at 119 ppm (4d) is correlated to the signal at 133 ppm. Carbons 4b and 4d are assigned to CH2 carbons, and carbons 4a and 4c are assigned to CH carbons via HET2DJ spectroscopy. These data suggest a CHXYCH2CHdCH2 structure. Calculated values of shift (Table 1) are consistent with the structure RSCHOHCH2CHdCH2. Assuming that RS ) N-acetylcysteine, this metabolite is assigned N-acetyl-S-(1-hydroxy-3-butenyl)-L-cysteine, a hemithioacetal. This metabolite has not been previously detected in urine from rats or mice administered BD. Metabolite 4 degrades upon storage at -80 °C. The CCC2DQ data for metabolite 5 have correlations for the carbon signal at 73 ppm (5b) to signals at 65 (5a) and 136 (5c) ppm as well as correlation between the signals at 117 ppm (5d) and 5c. The HET2DJ spectrum results in assignment of CH2 to carbons 5a and 5d and CH to carbons 5b and 5c. Combining these data suggests a structure of XCH(CH2Y)CHdCH2. An RHNCH(CH2OH)CHdCH2 structure (X ) RHN, Y ) OH) has calculated shift values similar to those observed for this metabolite. Reaction of BMO with amine compounds or amino acids has not previously been observed in urine from rats or mice administered BD. However, this type of reaction has been demonstrated between epoxides and the N-terminal valine in hemoglobin (28, 35). Another possible structure that also fits the same NMR data is CH2OHCHOHCHdCH2 (X ) Y ) OH, 3-butene-1,2-diol). This metabolite (5*) has been detected in rat liver microsomes treated with BD (18). 13C NMR shifts for the butenediol (137, 117, 66, and 73 ppm) are consistent with those observed in urine, where minor differences in shift may be due to pH, ionic strength, temperature, concentration, or referencing variabilities. The urinary NMR signals are believed to arise from 3-butene-1,2-diol, since it is known to be formed as a metabolite of BD in vitro. Metabolite 6 has CCC2DQ connectivities between 54 (6b) and 63 ppm (6a). An additional correlation is present between 54 and 71 ppm (6c). The peaks at 71 ppm in the 1-D spectrum are unresolved due to metabolites present in higher concentrations. The signal at 54 ppm is assigned to a CH carbon via HET2DJ spectroscopy. The methine carbon at 54 ppm appears as an eightline pattern consistent with that produced by diastereomers. A structure with calculated values consistent with those observed in urine is RSCH(CH2OH)CHOHCH2OH. Assuming that R ) N-acetylcysteine, this metabolite is assigned N-acetyl-S-(1-(hydroxymethyl)-3,4-dihydroxypropyl)-L-cysteine. This metabolite has not been previously detected in rat or mouse urine.


Metabolite 7 has correlations between carbon signals at 34 (7c) and 27 ppm (7d), and between 34 and 65 ppm (7b). The HET2DJ spectrum led to the assignment of CH2 to all three signals, suggesting an XCH2CH2CH2Y structure. Calculated values of shift (Table 1) are consistent with the structure RSCH2CH2CH2OH, N-acetylS-(3-hydroxypropyl)cysteine. This metabolite has not been detected in rat or mouse urine following exposure to BD. However, it has been detected as a urinary metabolite in rats administered 13 mg/kg acrolein (29). 13 C NMR shifts for a synthetic standard of this metabolite in D2O are 29, 33, and 61 ppm (30). Differences in shift, particularly for the hydroxylated carbon, may be due to pH, concentration, ionic strength, temperature, or referencing variabilities. The CCC2DQ data for metabolite 8 have correlations between carbon signals at 37 (8c) and 29 (8d) ppm and between 37 and 181 (8b) ppm. The HET2DJ spectrum results in the assignment of CH2 to carbons 8c and 8d, and the shift position of 8b indicates a carbonyl structure. These data suggest an XCH2CH2COY structure. Calculated values of shift (Table 1) are consistent with the structure RSCH2CH2CO2H, N-acetyl-S-(2-carboxyethyl)cysteine. This metabolite has not previously been detected in urine of rats or mice administered BD but has been detected in the urine of rats administered acrylic acid (31). 13C NMR shifts for a synthetic standard of this metabolite in D2O are 27, 36, and 180 ppm (31). Differences in shift may be due to pH, concentration, ionic strength, temperature, or referencing variabilities. Small signals (metabolite 9) are present near 50 and 63 ppm with coupling constants identical to those observed for metabolite 2,2′. These signals may arise from the cysteine derivative that precedes metabolism to 2,2′. Metabolites in Rat Urine. The 13C NMR spectra of control rat urine and of urine collected for 24 h following inhalation of 800 ppm [1,2,3,4-13C]BD are shown in Figure 3 panels A, B, and C, respectively. Signals for metabolites 1, 2, 2′, 3, and 5 (5*) are present in spectra obtained from rat urine. An additional metabolite (10) is present in rat urine but not in mouse urine. Metabolite 10 has signals (coupling constants) at 67 (41 Hz) and 212 ppm (41, 41 Hz). The doublet of doublets at 212 ppm with a 41 Hz coupling indicates a ketone carbon (33). The HET2DJ spectrum results in the assignment of CH2 to the signals at 67 ppm. A possible structure that fits these data is HOH2CC(O)CH2OH, 1,3-dihydroxypropanone. This metabolite has not been previously detected in urine from rats or mice administered BD. 13C NMR shifts for a commercially available form of this metabolite dissolved in control urine are 64 and 211 ppm. Quantitation of Metabolites. Metabolites in urine from one rat and one mouse were quantitated using dioxane as an internal standard (Materials and Methods). The total metabolites excreted in the urine of the mouse (5 µmol) were consistent with the detection of 7.0 ( 0.4 µmol of 14C-labeled compounds following administration of 1000 ppm [14C]BD to mice (13). The total metabolites excreted in rat urine (13 µmol) were also consistent with the detection of 21 ( 4 µmol of radioactivity following administration of 1000 ppm [14C]BD to rats (13). Because the total micromoles recovered in this study agree with previous findings, the quantities are presented as a percentage of total excreted metabolites. Relative quantities (average ( standard deviation, n ) 3) for each metabolite are presented in Tables 2 and 3.


Figure 3. 13C NMR spectra of control rat urine (A) and urine collected for 25 h following inhalation administration of 800 ppm [1,2,3,4-13C]BD (B, C). Signals for metabolites are labeled according to metabolite number (see Table 1 and Scheme 1) and a letter designating the carbon derived from 3,4-epoxy-1-butene, a, b, c, and d representing the 4, 3, 2, and 1 carbons, respectively. Table 2. Quantitative Determination of Metabolites of [1,2,3,4-13C]BD Excreted in Mouse Urinea metabolite

chemical shift (ppm)b

% of total metabolitesc

1,1′ 2,2′ 3 4 5* 6 7 8 9

71.1 50.4 32.2 40.5 72.7 52.4 33.8 37.5 50.3

43.9 (42.9, 44.9) 21.6 (21.5, 21.7) 7.1 (6.8, 7.5) 3.7 (3.7, 3.8) 2.9 (1.7, 4.4) 4.6 (4.4, 4.8) 5.4 (5.1, 5.6) 4.8 (4.6, 5.0) 4.7 (4.6, 4.9)

a Mice were exposed to 800 ppm [1,2,3,4-13C]BD for 5 h, and metabolites were measured in the urine excreted during the exposure and for 20 h following exposure. b Chemical shift of signal used in quantitation. c The amount of each metabolite is expressed as a percentage of total metabolites excreted in the urine in 25 h. Values listed are the means (95% confidence interval) for n ) 3.

Table 3. Quantitative Determination of Metabolites of [1,2,3,4-13C]BD Excreted in Rat Urinea metabolite

chemical shift (ppm)b

% of total metabolitesc

1 2,2′ 3 5* 10

38.1 50.4 32.2 72.7 67.2

8.0 (7.9, 8.1) 52.8 (47.5, 58.0) 26.4 (23.7, 29.2) 5.0 (3.0, 7.5) 5.3 (4.5, 6.3)

a Rats were exposed to 800 ppm [1,2,3,4-13C]BD for 5 h, and metabolites were measured in the urine excreted during the exposure and for 20 h following exposure. b The chemical shift of signal used for metabolite quantitation. c The amount of each metabolite is expressed as a percentage of total metabolites excreted in the urine in 25 h. Values listed are the means (95% confidence interval) for n ) 3.

The metabolite detected in highest concentration in mouse urine was N-acetyl-S-(2-hydroxy-3-butenyl)-Lcysteine (metabolite 1,1′), which accounted for 44% of the

Nauhaus et al.

total metabolites excreted. N-Acetyl-S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine (metabolite 2,2′) was formed by the mouse in the second highest concentration (22%). Conversely, metabolite 2,2′ was detected in the highest concentration in rat urine (53% of total), while metabolite 1 accounted for only 8% of the total metabolites excreted. Both rats and mice produce metabolites 3 and 5*, which account for 10% of total in mouse urine and 31% of total in rat urine. Mouse urinary metabolites 1,1′, 2,2′, and 3 account for 73 ( 5% of total metabolites. These data are in agreement with these metabolites comprising 78 ( 6% of total radioactivity as previously reported for mice exposed by inhalation to 8000 ppm [14C]BD for 2 h (23). In rat urine, these three metabolites account for 87 ( 11% of total, consistent with 66 ( 6% of total radioactivity previously reported (23). Five metabolites are detected in mouse urine but not in rat urine, comprising 23% of the total excreted: N-acetyl-S-(1-hydroxy-3-butenyl)-L-cysteine (metabolite 4), N-acetyl-S-(1-(hydroxymethyl)-3,4-dihydroxypropyl)L-cysteine (metabolite 6), N-acetyl-S-(3-hydroxypropyl)L-cysteine (metabolite 7), N-acetyl-S-(2-carboxyethyl)-Lcysteine (metabolite 8), and S-(1-(hydroxymethyl)-2propenyl)-L-cysteine (metabolite 9). A metabolite detected in rat urine but not in mouse urine, 1,3-dihydroxypropanone (metabolite 10), accounts for 5% of the total metabolites excreted.

Discussion This study has demonstrated the utility of 13C NMR in the detection, identification, and quantitation of BD metabolites directly in the urine of rats and mice following administration of [1,2,3,4-13C]BD. The presence of three previously described metabolites has been verified, along with the identification of six new metabolites in mouse urine and two new metabolites in rat urine. General advantages of this method for characterizing the metabolism of small molecules include the simultaneous detection directly in the urine of all excreted metabolites, the fact that considerable structural information can be obtained from the spectral data without the need for metabolite standards, and the avoidance of chromatographic techniques that may alter metabolite structure. Although the sensitivity of NMR is much lower than that of HPLC and GC/MS, carbon-13 NMR provides resolution of signals for individual metabolites, including structural isomers and diastereomers, at both low and high concentrations. Metabolites that accounted for as little as 3% of the total metabolites excreted in mouse urine (5 µmol) and 5% of total metabolites excreted in rat urine (13 µmol) were characterized. Ten metabolites of BD were characterized based on NMR data obtained for the portion of each metabolite derived from the carbon-13 labels of BD, consideration of substituent effects on chemical shift, comparison with chemical shifts reported for previously assigned metabolites, and calculated values of shift for mechanistically feasible metabolites. Knowledge of the structures of these additional metabolites will aid in the determination of appropriate synthetic standards, enabling more comprehensive studies of BD metabolism to be performed, particularly for identification of metabolites in target tissues. The metabolite detected in highest concentration in mouse urine results from conjugation of BMO with glutathione at the one carbon and is assigned to N-acetyl-


S-(2-hydroxy-3-butenyl)-L-cysteine (metabolite 1,1′). This metabolite has previously been identified in rat and mouse urine (22-24) and accounts for 44% of the total metabolites excreted in mouse urine but only 8% of the total metabolites excreted in rat urine. The metabolite detected in highest concentration in rat urine arises from glutathione conjugation with BMO at the 2 carbon and is assigned to N-acetyl-S-(1-(hydroxymethyl)-2-propenyl)L-cysteine (metabolite 2,2′). This metabolite has been previously described in rat and mouse urine (22) and comprises 53% of the total metabolites excreted in rat urine but only 22% of the total metabolites in mouse urine. Mice exhibit a preference (2:1) toward glutathione conjugation with BMO at the 1 carbon (44%, metabolite 1,1′) over the 2 carbon (22%, metabolite 2,2′), consistent with the ratio observed in previous studies (1.85:1) (22). For rats, the ratio of metabolite 1 to metabolite 2 is 1:7, indicating a preference for glutathione conjugation at the 2 carbon of BMO. Previous studies with BMO in the rat have shown a lower ratio (1:3) for these conjugation reactions (22). The difference in ratios observed in the rat may be related to differences in administered chemical and dose (71.85-285 µmol/kg BMO vs 800 ppm BD) or route of administration (ip vs inhalation) between the two studies. One metabolite derived from glutathione conjugation with BMO is detected only in mouse urine. S-(1-(Hydroxymethyl)-2-propenyl)-L-cysteine (metabolite 9) may be a cysteine derivative and precursor to metabolite 2,2′ and accounts for 5% of total excreted. Because the percentage of this metabolite is small, the contribution to the ratios of metabolites derived from glutathione conjugation with BMO at the 1 or 2 carbon is negligible. The hydrolysis product of BMO, 3-butene-1,2-diol (metabolite 5*), was detected in rat and mouse urine and accounts for 5% and 3% of the total metabolites excreted, respectively. This compound has been detected as a metabolite of BD in in vitro metabolism studies using rat liver microsomes (18). Oxidation of 3-butene-1,2-diol followed by conjugation with glutathione and subsequent reduction produces another previously described metabolite, N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (metabolite 3), which is present in rat and mouse urine as 26% and 7% of the total metabolites excreted, respectively. Metabolite 4, identified as N-acetyl-S-(1-hydroxy-3butenyl)-L-cysteine, is a hemithioacetal product of 3-butenal. This product is detected only in mouse urine and accounts for 4% of the total metabolites excreted. 3-Butenal has been proposed as an intermediate in the oxidation of butadiene to crotonaldehyde (16, 19, 20). N-Acetyl-S-(3-hydroxypropyl)cysteine (metabolite 7, 5% of total excreted) and N-acetyl-S-(2-carboxyethyl)cysteine (metabolite 8, 5% of total excreted) are detected only in mouse urine and could be derived from several pathways. One pathway would involve further metabolism of metabolite 3, or intermediates involved in its syntheiss (23), resulting in the loss of a labeled carbon. An alternative pathway could involve conversion of butadiene to acrolein, which undergoes further metabolism by glutathione conjugation to S-(oxoethyl)glutathione, and following oxidation or reduction will produce metabolites 7 and 8, respectively (29, 30, 36-39). Metabolite 8 may also be formed as a metabolite of acrylic acid (31), which can be produced by oxidation of acrolein (36). The oxidation of BD to give 3-butenal and crotonaldehyde in vitro has been described previously (18, 19). Metabolism of crotonaldehyde in rats results in the


formation of N-acetyl-S-(3-hydroxy-1-methylpropyl)cysteine and N-acetyl-S-(2-carboxy-1-methylethyl)cysteine (40). Neither of these metabolites was detected in this study, suggesting that crotonaldehyde was not formed to a significant extent in vivo. This may result from metabolism and excretion of 3-butenal prior to its rearrangenment to give crotonaldehyde. The production of acrolein from BD by metabolic processes has not previously been reported. It is possible that acrolein could be derived from BD by a peroxidative cleavage. N-Acetyl-S-(1-(hydroxymethyl)-3,4-dihydroxypropyl)-Lcysteine (metabolite 6), detected only in mouse urine (5% of total excreted), could be derived from glutathione conjugation at the 2 carbon of BDE. 1,3-Dihydroxypropanone (metabolite 10), found only in rat urine (5% of total excreted), could be derived from phosphorylation of BDE or its hydrolysis product erythritol to give erythrose phosphate. Metabolism of erythrose phosphate in the pentose phosphate pathway (41) results in the formation of dihydroxyacetone (metabolite 10). Another possible pathway to produce this compound could involve cytochrome P450 oxidation and phosphorylation of 3-butene1,2-diol (metabolite 5*). However, cytochrome P450 conversion of the butenediol has not been demonstrated in vivo or in vitro. Exhaled 14CO2 has been detected from rats exposed to radiolabeled BD (13, 14). The identification of metabolites containing only three of the BD-derived carbons makes it possible to elucidate pathways that may lead to the formation of CO2 (Scheme 1). The terminal hydroxyl group of metabolite 3 may be oxidized to a carboxyl group and CO2 given off upon decarboxylation to form metabolite 8. If metabolite 7 is formed from acrolein, then acrolein production will release a onecarbon fragment which may be another source of CO2. Metabolism of BDE through the pentose phosphate pathway to form metabolite 10 may also generate CO2. Metabolites derived from pathways that generate CO2 account for 10% of the total in mice and 5% in rats. BDE metabolic products are excreted in the same relative quantities in both mice (metabolite 6, 5%) and rats (metabolite 10, 5%). However, the removal processes of BDE appear to be different for the two species. Rats metabolize BDE through hydrolysis and subsequent sugar metabolism to form metabolite 10. Mice, on the other hand, metabolize BDE through glutathione conjugation to form metabolite 6. This result supports in vitro studies that showed BDE conjugation with glutathione to be more efficient in mouse liver (Vmax/Km ) 25.3) and lung (Vmax/Km ) 21.0) cytosol than in rat liver (Vmax/Km ) 7.6) and lung (Vmax/Km ) 4.1) cytosol, respectively (25). Differences in the production and further metabolism of BDE have important consequences for the toxicity of BD. BDE is more genotoxic than BMO or 3,4-epoxy-1,2butanediol (42). Glutathione transferase 5-5 expression enhances the mutagenicity of BDE in a bacterial mutagenicity assay (43), suggesting that GSH conjugates of BDE may be genotoxic. With detection and quantitation of these additional metabolites, the flux through metabolic pathways can be determined. The two major groups of BD metabolites are those derived from glutathione conjugation with BMO (metabolites 1,1′, 2,2′, and 9) and those derived from hydrolysis of BMO (metabolites 3 and 5*). Significant species differences in the formation of products arising from glutathione conjugation with BMO were not observed (70% in mouse, 61% in rat). This observation


corroborates in vitro studies which reported similar rates for BMO conjugation with glutathione between rats and mice (11). Species differences in the formation of products derived from glutathione conjugation with BMO following 8000 ppm exposure to BD for 2 h (60% of total radioactivity in mouse urine, 38% in rat, p e0.05) have been reported (23). Differences between these studies could be attributed to differences in exposure concentration. The formation of N-acetyl-S-(3,4-dihydroxybutyl)-Lcysteine (metabolite 3) is significantly different (p ) 0.01) between rats and mice. Previous studies at high BD exposures have shown the formation of this metabolite to be similar in mice (21 ( 19% of total excreted metabolites) and rats (35 ( 3%) (23). Species differences in flux through hydrolysis were also determined considering all metabolites formed through this pathway. The formation of metabolite 3 and its precursor 1,2-hydroxy3-butene (metabolite 5*) is significantly different at p ) 0.09 between rats (31% of excreted metabolites) and mice (10%). However, if N-acetyl-S-(3-hydroxypropyl)-cysteine and N-acetyl-S-(2-carboxyethyl)cysteine (metabolites 7 and 8) are considered to be further metabolic products of metabolite 3, the total metabolites derived from hydrolysis of BMO in mice (20%) and in rats (31%) are not statistically significant (p ) 0.29). In summary, 13C NMR was used to characterize six metabolites in mouse and two metabolites in rat urine following exposure to BD. Metabolites were detected in mouse urine that are also seen following exposure to acrolein and acrylic acid, and it is possible that these compounds may arise directly from BD oxidation, or indirectly from further metabolism of crotonaldehyde. In either case, the issue of a potential overlap in biomarkers of exposure between BD, acrolein, and acrylic acid becomes important (29). Metabolites derived from BDE were similar in rats and mice when expressed as a percentage of dose or as a percentage of total metabolites. However, when normalized to body weight (µmol/kg body wt), the amount of BDE-derived metabolites was 4 times greater in mouse urine than in rat urine. The greater body burden of BDE in the mouse and the ability of rats to detoxify BDE through hydrolysis may be related to the greater toxicity of BD in the mouse. The metabolites of BD derived via aldehyde intermediates in the mouse also suggest a potential role for these reactive intermediates in the toxicity of BD.

Nauhaus et al.

(4)

(5) (6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

Acknowledgment. Ms. Glenda Jones and Mr. Horace Parkinson are acknowledged for their assistance in sample collection and Mr. Del Ponder for his aid in exposing animals. We are also grateful to Mr. Paul Ross and the animal care staff at CIIT. We thank Dr. Barbara Kuyper for her excellent editorial review of this paper and Dr. Derek Janzen for his help in the statistical analysis.

(19)

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(21)

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TX950196U

Characterization of Urinary Metabolites from Sprague-Dawley Rats

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