Chem. Res. Toxicol. 1992,5, 787-790 787

S-(2-Hydroxy-3-buten-1-yl)glutathione and S-( l-Hydroxy-3-buten-2-yl)glutathione Are in Vivo Metabolites of Butadiene Monoxide: Detection and

Quantitation in Bile Jane E. Sharer and Adnan A. Elfarra'

Department of Comparative Biosciences and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53706

Received June 8, 1992

Administration (ip) of butadiene monoxide, a toxic metabolite of 1,3-butadiene, to rata caused the appearance of two new biliary peaks when analyzed by HPLC chromatography. These peaks were isolated and identified as the regioisomeric glutathione conjugates, S42-hydroxy- 3-buten-l-y1)glutathione (I) and S-(l-hydroxy-3-buten-2-yl)glutathione (II), by comparison of their HPLC retention times and fast atom bombardment mass spectra to those of synthetic standards. S-(4-Hydroxy-2-buten-l-yl)glutathione, a rearrangement product formed during chemical synthesis or storage of I, was not detected. Whether butadiene monoxide was given a t a dose of 14.3 or 143 pmol/kg, the amount of conjugates excreted in 30 min was a t least 85 7% of that excreted in 120 min. Conjugate excretion in 60 min did not exhibit saturation when the butadiene monoxide dose was varied between 14.3 and 286 pmol/kg; the total amount of the butadiene monoxide dose excreted as combined I and I1 averaged only 7.6 f 4.2 7% (mean i SD, n = 12), with approximately a 3:l ratio of isomers 1:II being excreted at all butadiene monoxide doses. Whereas these results indicate a role for glutathione S-transferase-catalyzed reactions in butadiene monoxide metabolism in vivo, biliary excretion of I and I1 can only account for a small fraction of the butadiene monoxide dose given.

Introduction 1,3-Butadiene is used extensively in the industrial

production of synthetic rubber and plastics. Studies in mice and rata have demonstrated that 1,3-butadiene is toxic and carcinogenic with neoplasms of the lung, mammary gland, liver, forestomach, and ovaries, malignant lymphoma, hemangiosarcoma, pancreatic exocrine ade- noma, thyroid follicular cell adenoma, and Leydig cell tumors being most commonly observed (I, 2). Butadiene monoxide (BM;13,4-epoxy-l-butene) has been identified as the major metabolite of l,&butadiene in vivo and in vitro, and evidence has implicated both cytochrome P450- and myeloperoxidase-catalyzed reactions in BM formation from l,&butadiene (3-8). When incubations were carried out with rat liver microsomes, both enantiomers of BM were detected in a 1:l ratio (8). Racemic BM has been shown to be mutagenic and to react with DNA or 2'-deox- yguanosine to form the regioisomers 7-(2-hydroxy-3-buten- l-y1)guanine and 7-( l-hydroxy-3-buten-2-yl)guanine (9, 10). These studies suggest that l,&butadiene oxidation to BM and subsequent reaction of BM with critical cellular macromolecules are involved in the mechanism of 1,3- butadiene toxicity.

Previously, we have demonstrated that BM is a substrate for human placental glutathione S-transferase (GST) and identified the products as S-(2-hydroxy-3-buten-l-yl)glu- tathione (I) and S-(l-hydroxy-3-buten-2-yl)glutathione ~ ~~

* To whom correspondence should be addressed at the Department of Comparative Biosciences, University of Wisconsin School of Veterinary Medicine, 2015 Linden Dr. W., Madison, WI 63706.

1 Abbreviations: BM, butadiene monoxide; GST, glutathione S-trans- ferase; GSH, glutathione; I, S-(2-hydroxy-3-buten-l-yl)glutathione; 11, S-( l-hydroxy-3-buten-2-yl)glutathione; 111, S-(4-hydroxy-2-buten-l-y1)- glutathione; FAB-MS, fast atom bombardment mass spectrometry.

0893-228~/92/2705-0787$03.00/0

(II), formed by nucleophilic attack of glutathione (GSH) at either of the two carbon atoms of the oxirane ring (11). IH NMR analysis and stability studies of I indicated that it existed in a 1:l equilibrium with a stable sulfurane form (Figure l), which slowly rearranged to yield S-(bhydroxy- 2-buten-l-y1)glutathione (111). Whereas I11 can also be formed during the chemical reaction of BM and GSH, it was not detected enzymatically (6, 11). Formation of I and I1 in enzymatic incubations of BM with rat liver ho- mogenates has also been demonstrated, and the GSTs catalyzing the reaction were localized to the cytosol (6). Formation of I and I1 was further shown to be catalyzed by mouse liver cytosol as well as by rat and mouse lung, kidney, and testis cytosol (6). The rates of BM-GSH conjugation by either rat or mouse liver cytosol were much higher than rates determined with lung, kidney, or testis cytosol. Although these results suggest that GSH con- jugation may represent an important detoxification mech- anism for BM, the reaction or the extent to which it occurs was not characterized in vivo. The present study dem- onstrates the in vivo formation of the GSH conjugates I and I1 and characterizes their excretion in rat bile after BM administration.

Experimental Procedures Materials. Racemic BM and trifluoroacetic acid were ob-

tained from Aldrich Chemical Co. (Milwaukee, WI). HPLC- grade acetonitrile and trichloroacetic acid were obtained from EM Science (Gibstown, NJ). BM-GSH conjugates 1-111 were synthesized as previously described (11). All other chemicals were of the highest grade commercially available. Caution: EM is a known mutagen and carcinogen in laboratory animals and must be handled using proper safety measures.

Surgical Procedure and Sample Preparation. Surgical procedures for bile duct cannulation were followed as previously

0 1992 American Chemical Society

788 Chem. Res. Toxicol., Vol. 5, No. 6, 1992

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G S T I G S H

Sharer and Elfarra

eSG t OH

I A

SG

I 1

-S G I OH

I B Figure 1. GST-catalyzed conjugation of BM with GSH. The open form of I (IA) is depicted in equilibrium with the sulfurane form (IB).

described (12,13). Briefly, male Sprague-Dawley rats (200-315 g) (Sasco, Omaha, NE) were anaesthetized with a single pen- tobarbital dose (50 mg/kg, ip). The rat’s stomach was shaved and an incision made that would allow temporary removal of the gastrointestinal tract and easy access to the bile duct. The bile duct was isolated and cut to allow insertion of a piece of PE-10 polyethylene tubing (Clay Adams, Parsippany, NJ). When a steady flow of bile (1-2 mL/h) was achieved, the incision was closed. Up to 0.5 mL of bile was collected as a control before administration of BM in 0.5 mL of corn oil as a single ip injection. After BM administration, bile was collected for appropriate time periods. Bile proteins were precipitated with 50 pL of 50% (w/ v) trichloroacetic acid/l mL of bile (final pH <2) followed by centrifugation in an Eppendorf microcentrifuge. Low-dose samples (14.3 pmol/kg) were concentrated 3-fold by speed vacuum lyophilization and resuspended in one-third the original volume of water. The resulting supernatant was then filtered with 0.2- pm LC 13 acrodisc membrane filters (Gelman Sciences, Ann Arbor, MI) prior to HPLC analysis. HPLC Analysis. Samples were analyzed using a Gilson

gradient-controlled HPLC system (Model 302 pumps; Middle- ton, WI) equipped with a Bio-Rad AS-100 HRLC automatic sampling system (Richmond, CA), a 3-cm Brownlee ODS guard column (San Jose, CA), and a Beckman Model 167 scanning ab- sorbance detector (Irvine, CA) on a Beckman Ultrasphere 5-pm ODS reverse-phase analytical column (4.6 X 250 mm) (San Ra- mon, CA) with UV detection at 220 nm. Mobile phase was isocratic at 4 % acetonitrile and 0.1 % trifluoroacetic acid in water (pH 2) with a flow rate of 1 mL/min. Peaks with retention times identical to those of the BM-GSH conjugates I and I1 were quantitated by comparison to standard curves (r > 0.99) of the synthetic conjugates after subtraction of background from controls.

Mass Spectrometry. Positive ion fast atom bombardment mass spectrometry (FAB-MS) analysis was performed using a Kratos MS-5OTC ultrahigh-resolution mass spectrometer (Manchester, United Kingdom) equipped with a saddle field fast atom bombardment gun. Bile peaks with retention times equivalent to BM-GSH conjugates I and I1 were collected by semipreparative HPLC and lyophilized to dryness; purity was verified by analytical HPLC to be >95 % . Approximately 600 pg of conjugate I and 500 pg of conjugate I1 were dissolved in a

A lb rb Time (mi4

Figure 2. Typical HPLC chromatograms of rat bile (A) prior to BM administration and (B) after BM administration.

matrix of glycerol prior to mass spectrometry. Synthetic BM- GSH conjugates I and I1 were also analyzed in a glycerol matrix for comparison. A drop of 1 M HCl was added to all samples to remove heavy sodium contamination.

Results and Discussion Identification of BM-GSH Conjugates in Bile.

HPLC analysis of bile samples revealed two peaks with retention times (14.5 and 15.9 min) similar to those obtained with synthetic BM-GSH conjugates I and I1 (Figure 2); the shoulder on peak I, which was also present when the reference compound was analyzed, may have resulted from conjugate I being present in equilibrium with the sulfurane form (11). No peaks other than I and I1 were consistently detected in bile collected after rats were given BM. FAB-MS spectra of the isolated peaks I and I1 exhibited mlz ratios which correlated with those detected with the synthetic standards (Figures 3 and 4). The pseudomolecular ion at mlz 378 (M + 1) corresponds to the BM-GSH conjugate molecular weight of 377. The peaks at mlz 57, 75, and 115 correspond to respective portions of the glutamate moiety: C(O)CH2CH2 + 1, NH2- CHCOzH + 1, and C(O)CH2CH2CHC02H + 1. Glycine also accounts for mlz 75, and cysteine corresponds to mlz 102. In addition, the glycerol matrix will show peaks at mlz 115,185, and 207 for glycerol + Na+, a(glycero1) + 1, and 2(glycerol) + Na+, respectively. These results dem- onstrate that BM is conjugated with GSH in vivo to produce I and I1 and that these conjugates are excreted in the bile.

Characterization of BM-GSH Biliary Excretion. BM-GSH conjugates I and I1 were detected in the bile collected for 30 min after the administration of a 143 pmoV kg dose of BM (Figure 5); the amount excreted at 30 min represents 85% of that excreted in 120 min. Rata given a 10-fold lower dose of BM (14.3 Imollkg) gave a similar

In Vivo Butadiene Monoxide-GSH Conjugate Formation

80 -

60 -

4 0 -

Chem. Res. Toxicol., Vol. 5, No. 6, 1992 789

P) V C 0 -0 C 2 2 p 100 -

; 80 -

75

.- -w 0 -

51

100 200 300 400

5 B

M+ 1 378

100 200 300 100

m/z

Figure 3. Positive ion FAB-MS spectra of (A) isolated bile peak I and (B) synthetic I.

115

loo 1 I A

M+ 1 0 20 c 0

c o 100 200 300 400

IO0 200 300 400

4 2

Figure 4. Positive ion FAB-MS spectra of (A) isolated bile peak I1 and (B) synthetic 11.

excretion profile (data not shown). When the BM dose was varied between 14.3 and 286pmollkg and the combined amounts of conjugates I and I1 excreted in 60 min were determined, an apparent linear doseresponse relationship (r = 0.98) was obtained (Figure 6). Total BM-GSH conjugates excreted in 60 min averaged 7.6 f 4.2 76 (mean f SD, n = 12) of the BM dose administered. These results

n

-- 0 50 100 1 so

Time (min)

Figure 5. Time-dependent biliary excretion of BM (143 rmol/ kg) expressed as the percentage of the BM dose excreted as GSH conjugates I and 11. Values are from one typical experiment. h m ---- 5 30

s v O t

100 200 300 3 2 0

Butadiene Monoxide (u"ol/kp)

Figure 6. Total BM-GSH conjugates excreted at 60 min as function of the BM dose. Values represent means f SD of the results obtained from 3 experiments.

would indicate that BM hepatic uptake and metabolism to yield the GSH conjugates I and 11, and biliary excretion of these conjugates, occur readily with no saturation of any of these processes at the BM doses studied; however, the large standard deviation values associated with the higher doses may be obscuring a saturation effect. In previous in vitro experiments with rat liver cytosol, saturation of GSH conjugate formation [ Vm, was nearly 70 nmol/(mg-min)] was observed only at high BM con- centrations ( K m was nearly 3 mM; 6). In this study, higher BM doses were avoided to minimize acute BM toxicity, since BM LDw in rats has been determined to be 2.4 mmol/ kg ip (14). Since BM is a metabolic product of 1,3-bu- tadiene, profiles on quantities and product ratios excreted may differ from those obtained if the parent compound were administered.

Formation of the GSH conjugates I and I1 and their excretion into bile may prevent reactions of BM with nu- cleic acids and other critical cellular targets and may protect against toxicity. However, the findings that bil- iary excretion of conjugates I and I1 can only account for approximately 7 % of the administered BM dose and that the corresponding mercapturic acids were not detected in urine when rats were given BM at doses lower than 1.43 mmol/kg (15) suggest that GST-dependent metabolism of BM is not likely to play a major role in BM detoxifi- cation in vivo. Consistent with this hypothesis is our previous finding that mouse tissues, which were more susceptible to 1,3-butadiene toxicity compared to rat tissues (1,2), exhibited BM-GSH conjugation rates similar to or higher than those determined with rat tissues (6).

790 Chem. Res. Toxicol., Vol. 5, No. 6, 1992

Furthermore, only a small amount (15 pmol) of the BM mercapturic acids were detected in rat urine in 24 h after rata were exposed to l,&butadiene at lo00 ppm for 6 h (16). Thus, mechanisms other than GSH conjugation and mercapturic acid formation may play a major role in BM metabolism and disposition. For example, BM may alkylate RNA, DNA, and proteins, since BM was previously shown to react with DNA in vitro (10). In addition, alkylation of hemoglobin, nuclear proteins, and DNA was demonstration after l,&butadiene exposure (16,17). BM has also been shown to be excreted through expired air in rata exposed to l,&butadiene (3), and to be metabolized in vitro by rat microsomal epoxide hydrolase (4). Thus, it is possible that some of the BM in our study was eliminated through these pathways. However, previous findings that the BMK, value for the microsomal epoxide hydrolase reaction was 11.5 mM at the optimum pH of 9 (4 ) , along with our inability to obtain evidence for this reaction in rat or mouse liver microsomes at pH 7.4 (61, suggest that it is unlikely that in vivo BM metabolism by microsomal epoxide hydrolase may have significantly competed with BM metabolism by the GST in the rat. Further studies are needed to assess the role of cytosolic epoxide hydrolase in BM metabolism and disposition.

The ratio of conjugate I and I1 in bile was approximately 3 to 1. This finding is consistent with the previous finding that rat liver homogenate readily metabolized BM to I and I1 in a 3:l ratio (6). Taken together, these results suggest preference by the liver GST isozymes to form I over 11, rather than selective excretion of I over 11, may be responsible for the observed conjugate ratio in the bile.

In summary, our results provide clear evidence for the in vivo formation of the BM-GSH conjugates I and I1 after BM administration and show that biliary excretion of GSH conjugates I and I1 can only account for a s m d portion of the administered BM dose. Thus, additional studies will be required to determine the fate of BM in vivo.

Acknowledgment. This research was supported by NIH Grant GM 40375. J.E.S. was supported by National Institute of Environmental Health Sciences Institutional Grant T32 ES07015.

Sharer and Elfarra

Owen, P. E., Glaister, J. R., Grant, I. F., and Pullinger, D. H. (1987) Inhalationtoxicitystudieawith 1,3-butadiene. 3. Two year toxicity/ carcinogenicity study in rats. Am. Ind. Hyg. Assoc. J. 48,407-413. Bolt, H. M., Schmiedel, G. Filser, J., Rolzhawr, H., Lieser, K., Wistuba, D., and Schurig, V. (1983) Biological activation of 1,3- butadiene to vinyl oxirane by rat liver microsomes and expiration of the reactive metabolite by exposed rata. J. Cancer Res. Clin. Oncol. 106,112-116. Malvoisin, E., and Roberfroid, M. (1982) Hepatic microsomal metaboiim of 1,3-butadiene. Xenobiotica 12, 137-144. Elfarra, A. A., Duescher, R. J., and Pasch, C. M. (1991) Mechanisms of 1,3-butadiene oxidations to butadiene monoxide and crotonal- dehyde by mouse liver microsomes and chloroperoxidaee. Arch. Biochem. Biophys. 286, 244-251. 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: possiblerole in 1,3-butadieneinduced toxicity. DrugMetab. Dispm. (in press). Duescher, R. J., and Elfarra, A. A. (1992) 1,3-Butadiene oxidation by human myeloperoxidaae: role of chloride ion in catalysis of divergent pathways. J. Biol. Chem. (in press). Schurig, V., and Wistuba, D. (1984) Asymmetric microsomal eDoxidation of simDle Drochiral olefins. Anaew. Chem.. Int. Ed.

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Carcinogenicity of 1,3-butadiene in C57BL/6 X C3HFl mice at low exposure concentrations. Cancer Res. 50, 6592-6599.

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Mutagenicity of butadiene and butadiene monoxide. . Biochem. Biophys. Res. Commun. 80, 298-305. Citti, L., Gervasi, P. G., Turchi, G., Bellucci, G., and Bianchini, R. (1984) The reaction of 3,4-epoxy-l-butene with deoxyguanosine and DNA in vitro: synthesis and characterization of the main adducta. Carcinogenesis 5,47-52. Sharer, J. E., Duescher, R. J., and Elfarra, A. A. (1991) Formation, stability, and rearrangementa of the glutathione conjugates of butadiene monoxide: evidence for the formation of stable sulfurane intermediates. Chem. Res. Toxicol. 4, 430-436. Mulder, G. J., Scholtens, E., and Meijer, D. K. F. (1981) Collection of metabolites in bile and urine from the rat. Methods Enzymol.

Sausen, P. J., and Elfarra, A. A. (1991) Reactivity of cysteine S-conjugate sulfoxides Formation of S-[l-chloro-2-(S-glutathionyl)- vinyl]- cysteine sulfoxide by the reaction of S-(1,2-dichlorovinyl)- L-cysteine sulfoxide with glutathione. Chem. Res. Toxicol. 4,655- 660. US. Department of Health and Human Services (1981-1982) in Registry of ToxicEffects of ChemicalSubstances, Volume 1 (Tatken, R. L., and Lewis, R. J., Eds.) p 770, National Institute for Occupational Safety and Health, Cincinnati. Sharer, J. E., and Elfarra, A. A. (1992) Rat urinary and biliary excretion of glutathione conjugates and mercapturic acids of butadiene monoxide. Toxicologist 12,414. Osterman-Golkar, S., Kautiainen, A., Bergmark, E., Hakansson, K., and Maki-Paakkanen, J. (1991) Hemoglobin adducta and urinary mercapturic acids in rats as biological indicators of butadiene exposure. Chem.-Biol. Interact. 80,291-302. Kreiling, R., Laib, R. J., and Bolt, H. M. (1986) Alkylation of nuclear proteins and DNA after exposure of rata and mice to [1,4-W11,3- butadiene. Toxicol. Lett. 30, 131-136.

77,21-30.

Regietry No. I, 133872-48-7; 11,133872-49-8; BM, 930-22-3; GST, 50812-37-8.