Chem. Res. Toxicol. 1995, 8, 68-76
68
Synthesis and Characterization of N-Acetyl-L-cysteine S-Conjugates of Butadiene Monoxide and Their Detection and Quantitation in Urine of Rats and Mice Given Butadiene Monoxide Adnan A. Elfarra,” Jane E. Sharer, and Renee J. Duescher Department of Comparative Biosciences and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53706 Received May 13, 1994@ Butadiene monoxide (BM), a mutagen and carcinogen, is the major metabolite of 1,3butadiene in rats and mice. Because mercapturic acids (N-acetyl-L-cysteineS-conjugates) were expected in vivo metabolites of BM, reference BM-mercapturic acids were prepared by the reaction of racemic BM with N-acetyl-L-cysteine. Four isomers were purified and characterized as diastereomeric pairs of S-(2-hydroxy-3-buten-l-yl)-N-acetyl-~-cysteine (I) and S-(l-hydroxy3-buten-2-yl)-N-acetyl-~-cysteine (11) based on analyses by lH NMR, fast atom bombardment mass spectrometry, and high resolution electron impact mass spectrometry. Regioisomers I and I1 were identified in the urine of rats and mice administered (ip) BM based on GC/MS analyses performed after HPLC fractionation followed by esterification and silylation of the carboxyl and hydroxyl groups, respectively, and comparison of GC retention times with synthetic standards. S-(4-Hydroxy-2-buten-l-yl)-N-acetyl-~-cysteine, a rearrangement product formed during chemical synthesis or storage of both I and 11under acidic conditions, was not detected; no other BM metabolites were evident in urine samples using this method. When rats were given BM at a dose of 71.5 to 285 pmol/kg, their urinary excretion of I and I1 within 8 h of BM administration exhibited linear relationships with the administered BM dose; the total amount of the BM dose excreted as combined I and I1 averaged 17 f 4% (mean f SD, n = 15). No metabolites were detected in urine samples collected between 8 and 24 h after BM dosing. Mice, which are known to be more sensitive to 1,3-butadiene carcinogenicity than rats, excreted similar amounts of mercapturic acids (26 f 13%)at the 285 pmol/kg BM dose within 24 h of BM administration, however, at the 143 and 71.5 pmoVkg BM doses, they excreted only 7 f 3% and 9 f 3%of the BM dose as mercapturic acids, respectively. Thus, the ability of the rat to excrete higher levels of BM-mercapturic acids compared t o the mouse at low BM doses may partially explain the lower sensitivity of the rat to 1,3-butadiene-induced carcinogenicity. Furthermore, the BM-mercapturic acid analysis method, which has limits of detection of 25 and 40 pM in rat and mouse urine, respectively, may be used to assess human exposure to 1,3-butadiene.
Introduction
Butadiene monoxide (BM;’ 1,2-epoxy-3-butene) has been identified as the major in vitro metabolite of 1,31,3-Butadiene, a colorless gas used in the industrial butadiene in human, mouse, and rat liver microsomes production of synthetic rubbers and plastics and detected (8-12). BM has also been recognized as the primary 1,3in gasoline vapor, automobile exhaust, and cigarette butadiene in vivo metabolite in the rat (11). In rat liver smoke, has been found to be more toxic and carcinogenic microsomal incubations, both enantiomers of BM were in mice compared to rats in chronic exposure studies (1produced (12). BM was shown to be mutagenic in the 5). Whereas the International Agency for Research on Ames test as well as an effective inducer of sister Cancer has classified 1,3-butadiene as “possibly” carcichromatid exchange and chromosome aberrations in vivo nogenic t o humans (6)and epidemiological studies have (13, 14). Furthermore, BM was demonstrated t o be suggested an increase in lymphatic and hematopoietic carcinogenic in mouse skin painting studies (15). Becancers in occupationally exposed workers (71, the biocause these studies suggest that BM may play a role in chemical basis for species differences in 1,3-butadiene 1,3-butadiene-induced carcinogenicity, the metabolism carcinogenicity remains unclear. and disposition of BM was investigated. In previous studies (16-18), we have shown that cytosolic glu* To whom correspondenceshould be addressed at the Department tathione 5’-transferase catalyzed the conjugation reaction of Comparative Biosciences, University of Wisconsin School of Veteriof BM with glutathione (GSH) (Figure 1)in vitro t o form nary Medicine, 2015 Linden Dr. W., Madison, WI 53706. Abstract published inAduance ACSAbstracts, November 15,1994. S-(2-hydroxy-3-buten-l-yl)glutathione (BM-GSH I) and 1 Abbreviations: BM, butadiene monoxide (1,2-epoxy-3-butene); S-( l-hydroxy-3-buten-2-yl)glutathione (BM-GSH 11). PuGSH, glutathione; BM-GSH I, S-(2-hydroxy-3-buten-l-yl)glutathione; rified human placental glutathione S-transferase cataBM-GSH II,S-(l-hydroxy-3-buten-2-yl)glutathione; DNFB, 2,4-dinitroflnorobenzene;ACN, acetonitrile;I, S-(2-hydroxy-3-buten-l-yl)-N- lyzed the reaction with a Vmaxof 500 nmol/(mg.min), and acetyl-L-cysteine; 11, S-(l-hydroxy-3-buten-2-yl)-iV-acetyl-~-cysteine; mouse liver, lung, kidney, and testis cytosolic fractions FAB/MS, fast atom bombardment mass spectrometry;EI-MS, electron were also shown to catalyze the reaction a t rates equal impact mass spectrometry. @
0893-228d95/2708-0068$09.00/00 1995 American Chemical Society
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 69
Butadiene Monoxide Mercapturic Acids
1 CST I G S H
BY-CSH
I
BY-GSH
II
TOOH
C H C HN H C 0 C H
-/S
t
/
OH
I
4"
0 OH
S CH,CHNHC OCH,
I1 Figure 1. Pathway of BM-mercapturic acid biosynthesis. GST, glutathione S-transferase; GSH, glutathione; BM-GSH I, S42hydroxy-3-buten-1-y1)glutathione; BM-GSH 11,S-(l-hydroxy-3buten-2-y1)glutathione; I, S-(2-hydroxy-3-buten-l-yl)-N-acetylL-cysteine; 11, S-(l-hydroxy-3-buten-2-yl)-N-acetyl-~-cysteine. "he three consecutive arrows represent metabolism of the GSH conjugate by y-glutamyl transpeptidase, followed by dipeptidase, and then acetylation by N-acetyltransferase.
I 0
I
I 10
I
I 20
1
I 30
Time (min) Figure 2. Reverse-phase HPLC chromatogram of BM-Nacetyl-L-cysteine S-conjugate crude reaction mixture showing the formation of five products (peaks 1-5).
wks) were obtained from Jackson Laboratories (Bar Harbor, ME). Rats were housed in Nalgene metabolic cages (Rochester, NY) (l/cage) which were later adapted for use with mice (Ucage). to or greater than those of the respective rat tissues. In Animals were kept on a 12 h light cycle and given food and water initial in vivo studies in which rat bile ducts were ad libitum. surgically cannulated, bile collected for 2 h after ip Synthesis of BM-N-Acetyl-L-Cysteine S-Conjugates. administration of BM (14.3-285pmoVkg) contained only Conjugates were synthesized under conditions similar to those 8%of the total molar dose given as BM-GSH conjugates. used to synthesize BM-GSH conjugates (17). Briefly, N-acetylBecause GSH conjugates of several xenobiotics are L-cysteine (280 mg, 1.7 mmol) was dissolved in 10 mL of acetone known to be further metabolized in vivo to form N-acetyland water (1:l v/v) and the pH adjusted t o 8.5 with triethylL-cysteine S-conjugates (mercapturic acids) which are amine. A molar excess of BM (0.17 mL, 2.1 mmol) was added, then excreted in urine, metabolism of BM via the merand the solution was held a t reflux for 5.5 h. Excess BM was removed by extracting three times with ethyl ether in a 1:2 capturic acid pathway (Figure 1) was characterized volume ratio and the aqueous solution rotary evaporated to near further in this study. Five conjugates from the chemical dryness. Distilled deionized water (10 mL) was added and the reaction of racemic BM with N-acetyl-L-cysteine,including two stereoisomers each of S-(2-hydroxy-3-buten-l-y1)- pH adjusted to 3.5 with acetic acid. "he solution was then N-acetyl-L-cysteine(I) and S-(l-hydroxy-3-buten-2-ylI-N- passed through two Bio-Rad AG ll-A8 ion retardation columns (Richmond, CA) to remove metal ions followed by rotary acetyl-L-cysteine (II), were characterized. After the evaporation and lyophilization to dryness. A white crystalline development of a sensitive GC method to detect and substance was recovered, giving a crude material yield of 148%, quantitate BM-mercapturic acids in control mouse and indicating the sample was highly hygroscopic. rat urine, I and I1 were then identified and quantitated HPLC Analysis and Purification of Synthetic BM-Nin the urine of rats and mice given BM (71.5-285pmol/ Acetyl-L-cysteine S-Coqjugates. HPLC analysis of crude material was performed using a Beckman gradient-controlled kg). HPLC system (pump A, Model llOB; pump B, Model 114M, San Ramon, CA) equipped with a 3-cm Brownlee ODS guard column Experimental Procedures (San Jose, CAI, and a Spectroflow 757 variable wavelength Chemicals. Racemic BM, 1-methyl-3-nitro-1-nitrosoguani- detector (Kratos Analytical Inc., Ramsey, NJ) on a Beckman dine, chlorotrimethylsilane, HPLC-grade pyridine, 2,4-dinitrofUltrasphere 5-pm ODS reverse-phase analytical column (4.6 x 250 mm) with UV detection a t 210 nm. Samples (20 pL) were luorobenzene (DNFB), trifluoroacetic acid, DzO (99.96%D), and injected and separated using a mobile phase of 1% ACN and MezSO-de (99.9% D) were obtained from Aldrich Chemical Co. 0.1% trifluoroacetic acid, pH 2.5, at a flow rate of 1 mUmin. (Milwaukee, WI). N-Acetyl-L-cysteine and porcine kidney acylase I (grade 1, specific activity = 2125 unitdmg of protein) were Five major peaks were separated with retention times of 21.8, purchased from Sigma Chemical Co. (St. Louis, MO). HPLC22.9, 23.6, 26.2, and 28.3 min and designated with labels 1-5, respectively (Figure 2). Bulk separation of crude material was grade and bioanalytical-grade acetonitrile (ACN) was obtained performed using a Gilson gradient-controlled HPLC system from EM Science (Gibstown, NJ). GC-grade carbonyl-free ethyl (Model 302 pumps; Middleton, WI) equipped with a Bio-Rad ASacetate was obtained from Baxter-Burdick & Jackson (Muskeg100 HRLC automatic sampling system, a 3-cm Brownlee ODS on, MI). N M R supplies were purchased from Wilmad Glass Co. guard column, and a Beckman Model 167 scanning absorbance (Buena, NJ). All other chemicals were of the highest grade commercially available. Caution: BM is a known mutagen and detector (Irvine, CA) on a Beckman Ultrasphere 5-pm ODS carcinogen in laboratory animals and must be handled using reverse-phase semipreparative column (10 x 250 mm) with UV detection at 210 nm. Injection volumes of 1mL were separated proper safety measures. Animals. Male Sprague-Dawley rats (230-290 g) were with a gradient of 3.5% ACN, pH 2.5, adjusted with trifluoroacetic acid for 8 min, decreased to 1.0%ACN in 6 min where it obtained from Sasco (Omaha, NE). Male B6C3F1 mice (6-8
70 Chem. Res. Toxicol., Vol. 8, No. 1, 1995 remained for 18 min, and then returned to 3.5% ACN in 6 min with a constant flow rate of 3 mumin. Peaks 1-5, which had retention times of 24.8, 26.5, 27.9, 31.3, and 34.3 min, respectively, were collected with a Gilson 203 microfraction collector. Purified solutions were each rotary evaporated and dried as described for the crude reaction mixture. Purities of each of the conjugates were greater than 95% as determined by HPLC. MS Analyses of Synthetic Conjugates. Positive ion fast atom bombardment mass spectrometry (FAB/MS) analyses of synthetic BM-mercapturic acids were performed using a Kratos MS-5OTC ultrahigh-resolution mass spectrometer (Manchester, United Kingdom) equipped with a saddle field fast atom bombardment gun. Conjugate peaks 1-4 (1-4 mg each) were dissolved in a glycerol matrix and analyzed with a spectrum ranging from m/z 100 t o m/z 300. When necessary, HCl was added to the sample to remove heavy Na+ contamination. Highresolution electron impact mass spectrometry (EI-MS) analyses of BM-mercapturic acids (conjugate peaks 1-5) were performed using a Kratos MS-80. Samples were placed directly on the probe before spectra were obtained a t 44 eV ionization energy. NMR Spectroscopy. Proton NMR spectra of components 1-5 dissolved in DzO or MezSO-ds were obtained on a Bruker spectrometer (Karlsruhe, Germany) a t 500 MHz with chemical shifts reported in ppm from sodium 3-(trimethylsilyl)tetradeuteriopropionate. In samples analyzed in DzO, irradiation a t 4.8 ppm was performed to minimize the residual water peak.
HPLC Separation and Direct W Detection of BM-NAcetyl-L-CysteineS-Conjugates in Urine. Urine samples containing various concentrations of synthetic BM-N-acetylL-cysteine S-conjugates were separated using the same HPLC system described above for the analysis of the synthetic conjugates alone. The gradient was changed, however, to minimize background. Here, mobile phase was held a t 1%ACN for 27 min, after which it was increased to 50%ACN in 10 min, held at 50% ACN for 3 min, and returned to 1%ACN in 10 min. Peaks 1-5 eluted with retention times of 23.4, 24.4, 25.3, 28.9, and 31.1 min, respectively. Deacetylation and Derivatization of BM-N-Acetyl+ cysteine S-Conjugates for HPLC Analysis. Enzymatic deacetylation using acylase I was done in a fashion similar t o that of Osterman-Golkar et al. (29).BM-N-acetyl-L-cysteine S-conjugate reaction mixture (1.4 mg, 6 pmol) was incubated a t 30 "C with 3790 units of acylase I in 1.0 mL of phosphate buffer (0.1 M K&Po4, 0.15 M KCl, 1.5 mM EDTA, pH 7.4) for up to 9.3 h. At various time points, aliquots of 100 pL were removed and the reaction was stopped with 5 pL of trifluoroacetic acid. Samples were filtered with 0.2-pm LC 13 acrodisc membrane filters (Gelman Sciences, Ann Arbor, MI) and analyzed directly by HPLC as described above for the synthetic mixture. Nonenzymatic deacetylation was performed by incubating BM-N-acetyl-L-cysteine S-conjugates in 0.5 mL of 7 M HCl for 8 h a t 50 "C. Samples were then transferred to ice, and the pH was adjusted slowly to 2-3 with 7 M NaOH, filtered, and analyzed directly by HPLC. Samples deacetylated nonenzymatically were derivatized with DNFB. An aliquot (0.5 mL) of 1.0 mM deacetylated BMN-acetyl-L-cysteine S-conjugate I1 was mixed with 0.5 mL of 3% DNFB in ethanol and 100 pL of 1 M Na~C03.The pH was then adjusted to 2 9 with 5 pL of 4 N NaOH, and the sample was kept a t 25 "C in the dark for 24 h. The solution was filtered and analyzed by HPLC at 365 nm using the Beckman system described above. The gradient was varied beginning at 37.5% ACN for 18 min, followed by an increase t o 75% ACN in 8 min. In Vivo Experiments. Doses of 285,143, and 71.5 pmolkg BM in 3 m u g corn oil o r corn oil alone were given ip t o Sprague-Dawley rats or B6C3F1 mice. Rat urine was collected a t 0-8 and 8-24 h; 0-8 h urine was diluted to 5.0 mL with deionized HzO and stored a t 0 "C until analysis, whereas 8-24 h urine was stored a t 0 "C without dilution. Due to smaller urine volumes and less frequent urination, mouse urine was collected a t 0-24 h, after which it was diluted to 4.0 mL with deionized HzO and stored a t 0 "C until analysis.
Elfarra et al. GC Analysis of BM-N-Acetyl-L-cysteineS-Conjugates. Aliquots of 1.0 mL of urine were acidified with 3 drops of 10 M HCl, vortexed, and centrifuged. Supernatants of rat urine were filtered through 0.2-pm LC 13 acrodisc membrane filters, and 500 pL was injected on a Beckman Ultrasphere 5-pm ODS reverse-phase semipreparative column (10 x 250 mm) installed on the Beckman HPLC system described above. The detection wavelength was 210 nm, and flow rate was 3 m u m i n . A gradient containing 1%ACN, pH 2.5, in pump A and 75% ACN, pH 2.5, in pump B was used to separate the mercapturic acids away from a large portion of the other urine components. Initially, the system increased from 0% to 30% B over 12 min when it was held for 3 min. The gradient was then increased to 100% B over 7 min, where it was held for an additional 2 min. It was then decreased to 0% B for a total run time of 40 min. The mercapturates eluted between 10 to 12 min. Collection of the desired fraction was from 7 to 14 min to ensure that the mercapturates were collected. The collected fraction was lyophilized to dryness. Deionized water (100 pL) was added to the lyophilized sample to dissolve it, and the sample was transferred to a diazomethane generator (Aldrich Chemical Co.) where it was mixed with 2 mL of ACN. Esterification was begun with the addition of 0.5 mL of 5 M NaOH to 100 mg of l-methyl3-nitro-1-nitrosoguanidine in the upper chamber of the generator, and the reaction was allowed to proceed for 90 min. Chambers were opened to terminate the reaction, and excess diazomethane was evaporated from the ACN solution with N2 flushing. The solution was then dried completely by speed vacuum lyophilization. Samples were dissolved in 40 pL of 1:3 H20/ACN solution and transferred to screw-cap glass vials containingmagnetic stir bars. Ethyl acetate (1.0 mL) was added to samples followed by 1.0 mL of 20% pyridine in ethyl acetate and 0.75 mL of 20% chlorotrimethylsilane in ethyl acetate with constant stirring. After 15 min, silylation was terminated with the addition of 1mL of H2O and stirred rapidly until cloudiness disappeared. The organic layer was then removed and concentrated by speed vacuum lyophilization, dried with a Na2S04 sample drying device (Whatman, Clifton, NJ), and the volume was adjusted to 0.5 mL with ethyl acetate or further evaporation with a Nz stream. Samples (3 pL) were analyzed on a HewlettPackard 5890A gas chromatogram (Avondale, PA) equipped with a split injector, a DB-1 15 m x 0.32 mm i d . , 3-pm film thickness column (J&W Scientific, Folsom, CAI, and a flameionization detector with a Hewlett-Packard 3393A integrator. The injector temperature was 175 "C; the detector temperature was 250 "C; the helium carrier gas flow was 2.9 mL/min. A temperature gradient was used of 175 "C initial temperature followed by an increase to 240 "C a t a rate of 10 "C/min, where it was held for 11 min. Peaks eluting at 8.4 and 8.6 min were quantitated by comparisons of their heights with standard curves obtained using equal amounts of synthetic stereoisomers of I1 and I, respectively, dissolved in urine and subjected to similar fractionation and derivatization procedures. The standard curves in rat and mouse urine exhibited excellent correlation coefficients (0.997 and 0.999, respectively). Good reproducibility was also observed over the entire length of the study. S-(4-Hydroxy-2-buten-l-yl)-N-acetyl-~-cysteine (peak 5) was found to elute at 10.9 min when derivatized and analyzed in the same manner.
GC/MS Characterization of the Derivatized BM-NAcetyl-L-cysteineS-Conjugates. Urine collected from three rats dosed with 285 pmolkg BM was fractionated and derivatized as described above. The ethyl acetate solutions obtained after the silylation reactions were combined and concentrated to 0.5 mL under a N2 stream. GC/MS analyses were carried out using a Kratos MS25 with a Carlo Erba GC/mass spectrometer in the electron impact mode. The injector was set a t 175 "C and the ion source a t 250 "C. A split injection (0.1 pL) was run on a 60-m Supelco DB-5 capillary column with helium as the carrier gas. Initial oven temperature was 175 "C, which was increased a t a rate of 10 W m i n to 240 "C, where it was held for 5 min. Retention times of the derivatized BMmercapturates I1 and I were 6.80 and 6.87 min, respectively.
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 71
Butadiene Monoxide Mercapturic Acids
Table 1. SO0 M H z 'H NMR Data for Peaks 1-S in D2O (J Values Reported in Hz) 2.06 ppm, s, 3H,CH3; 2.88 ppm, dd, lH,Cyss (J= 14.1,8.3);3.06ppm, dd, lH,Cysp (J= 14.1,4.5); 3.49 ppm, dt, lH,H4 (J= 9.0,6.4);3.69ppm, d, 2H,H& (J = 5.8);4.53 ppm, Cysap 5.24ppm,d,0.5H,nH~(J=17.9);5.27ppm,d,0.5H,nHz(J= 10.5);5.70ppm,ddd, 1H,H3(J=17.0,lO.1,g.O) 2 2.05 ppm, s,3H,CH3; 2.73ppm, dd, lH,HdH6 (J= 13.6,7.3);2.79ppm, dd, IH,HdH6 (J = 13.6,5.2); 2.95ppm, dd, lH,Cysj3 (J = 14.0,8.1);3.10 ppm, dd, lH,Cyss (J = 14.0,4.6);4.29ppm, H4;a 4.52 ppm, Cysa;a 5.22 ppm, d, 0.5H,"HZ(J= 10.5);5.30 ppm, d, 0.5H,"Hi (J= 17.3); 5.88ppm, ddd, lH,H3 (J = 17.1,10.4,6.5) 3 2.05ppm, s,3H,CH3; 2.72ppm, dd, lH,HdH6 (J = 13.6,7.2);2.82 ppm, dd, lH,HdH6 (J = 13.4,5.2); 2.93 ppm, dd, 1H,Cy@ (J = 13.9,8.2);3.11ppm, dd, lH,Cysj3 (J= 14.0,4.2);4.30ppm, dd, H4;a4.45ppm, dd, Cysa;n 5.23ppm, d, 0.5Hp HZ(J= 10.9);5.31ppm, d, 0.5H,"Hi (J = 17.7);5.89 ppm, ddd, lH,H3 ( J = 17.2,10.6,6.3) 4 2.04ppm, a, 3H,CH3; 2.93ppm, dd, lH,Cyes (J= 13.9,7.4);3.00 ppm, dd, lH, Cysg (J = 13.8,4.8); = 6.2);4.46ppm, Cysa;a 3.48 ppm, dt, lH,H4 (J = 8.5,6.6);3.70 ppm, AB ofAFJX,2H,H a 6 (JAB= 11.3,JAX= 6.5,JBX 5.24ppm,d,0.5H,OH~(J=16.1);5.25ppm,d,0.5H,"H~(J=11.2);5.71ppm,ddd,1H,H3(J=17.7,9.0,9.0) 5 2.06ppm, s,3H,CH3; 2.83 ppm, dd, Cy@ (J= 14.0,7.8);2.99 ppm, dd, lH,Cysj3 (J = 13.8,4.3); 3.22ppm, d, 2H,HE& (J= 7.0);4.11 ppm, d, 2H, (J= 5.5);4.36 ppm, dd, lH,Cysa (J = 7.7,4.2); 5.71ppm, dt, lH,H4 (J values unavailable); 5.81 ppm, dt, lH,H3 (J values unavailable) a Interference exists with signals and/or integrations due to proximity to irradiated DOH peak. 1
)=W3
"2
IH 3
NI H C O C H 3
'( I
H,
OH
SCH,CH
acetyl
H
COOH
A
B
.
-7.
6.0
. .
#
5.5
..
.
.
I. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.0
4.5
4.0
PPI4
3.5
3.0
2.5
7-7 2.0
Figure 3. 500 MHz 'H NMR spectra of reaction mixture peaks 2 (A) and 3 (B),corresponding to the two possible stereoisomers of BM-N-acetyl-L-cysteine S-conjugate I. These spectra were recorded in DzO.
Results Identification and Characterization of Synthetic N-Acetyl-L-cysteine S-Conjugates of BM. The reaction of racemic BM with N-acetyl-L-cysteine yielded five peaks of unknown identity separable by reverse-phase HPLC (Figure 2). These peaks were designated 1-5 in order of their elution. Peaks were individually collected with HPLC preparative chromatography and characterized by lH N M R spectroscopy (see Table 11,FABMS, and high-resolution EI-MS. Peaks 1and 4 were determined to be stereoisomers of 11 and peaks 2 and 3 to be stereoisomers of I, based on expected proton chemical shifts and integrations, and comparisons with previouslyassigned protons in the lH NMR spectra of BM-GSH conjugates I and I1 (Figure 1) (17).Regioisomers of I and I1 were distinguished by chemical shifts of protons €&and H5H6; the & signal was d o d e l d (4.30 ppm) and H5H6 upfield in the spectra of peaks 2 and 3 as would be expected if the 0 and S atoms were bonded to their respective carbons (Figure 3). Likewise, chemical shiRs of H4 (3.50 ppm) and H& (3.70 ppm) in spectra of peaks 1and 4 indicate S is bonded to the I& carbon and 0 to the H5H6 carbon (Figure 4). The weak signals of HIHz and Cysa in all four spectra and I& in spectra of peaks 2 and 3 appear to be consequences of the strong irradia-
tion necessary to suppress the water peak at 4.8 ppm. When samples were analyzed in MezSO-d6, H1Hz and H3 displayed the expected 2:l ratio (Figure 5). Peak 5 was assigned the structure of S-(4-hydroxy-2-buten-l-yl)-Nacetyl-bcysteine based on expected proton chemical shifts and integrations, and comparison with previously-assigned signals of BM-GSH conjugate 111,S-(4-hydroxy2-buten-1-yl)glutathione (17). Further analysis of the lH NMR spectra gave insight into other structural features of the conjugates. All conjugates showed coupling between the individual Cysp protons, indicating a rigid cyclic conformation including the Cysp protons. This may be caused by hydrogenbonding between the sulfur atom and a proton on the N-acetyl-L-cysteine moiety, most likely the N-proton, since this splitting was also seen in the GSH conjugates and the carboxyl proton would not be available in these conjugates. Peaks 2,3, and 4 also show splitting between the individual H& protons, indicating another possible cyclic conformation, formed by a probable H-bonding interaction between the OH group and the N-acetyl-Lcysteine moiety. The finding that peak 1did not display this splitting suggests that the C3-C4 bond was rotating freely. The increased hydrophilicity from the free OH group would explain why peak 1 elutes relatively early in reverse-phase chromatography.
Elfarra et al.
12 Chem. Res. Toxicol., Vol. 8, No. 1, 1995 I!
NHCOCI13
SCH,CH "1
OH
\ COOH
acetyl H
",",
I
B
...
.---7
6 0
5 5
~
5.0
....,.,..
. . . , . . . . 4 5
4 0
PPI4
3.5
. . . . , . . ,. 2 5
3.0
. . . . , 2.0
Figure 4. 500 MHz lH NMR spectra of reaction mixture peaks 1 (A) and 4 (B), corresponding to the two possible stereoisomers of BM-N-acetyl-L-cysteine S-conjugate 11. These spectra were recorded in DzO.
L
100
-
80
-
60
-
40
-
IO2
A
M+ 1 234
136
,
256
0 150
100
B loo 80
11
200
250
B lo2
,136
60
40
M+ 1
256
234
r- 1 6.0 5.5
I
I
I
5.0
4.5
4.0
20
PPm
Figure 5. 500 MHz lH NMR spectra of reaction mixture peaks 2 (A) and 4 (B), corresponding to BM-N-acetyl-L-cysteine S-conjugates I and 11,respectively, in the 4-6 ppm region. The spectra were recorded in MezSO-ds.
FAB/MS analyses of peaks 1-4 yielded a pseudomolecular ion of mlz 234, consistent with the expected molecular weight of an N-acetyl-L-cysteine conjugate of BM 1 (Figure 6). The peak at mlz 256 corresponds to M Na, whereas the fragments a t mlz 219, 216, and 192 correspond to loss of a methyl group, water, and a ketene moiety from the M 1 peak, respectively. The fragment at mlz 162 (Figure 6A) indicates cleavage of the C-S linkage of the butadiene moiety. The fragments a t
+ +
+
-
0 100
150
200
250
M/Z Figure 6. Positive ion FABMS of a stereoisomer of mercapturic acid conjugates I (A)and I1 (B).
mlz 154 and 102 correspond to the two fragments expected by cleavage of the C-S linkage of the cysteine Na ion (mlz 256), whereas the moiety of the M fragment at mlz 136 may have resulted from the loss of water from the 154 ion. High-resolution EI-MS analyses of peaks 1-5 provided further evidence for the identity of these peaks. The
+
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 73
Butadiene Monoxide Mercapturic Acids
+
mass of the protonated molecular ions (M H) obtained with peaks 1-5 were 234.0810, 234.0801, 234.0810, 234.0795, and 234.0804, respectively. The calculated A mass for the protonated BM-N-acetyl-L-cysteine Sconjugates is 234.0800. Peaks 2 and 3, which correspond to the two possible stereoisomers of BM-N-acetyl-Lcysteine S-conjugate I, also exhibited molecular ion (M) peaks at 233.0674 and 233.0597, respectively. These peaks are in agreement with the calculated mass (233.0722) for BM-N-acetyl-L-cysteine S-conjugate I. Ratios of peaks 1-5 immediately following BM-Nacetyl-L-cysteine synthesis were approximately 7:6:6:7: a 1, respectively. The amount of peak 5 varied depending 8 a on reaction conditions (length of reaction and reagent 9 concentrations). After storage of the reaction mixture at c* pH '3, 25 "C, for 5 days, peaks 1-4 each lost 21-28% area whereas peak 5 increased 4.5-fold. Conjugates stored in acidic solution at 0 "C for 9 weeks showed a 6-7-fold increase in peak 5 and a parallel decrease in peaks 1-4. Peaks 1 and 4 (If) invariably decreased a t faster rates than 2 and 3 (I), and peak 4 decreased a t a faster rate than peak 1under the same conditions. When conjugates were stored at 0 "C in HzO, pH 7, or at -30 "C or colder under acidic conditions for up to 9 weeks, I I I I I no significant changes in peak areas were observed. 7 9 11 13 15 Detection of BM-N-Acetyl-L-cysteine S-CoqjuTime (min) gates in Urine. GC analysis of derivatized mercapturates was investigated since many N-acetyl-L-cysteine Figure 7. Gas chromatograms of typical extracted, derivatized urine samples obtained from (A) rat dosed with corn oil alone S-conjugates had previously been analyzed by this method and (B)rat dosed with corn oil and 285 pmolkg BM exhibiting (20,21). Upon GC analysis of the methylated BM-Nconjugate I and Il after esterification with diazomethane and acetyl-L-cysteine S-conjugate reaction mixture, a broad silylation with chlorotrimethylsilane. unresolved peak was observed with a retention time of 7.6 min that was not seen when the reaction was run in lowing isolation by HPLC and derivatization with dithe absence of the conjugates (data not shown). Derivaazomethane and chlorotrimethylsilane (Figure 8). The tization of the hydroxyl groups with chlorotrimethylsilane expected molecular ion (mlz 319) was not detected, but in pyridine was then performed to improve resolution. ions at mlz 304 (M - 15; loss of a methyl group), 260 (M Under identical GC conditions as the analysis of the - 59; loss of COOCH3), and 229 (M - 90; loss of HOSimethylated mercapturates, two sharp peaks were ob(CH&) were detected. This fragmentation pattern is served with retention times of 8.4 and 8.6 min as well as common for silylated hydroxyl and methyl ester derivaa smaller sharp peak at 10.9 min. Later analysis with tives. The base peak (mlz 73) corresponds to the expected the individual synthetic conjugates revealed the peak at Si(CH& fragment. The ions a t 176 (M - 143; C-S 8.4 min to be both stereoisomers of conjugate I1 and that cleavage of the cysteine moiety), 170 (229 - 59; loss of at 8.6 min to be the stereoisomers of conjugate I. The COOCH& 144 (M - 175; C-S cleavage of the cysteine peak at 10.9 min was determined to be the esterified, moiety), 129 (144 - 15; loss of a methyl group), and 103 silylated derivative of S-(4-hydroxy-2-buten-l-yl)-N-acetyl- (176 - 73; loss of Si(CH3)3) were also detected. These L-cysteine. This method of derivatization and analysis results provide strong evidence for the chemical structure gave limits of detection of 2 pg/mL (9 pM) for all of the derivatives and, thus, provide further evidence for conjugates. the identity of the urinary metabolites. Characterization and Quantitation of BM-NThe dose-response of BM-N-acetyl-L-cysteine S-conAcetyl-L-Cysteine S-Coqjugates in Urine of Rat and jugates measured in the urine of rats 8 h after dosing is Mice Dosed with BM. Upon GC analysis of urine of shown in Figure 9A. Urinary excretion of I and I1 rats treated with a high BM dose (285 pmolkg), the two exhibited linear relationships with the administered BM peaks at 8.4 and 8.6 min were prominently visible (Figure dose; the total amount of the BM dose excreted as 7B). These peaks were not seen in rats dosed with corn combined I and I1 averaged 17 f 4% (mean f SD, n = oil alone. Further, no background peaks were observed 15). No BM-N-acetyl-L-cysteine S-conjugates were dein the 8.4-8.6 min elution region of the control urine, tectable in rat urine collected 8-24 h after dosing. All indicating little potential for interference from backdoses taken together, the excretion of I1 over I was preferred by nearly 3 to 1. Figure 9B shows the doseground at low doses. This was also true for control mouse urine. No other peaks were observed up to 15 min that response profile for recovery of BM mercapturates from were not apparent in the urine of control rats or mice, mice at 24 h. BM-N-acetyl-L-cysteine S-conjugates including the expected retention time of S-(Chydroxy-2accounted for 25.7 f 13.0% of the 285 pmoVkg BM dose buten-1-y1)-N-acetyl-L-cysteine (10.9 min). The limits of and only 7.7 f 2.6% and 9.6 f 4.7% of the 143 and 71.5 detection in rat urine determined by standard curves pmolkg BM doses, respectively. In contrast to rats, mice were 25 pM for conjugate I or 11. In mouse urine, limits preferentially excreted regioisomer I (1.85-fold) over of detection for conjugate I or I1 were 40 pM. regioisomer I1 with no changes in regioisomeric selectivity as the BM dose was vaned from 71.5 to 285 pmolkg The BM-mercapturic acid metabolites present in rat urine were also characterized by GCMS analyses fol(Figure 9).
74 Chem. Res. Toxicol., Vol. 8, No. 1, 1995 100
-
80
-
60
-
Elfarra et al.
73
103
176
0
100
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0
100
200
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229 217
100
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IO0
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73
B
FOOCH3 SCH~CHNHCOCH,
I
O S i ( CH,)3 129
100
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;I
0 50
I
i
:03 20
1
I I I
144
'70
I
b 100
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260 300
M/Z
Figure 8. GC/mass spectra of urinary metabolites of BM; BM conjugates I1 (A) and I (B) were isolated and derivatized as described under Experimental Procedures.
0
100
200
300
B M ("Vkd Figure 9. Dose-response relationship of BM-N-acetyl& cysteine S-conjugate excretion in animals dosed with 5-20 mg/
Discussion
kg (71.5-285 pmollkg) BM: (A) rat, (B)mouse. Closed circles represent BM-N-acetyl-L-cysteine S-conjugate I; open circles represent BM-N-acetyl-L-cysteine S-conjugate II. (C) Combined I and 11of rat (open squares) and mouse (closed squares). Values represent the means f SD of the results obtained from 5 (rat) or 4 (mouse) experiments.
In this paper we report the synthesis and characterization' of five N-acetyl-L-cysteine S-conjugates of BM. These conjugates were determined to consist of the two stereoisomers of the product of N-acetyl-L-cysteine nuThe data described in this manuscript provide the first cleophilic attack on C4 of BM, conjugate I, and the two conclusive characterization of the N-acetyl-L-cysteine stereoisomers of the product of nucleophilic attack on C 3 S-conjugates of BM. Osterman-Golkar et al. reported the of BM, conjugate 11. The fifth mercapturic acid was characterization of synthetic BM cysteine conjugates characterized as S-(4-hydroxy-2-buten-l-yl)-N-acetyl-~- based on positive ion chemical ionization mass spectromcysteine. S-(4-Hydroxy-2-buten-l-yl)-N-acetyl-~-cysteineetry of the heptafluorobutanoic acid anhydride derivamay be formed from conjugate I or I1 by a mechanism tives of the conjugates which were assumed to be which involves the formation of a cyclic sulfonium by predominantly S-(2-hydroxy-3-buten-l-yl)-~-cysteine intermediate (17)) since this conjugate can also be formed analogy to the reaction of BM with valine (19).No N M R from purified conjugates I or I1 under synthetic condidata were provided to confirm this structure. Sabourin tions and during storage a t low pH over time. et al. described the characterization of a synthetic BMN-acetyl-L-cysteine S-conjugate, presumed to be a mixUnlike the previous analysis of BM-GSH S-conjugates ture of I and 11, by G C N S of the bis(trimethylsily1) (171,we were able to separate the stereoisomers of each BM-N-acetyl-L-cysteine regioisomer formed and charderivatives (21).No NMR analyses were reported for the synthetic conjugates; however, the group did attempt IH acterize them separately by NMR. These analyses demonstrated that our previous characterization of BMNMR analysis of a urine metabolite that eluted with an GSH conjugate I (Figure 11, an open form in a 1:l identical HPLC retention time as the synthetic mixture of I and 11, but the NMR results were consistent with equilibrium with its sulfurane form ( I 71, may have been misinterpreted. To verify this, an attempt was made to the presence of only conjugate I1 in the urine sample. It separate the shoulder from the main portion of our is unclear why NMR peaks corresponding to conjugate I original BM-GSH conjugate I peak. Indeed, when anawere not detected since it was found in the urine of both lyzed by 'H NMR, the equilibrium had shifted; the species in our investigation and predominated over spectra of the main portion of the peak appeared to be a conjugate I1 in mouse urine. Sabourin et al., however, stereoisomer of BM-GSH conjugate I1 whereas the shoulalso described the identification of another mercapturic der appeared to be a mixture of the two stereoisomers of acid metabolite, 1,2-dihydroxy-4-(N-acetyl-~-cysteinyl)the BM-GSH conjugate I open form. butane, in rat and mouse urine following exposure to 1,3-
Butadiene Monoxide Mercapturic Acids butadiene. The latter conjugate was suggested to be formed through further metabolism of the epoxide hydrolase BM metabolic product, 3-buten-1,2-diol. If it is thus formed, it should be a product in animals dosed with BM. In addition, it would be expected to be detectable by the method described in this paper, since its structure is similar to the BM-N-acetyl-L-cysteine S-conjugates characterized in this study. The apparent absence of this metabolite in the urine of rats or mice given BM, whether HPLC fractionation or CU solid phase extraction was used to isolate the metabolites, suggests that 1,2-dihydroxy-4-(N-acetyl-~-cysteinyl)butane may be formed from 1,3-butadieneby a mechanism which does not involve BM formation. Alternatively, this metabolite could be a rearrangement product of the undetected mercapturic acid I under the conditions used by Sabourin et al. to isolate the [14C]-labeled 1,3-butadiene metabolites. Direct detection by HPLC of conjugates I and I1 at 210 nm gave limits of detection of 0.04 and 0.03 mM, respectively. However, when analyzed in rat or mouse urine, background peaks interfered so severely that concentrations of 1 mM or higher were necessary to distinguish BM-N-acetyl-L-cysteine conjugates from other urine components. Deacetylation of the BM mercapturates followed by derivatization with DNFB was attempted to improve HPLC sensitivity. Initially, the method of Osterman-Golkar et al. utilizing acylase I was undertaken to deacetylate the BM-N-acetyl-L-cysteine S-conjugates (19). During the incubation of acylase I with the BM-N-acetyl-L-cysteine conjugates, the disappearance of peaks 2,3, and 5 was observed upon HPLC analysis. Within 1 h of incubation, peaks 3 and 5 disappeared completely, whereas peak 2 was diminished by 50%. Peak 2 eventually disappeared by 9.3 h. Peaks 1and 4 remained unaffected by acylase I, even after 9.3 h. Thus, acylase I appears to be regiospecific for BMN-acetyl-L-cysteine S-conjugates and, therefore, not an effective method of deacetylation. When incubated in 7 M HC1, however, all peaks completely disappeared within 8 h, with peaks 1and 4 dissipating at a faster rate than peaks 2, 3, and 5. Following acidic deacetylation and derivatization with DNFB, BM-N-acetyl-L-cysteine, one isomer of conjugate I1 (peak 11, was analyzed by HPLC with W detection at 365 nm. A peak was observed at 24.5 min that was not seen in samples of water alone derivatized with DNFB. From this peak, the limit of detection of conjugate I1 for this method was estimated to be 0.04 mM. However, when a concentration of 1.2 mM of I1 in urine was taken through the same procedure, the conjugate peak could not be distinguished from the background urine peaks. Therefore, it was concluded that these HPLC methods would be inadequate for sensitive analysis of BM-N-acetyl-L-cysteine S-conjugates in urine. Several mercapturic acid metabolites have been quantitated in the urine through derivatization with diazomethane and GC-flame-ionization detection analysis with detection limits reported as low as 0.1 pg/mL as reviewed by van Weilie et al. (22). Mercapturic acids containing a polar hydroxy group have varied in their limit of detection; (3-hydroxypropyl)-N-acetyl-~-cysteine has been reported to be detectable at 0.2 pg/mL in urine, whereas the regioisomers N-acetyl-S-(2-hydroxy-1-phenylethy1)-L-cysteineand N-acetyl-S-(2-hydroxy-2-phenylethyl)-Lcysteine could be detected no lower than 0.35 mM (nearly 100 ,ug/mL) (23, 24). We found esterification alone to be insufficient to obtain resolution of conjugates
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 76
I and 11. Therefore, a further derivatization step was performed to make the compounds less hydrophilic. The products of this two-step derivatization eluted cleanly and made possible the resolution of the two regioisomers of the BM mercapturates. The limits of detection, however, were significantly higher than those of the methods discussed in the van Weilie review (22): 6.25 pg/mL for BM mercapturates in rat urine and 10 pg/mL in mouse urine. Whether this method is sufficiently sensitive to monitor human worker exposure to 1,3-butadiene is unknown. However, Osterman-Golkar et al. were able to use the acylase-based method described above, which has limits of detection similar to our GC method, to detect BM mercapturates in urine of rats exposed to 1,3-butadiene concentrations at 250 ppm after 6 h of exposure (19). These results suggest that our method may also be useful a t similar 1,3-butadiene concentrations. The lower BM-N-acetyl-L-cysteine S-conjugate output at low BM doses observed in mice compared to rats is not likely to be due to a lower capacity of mouse glutathione S-transferase to mediate the conjugation reaction since earlier results indicate mouse tissue cytosolic incubates catalyzed the reaction to an equal or greater extent than cytosolic incubates of the corresponding rat tissues (16). One possible explanation for the species difference in BM-N-acetyl-L-cysteine S-conjugate excretion is a difference in exhalation rates of BM between rats and mice. Kreiling et al. reported the kinetic constant, k21, for BM elimination via the lungs in mice as 0.79 compared to 0.37 for rats (25). When ventilation rates are taken into account, however, rats are predicted to exhale BM a t 3 times the rate of mice (26). Therefore, exhalation of BM cannot explain the observed species differences in BM-mercapturic acid excretion. Species differences in BM pharmacokinetics, BM tissue distribution, and/or BM metabolism by enzymes other than GSH S-transferases may play a role in the observed species differences in BM-mercapturic acid excretion at the low BM doses. Consistent with this hypothesis, mice exhibited a 2-fold higher alkylation of liver nuclear proteins than rats following uptake of equal amounts of [1,4-l4C11,3-butadiene(27). A species difference in the regioselectivity of BMmercapturic acid excretion was observed between the mouse and the rat (Figure 9). While this difference may be attributed to differences in the regioselectiveformation of the GSH conjugates of BM by the rat and mouse glutathione S-transferases, it is unlikely that this difference would have an effect on BM carcinogenicity since the GSH conjugates of BM are unreactive detoxication products (17). However, the lower ability of the mouse to detoxify BM as mercapturic acids at low BM doses as compared t o the rat may contribute to the higher sensitivity of the mouse to 1,3-butadiene carcinogenicity (5). Another factor that has previously been suggested to play a role in species differences in lB-butadiene toxicity is the rate of BM formation (8, 16). In summary, this study presents the synthesis and characterization of five BM-N-acetyl-L-cysteine conjugates. A sensitive method to measure these BM-Nacetyl-L-cysteine conjugates in urine without the use of radiolabeled isotopes is described. Excretion of BMmercapturic acids in rats was similar to that in mice at a dose of 285 pmoVkg; however, at 71.5 and 143 pmol/ kg, excretion of the mercapturates by rats was greater than in mice. Whereas these results are compatible with
76 Chem. Res. Toxicol., Vol. 8,No.1, 1995
the lower sensitivity of the rat to 1,3-butadiene carcinogenicity compared to the mouse, the biochemical basis for this species difference in BM-mercapturic acid excretion remains unknown. In addition, urinary excretion of mercapturic acids I and II can only account for a small portion of the administered BM dose. Thus, additional studies will be required to determine the fate of BM in vivo.
Acknowledgment. This work was supported by Grants GM 40375 from the National Institute of General Medical Sciences, NIH, and ES 06841 from the National Institute of Environmental Health Sciences. J.E.S. was supported by Institutional Grant T32 ES07015 from the National Institute of Environmental Health Sciences.
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Suppl. 7, World Health Organization, Lyon. (7) Matanoski, G. M., Santos-Burgoa, C., and Schwartz, L. (1990) Mortality of a cohort of workers in the styrene-butadiene polymer manufacturing industry (1943-1982). Environ. Health Perspect. 86, 107-117. (8) Deuscher, R. J., and Elfarra, A. A. (1994) Human liver microsomes are efficient catalysts of 1,3-butadiene oxidation. Evidence for major roles by cytochromes P450 2A6 and 2E1. Arch. Biochem. Biophys. 311,342-439. (9) Elfarra, A. A., Duescher, R. J., and Pasch, C. M. (1991) Mechanisms of 1,3-butadiene oxidations to butadiene monoxide and crotonaldehyde by mouse liver microsomes and chloroperoxidase. Arch. Biochem. Biophys. 286,244-251. (10) Duescher, R. J.,and Elfarra, A. A. (1992)1,IButadiene oxidation by human myeloperoxidase: role of chloride ion in catalysis of divergent pathways. J . Biol. Chem. 267, 19859-19865. (11) Bolt, H. M., Schmiedel, G., Filser, J., Rolzhauser, H., Lieser, K., Wistuba, D., and Schurig, V. (1983) Biological activation of 1,3butadiene to vinyl oxirane by rat liver microsomes and expiration of the reactive metabolite by exposed rats. J . Cancer Res. Clin. Oncol. 106,112-116. (12) Schurig, V., and Wistuba, D. (1984) Asymmetric microsomal epoxidation of simple prochiral olefins. Angew. Chem., Int. Ed. Engl. 23, 796-797.
Elfarra et al. (13) de Meester, C., Poncelet, F., Roberfkoid,M., and Mercier, M. (1978) Mutagenicity of butadiene and butadiene monoxide. Biochem. Biophys. Res. Commun. 80, 298-305. (14) Sharief, Y., Brown, A. M., Backer, L. C., Campbell, J. A., Westbrook-Collins, B., Stead, A. G., and Allen, J. W. (1986)Sister chromatid exchange and chromosome aberration analyses in mice after in vivo exposure to acrylonitrile, styrene, or butadiene monoxide. Environ. Mutagen. 8,439-448. (15) Van Duuren, B. L., Nelson, N., Orris,L., Palmes, E. D., and Schmitt, F. L. (1963) Carcinogenicity of epoxides, lactones, and peroxy compounds. J . Natl. Cancer Inst. 31, 41-55. (16) Sharer, J. E., Duescher, R. J., and Elfarra, A. A. (1992) Species and tissue differences in the microsomal oxidation of 1,3-butadiene and the glutathione conjugation of butadiene monoxide in mice and rats: possible role in 1,3-butadiene-induced toxicity. Drug Metab. Dispos. 20, 658-664. (17) Sharer,J. E., Duescher, R. J., and Elfarra, A. A. (1991)Formation, stability, and rearrangements of the glutathione conjugates of butadiene monoxide: evidence for the formation of stable sulfurane intermediates. Chem. Res. Toxicol. 4, 430-436. (18) Sharer, J. E., and Elfarra, A. A. (1992) S-(2-Hydroxy-3-buten-ly1)glutathione and S-(l-hydroxy-3-buten-2-yl)glutathione are in vivo metabolites of butadiene monoxide: detection and quantitation in bile. Chem. Res. Toxicol. 5 , 787-790. (19) Osterman-Golkar, S., Kautiainen, A., Bergmark, E., Hakansson, &, and Maki Paakkanen, J. (1991) Hemoglobin adducts and urinary mercapturic acids in rats as biological indicators of butadiene exposure. Chem.-Biol. Interact. 80, 291-302. (20) Slatter, J. G., Rashed, M. S., Pearson, P. G., Han, D., and Baillie, T. A. (1991) Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and imdications for isowanate-mediated toxicities. Chem. Res. Toxicol.*4,157-161. (211 Sabourin. P. J.. Burka. L. T.. Bechtold, W. E., Dahl, A. R., Hoover, M. E., Chang, I. Y.; and Henderson, R. F. (1992) Species differences in urinary butadiene metabolites; identification of 1,2d i h y d r o x y - 4 - ( N - a c e t ) b u ~ ea,novel metabolite of butadiene. Carcinogenesis 13, 1633-1638. (22) van Weilie, R. T. H., van Dijck, R. G. J. M., and Vermeulen, N. P. E. (1992)Mercapturic acids, protein adducts, and DNA adducts as biomarkers of electro~hilicchemicals. CRC Crit.Rev. Toxicol. 22,271-306. (23) Onkenhout. W.. Van Bereen, E. J. C., Van der wart, J. H. F., Vos, G. P., Buijs, W., and Ve-rmeulen, N. P. E. (1986) Identification and quantitative determination of four different mercapturic acids formed from 1,3-dibromopropane and its 1,1,3,3-tetradeutero analogue by the rat. Xenobiotica 16, 21-33. (24) Zoetemelk, C. E. M., van Hove, W., van der Laan, W. L. J., van Meeteren-Wdchli, B., van der Gen, A,, and Breimer, D. D. (1987) in rats in Glutathione conjugation of 1,2-dibromo-l-phenylethane vivo. Drug Metab. Dispos. 15, 418-425. (25) Kreiling, R., Laib, R. J., Filser, J. G., and Bolt, H. M. (1987) Inhalation pharmacokinetics of 1,2-epoxybutene-3reveal species differencesbetween rats and mice sensitive to butadiene-induced carcinogenesis. Arch. Toxicol. 61, 7-11. (26) Bond, J. A., Dahl, A. R., Henderson, R. F., Dutcher, J. S., Mauderly, J. L., and Birnbaum, L. S. (1986) Species differences in the disposition of inhaled butadiene. TOX.Appl. Pharmacol. 84, 617-627. (27) Kreiling, R., Laib, R. J., and Bolt, H. M. (1986) Alkylation of nuclear proteins and DNA after exposure of rats and mice to [1,4-14C]1,3-butadiene.Toxicol. Lett. 30, 131-136.
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