Identification of S-(n-Butylcarbamoyl)glutathione, a Reactive

In support of this hypothesis, S-(N-methylcarbamoyl)glutathione (SMG, Figure 1), the GSH conjugate of methyl isocyanate and an effective inhibitor of ...
0 downloads 0 Views 173KB Size
Download PDF
1138

Chem. Res. Toxicol. 1999, 12, 1138-1143

Identification of S-(n-Butylcarbamoyl)glutathione, a Reactive Carbamoylating Metabolite of Tolbutamide in the Rat, and Evaluation of Its Inhibitory Effects on Glutathione Reductase in Vitro Xiangming Guan,*,† Margaret R. Davis,‡ Cuyue Tang,§ Claudia M. Jochheim,| Lixia Jin,§ and Thomas A. Baillie§ Pharmaceutical Sciences Department, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007, Cerep Corporation, 15318 Northeast 95th Street, Redmond, Washington 98052, Department of Drug Metabolism, Merck Research Laboratories, WP75A-303, West Point, Pennsylvania 19486, and Immunex Corporation, 51 University Street, Seattle, Washington 98101 Received May 17, 1999

Tolbutamide (TOLB), a widely used hypoglycemic agent in the therapy of non-insulindependent diabetes mellitus, has been reported to be teratogenic and/or embryotoxic in several animal species and humans. It has been proposed that the teratogenic effects of TOLB are linked to drug-mediated depletion of glutathione (GSH) through inhibition of the enzyme glutathione reductase (GR), although the mechanism by which this inhibition occurs remains unknown. In the study presented here, rats were injected with TOLB (200 mg/kg ip), and bile was collected for analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This led to the identification of S-(n-butylcarbamoyl)glutathione (SBuG), a reactive GSH conjugate derived from n-butyl isocyanate, as a minor metabolite of TOLB in bile. Upon incubation of SBuG (0.25-1.0 mM) with GR from either yeast or bovine intestinal mucosa in the presence of NADPH (0.20 mM), enzyme activity was lost in a time- and concentrationdependent manner. No inhibition was observed when NADPH was omitted from incubations, or when the natural substrate for the enzyme, glutathione disulfide (GSSG, 0.05 mM), was added. TOLB itself did not inhibit GR over the concentration range of 0.8-2.0 mM. It is concluded that metabolic activation of TOLB in vivo leads to the generation of reactive intermediates (n-butyl isocyanate and SBuG) which carbamoylate and thereby inhibit GR. At critical periods of organogenesis, the resulting perturbation of GSH homeostasis in exposed tissues may play a key role in the teratogenic and/or embryotoxic effects of TOLB.

Introduction Tolbutamide [1-n-butyl-3-(p-tolylsulfonyl)urea, TOLB,1 Figure 1] has been employed widely as a hypoglycemic agent in the treatment of non-insulin-dependent diabetes mellitus. However, TOLB has been shown to be teratogenic and/or embryotoxic in several animal species and humans (1-4), and its use is contraindicated in pregnant diabetic patients. The basis for these adverse effects remains unknown, although recent in vitro experiments with mouse embryos (3) and rat embryos (4) have demonstrated that the embryotoxicity of TOLB is not merely a consequence of hypoglycemia. * To whom correspondence should be addressed. Phone: (605) 6885314. Fax: (605) 688-6232. E-mail: [email protected]. † South Dakota State University. ‡ Cerep Corp. § Merck Research Laboratories. | Immunex Corp. 1 Abbreviations: TOLB, tolbutamide; GSSG, glutathione disulfide; GR, glutathione reductase; SBuG, S-(n-butylcarbamoyl)glutathione; SPrG, S-(n-propylcarbamoyl)glutathione; SMG, S-(N-methylcarbamoyl)glutathione; BCNU, N,N′-bis(2-chloroethyl)-N-nitrosourea; BSA, bovine serum albumin; BSO, buthionine sulfoximine; kinact, rate constant for enzyme inactivation; LC/MS/MS, liquid chromatography/ tandem mass spectrometry; CID, collision-induced dissociation; SRM, selected reaction monitoring.

TOLB has been reported to inhibit glutathione reductase (GR) from yeast (5) and to inhibit the corresponding enzyme in rat embryos (4). GR catalyzes the reduction of the oxidized form of glutathione (GSSG) to the reduced form of glutathione (GSH). As a major endogenous protective agent, GSH protects cells from endogenous and xenobiotic reactive species, and GSSG is a major product generated from the protection reactions (6). In view of the key role of GR in maintaining intracellular GSH levels, which is critical to cell viability, it has been proposed that inhibition of GR by TOLB may be responsible for the teratogenic effects of this drug (4). Consistent with this hypothesis, it has been established that a certain basal level of GSH is essential for normal development in the whole rat embryo culture system (7), and depletion of GSH by buthionine sulfoximine (BSO) has been reported not only to enhance the embryolethal, teratogenic, and growth-retarding properties of various chemicals in vitro (8, 9) but also to cause an increase in the number of dead and malformed rat embryos in vivo (10). Recent reports further demonstrate that the preimplantation stage of mouse embryo development appears to be most sensitive to GSH depletion since the embryo cannot synthesize GSH at this stage (11, 12). As

10.1021/tx990086d CCC: $18.00 © 1999 American Chemical Society Published on Web 11/10/1999

Metabolic Activation of Tolbutamide

Figure 1. Structures of compounds mentioned in the text.

a result, GR has been shown to play a major protective role in the preimplantation embryo by recovering GSH via reduction of GSSG (13). Therefore, the issue of establishing the mechanism by which TOLB inhibits GR becomes important in terms of understanding the biochemical mechanism of TOLB-associated teratogenicity. It has been known for some time that certain substituted ureas, e.g., the antitumor nitrosourea N,N′-bis(2chloroethyl)-N-nitrosourea (BCNU, Figure 1), serve as potent inhibitors of GR in vitro and in vivo (14). These inhibitory properties are believed to result from decomposition of the parent nitrosourea to a reactive alkyl isocyanate (Figure 1) which carbamoylates a key cysteinyl sulfhydryl group at the active site of the enzyme (14, 15). Although isocyanates readily form adducts with GSH in vivo, it has been shown that the resulting S-linked conjugates (Figure 1) are themselves carbamoylating agents which inhibit GR by a similar mechanism to the free isocyanates (16-18). In light of these findings, it appeared to be possible that TOLB, a sulfonylurea derivative, also may undergo metabolism to an alkyl isocyanate (n-butyl isocyanate) (Figure 1), and that this species and/or the corresponding GSH conjugate, S-(nbutylcarbamoyl)glutathione (SBuG, Figure 1), might carbamoylate and thereby inhibit GR. The objectives of this study, which was designed to test this hypothesis using the rat as an animal model, were as follows: (i) to determine whether TOLB undergoes metabolism in vivo to SBuG, (ii) to establish the quantitative importance of this putative pathway of TOLB metabolism in vivo, (iii) to determine whether SBuG inhibits GR in vitro, and (iv) to compare the inhibitory properties of TOLB and SBuG on GR in vitro.

Experimental Procedures Materials. Tolbutamide was a gift from the Upjohn Co. (Kalamazoo, MI). GSSG, reduced nicotinamide adenine dinucle-

Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1139 otide phosphate (NADPH), and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). Baker’s yeast GR (EC 1.6.4.2) was obtained from Fluka Chemical Corp. (St. Louis, MO), and bovine intestinal mucosa GR (EC 1.6.4.2) was purchased from Sigma. Units of activity for these enzymes were reported by the respective manufacturers as follows. One unit of baker’s yeast GR will reduce 1.0 µmol of GSSG per minute at pH 7.6 and 25 °C, and 1 unit of the bovine intestinal mucosa GR will reduce 1.0 µmol of the glutathionylcoenzyme A mixed disulfide per minute at pH 5.5 and 25 °C. Polyethylene-10 (PE-10) tubing was purchased from Becton Dickinson & Co. (Parsipany, NJ). SBuG and S-(n-propylcarbamoyl)glutathione (SPrG) were synthesized by the reaction of n-butyl and n-propyl isocyanate, respectively, with GSH (19) and exhibited NMR and mass spectral characteristics which were fully consistent with their assigned structures (20). All other chemicals and solvents were reagent or HPLC grade, and were used as received. Instrumentation. Tandem mass spectrometry (MS/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were carried out on a Perkin-Elmer-Sciex API III triplequadrupole mass spectrometer, equipped with an atmospheric pressure ion source and an ion-spray interface, as described previously (16). MS/MS experiments were performed using collision-induced dissociation (CID) of precursor ions in the rfonly quadrupole, where Ar was used as the target gas. Assay of GR activity, which was based on the loss of the NADPH chromophore at 340 nm, was performed on a Cary 3E UVvisible spectrophotometer equipped with a PC central controller (Varian Instruments, Sugar Land, TX). Identification and Quantitative Analysis of SBuG in Rat Bile. Male Sprague-Dawley rats (200-300 g) were purchased from Charles River Laboratories (Wilmington, MA) and were allowed free access to food and water prior to use. The procedure adopted for the identification of SBuG in bile was similar to that described in detail elsewhere (19). Briefly, four rats were anesthetized with urethane (1.4 g/kg ip), and the bile duct of each rat was isolated and cannulated with PE-10 tubing. TOLB was then administered (200 mg/kg ip) as a suspension in 0.5% methyl cellulose (40 mg/mL), and bile was collected over ascorbic acid at 0 °C for 5 h. A control experiment was conducted in parallel in which vehicle only was administered. Specimens of bile were passed through a 0.2 µm nylon membrane filter and analyzed directly by LC/MS/MS using a narrow bore (15 cm × 2.0 mm i.d.) C18 HPLC column for sample introduction (16). Candidate GSH conjugates were detected by constant neutral loss scanning (129 Da) and, in the case of SBuG, by selected reaction monitoring (SRM) of the transition from m/z 407 to 278 which corresponds to elimination of 129 Da (pyroglutamic acid) from the MH+ ion (21). Identification of SBuG in bile was based on a comparison of both the LC retention properties and MS/MS characteristics (product ions of m/z 407) with those of the corresponding reference material prepared by synthesis. Quantitative analyses were carried out by SRM (again using the m/z 407 to 278 transition for SBuG) and employed SPrG as an internal standard (detected by monitoring the corresponding transition, from m/z 393 to 264). For these experiments, specimens of bile were filtered and infused directly into the mass spectrometer (i.e., without chromatographic separation). The concentration of SBuG in bile was determined by reference to a standard curve which was prepared by analyzing specimens of drug-free rat bile to which had been added different amounts of SBuG together with a fixed amount of SPrG. Inhibition of GR by SBuG. SBuG (0.25-1.0 mM) was incubated with GR from bovine intestinal mucosa (0.016 unit/ mL) in 0.1 M phosphate buffer (pH 7.4) containing BSA (1.0 mg/mL) and NADPH (0.20 mM) at 25 °C in a total volume of 10 mL. Aliquots (0.9 mL) were withdrawn at intervals for measurement of the remaining GR activity, which was performed by adding an aqueous solution of GSSG (0.1 mL, final concentration of 0.52 mM) and determining the initial rate of

1140

Chem. Res. Toxicol., Vol. 12, No. 12, 1999

disappearance of NADPH spectrophotometrically at 340 nm. Parallel experiments carried out in the absence of the inhibitor served as controls. Inhibition of GR by TOLB. A similar protocol was employed to study the time course of TOLB-dependent inhibition of GR. For reference purposes, yeast GR was used in the experiments. The concentrations of TOLB that were studied were 0.8 and 2.0 mM. Inhibition of GR by n-Butyl Isocyanate. A freshly prepared solution of n-butyl isocyanate (20 mM in DMSO, 12 µL) (highly toxic, handle with caution) was added to a phosphate buffer (pH 7.4, 0.1 M, 948 µL) containing GR (0.019 unit/mL), BSA (1.0 mg/mL), and NADPH (0.20 mM). The solution was left at 25 °C for 30 min. An aqueous solution of GSSG (13 mM, 40 µL) was added to the solution to determine the remaining GR activity as described above. Parallel experiments carried out in the absence of the inhibitor served as controls. Effect of NADPH on the Inhibition of GR by SBuG. These studies were performed in a fashion similar to those described above, except that NADPH was omitted from the initial incubations of SBuG with GR. However, at the end of the incubation period, NADPH and GSSG were added to determine the residual GR activity. Effect of Substrate (GSSG) on the Inhibition of GR by SBuG. GR from bovine intestinal mucosa (0.016 unit/mL) was incubated for 10 min with SBuG (0.25-1.0 mM) and various concentrations (5, 30, and 50 µM) of GSSG in 0.1 M phosphate buffer (pH 7.4) containing BSA (1.0 mg/mL) and NADPH (0.20 mM) at 25 °C. Residual enzyme activity then was determined as described previously, and compared to control values obtained from parallel incubations to which the inhibitor had not been added.

Results Identification and Quantitative Analysis of SBuG in Rat Bile. When a sample of bile from a rat which had been treated with TOLB (200 mg/kg ip) was analyzed by LC/MS/MS with constant neutral loss scanning for 129 Da [a characteristic pathway of fragmentation of GSH conjugates under CID conditions (21)], a single metabolite was detected at a retention time of 6.3 min (Figure 2A). This material, which was not present in the bile of control rats, exhibited a parent ion at m/z 407, corresponding to the MH+ value of SBuG. Analysis of an authentic specimen of SBuG under identical LC/MS/MS conditions afforded the chromatogram reproduced in Figure 2B, which also exhibited a single peak at 6.3 min. A product ion spectrum of the metabolite (obtained upon CID at m/z 407) then was recorded by direct infusion of the bile specimen, and the result is shown in Figure 3A. Comparison of this spectrum with that of a synthetic sample of SBuG (Figure 3B) confirmed that all of the major fragments present in the standard (at m/z 278, 179, 175, and 162) were present in the biliary metabolite. (The ions at m/z 231 and 214 in the spectrum of the biological sample were ascribed to interfering endogenous constituents of bile, since no chromatographic separation was employed in this analysis.) Thus, on the basis of the fact that the biliary metabolite exhibited both LC and MS/ MS characteristics which were essentially identical to those of the corresponding reference material prepared by synthesis, the metabolite of TOLB was identified as SBuG. Quantitative studies on the excretion of SBuG in the bile of rats treated with TOLB (200 mg/kg ip) revealed that this GSH conjugate was a minor metabolite, accounting for 0.21 ( 0.17% (mean ( SE, n ) 4) of the dose eliminated over the course of 5 h.

Guan et al.

Figure 2. Ion current chromatograms depicting the detection by LC/MS/MS of SBuG using SRM of the transition from m/z 407 to 278. Chromatogram A was obtained during the analysis of a specimen of bile from a rat which had been treated with TOLB (200 mg/kg ip), while chromatogram B was recorded with an authentic sample of SBuG analyzed under identical conditions.

Inhibition of GR by SBuG. Incubation of SBuG with GR from bovine intestinal mucosa in the presence of NADPH led to loss of enzyme activity, as depicted in Figure 4A. This inhibition was both time- and concentration-dependent. Figure 4B shows a plot of the reciprocal of the slopes (Kapp) of the lines in Figure 4A versus the reciprocal of the corresponding inhibitor concentrations, from which the kinetic constants Ki (2.1 mM) and kinact (0.27 min) for SBuG were derived from the relationship (22)

Ki 1 1 1 ) + × Kapp kinact kinact [I] Inhibition of GR by n-Butyl Isocyanate. n-Butyl isocyanate appeared to be a much more potent inhibitor than SBuG. At a concentration of 0.25 mM, n-butyl isocyanate caused almost a complete loss of GR activity while SBuG produced ∼50% GR inhibition (Figure 5). Effect of Substrate (GSSG) on the Inhibition of GR by SBuG. When GSSG (the natural substrate for GR) was added to incubation media, a strong protective effect against inhibition by SBuG was observed, even at GSSG concentrations as low as 5 µM (less than 2% of the inhibitor concentration) (Figure 6). At a level of 50 µM, GSSG completely blocked the inhibitory effects of SBuG (0.25-1.0 mM). Effect of NADPH on the Inhibition of GR by SBuG. When SBuG (0.25 mM) was incubated with GR in the presence of NADPH (0.20 mM), enzyme activity

Metabolic Activation of Tolbutamide

Figure 3. Product ion spectrum obtained by CID of the parent ion (MH+) of SBuG at m/z 407. The spectrum in panel A was recorded from a sample of crude bile from a rat which had been treated with TOLB, while that depicted in panel B was obtained from an authentic specimen of SBuG. The origins of characteristic product ions are as indicated. In each case, the spectra were recorded by infusion of the sample directly into the mass spectrometer, without prior chromatographic separation.

fell to ca. 25% of control values over the course of 60 min, as shown in Figure 7. However, when NADPH was omitted from incubation media, and added only at the point of GR assay, no inhibition of the enzyme was detected. Comparison of SBuG and TOLB as Inhibitors of GR. For reference purposes, TOLB was incubated with yeast GR at a concentration (0.8 mM) which had been reported to be inhibitory (5), and no effect was observed on GR activity over the course of 80 min (Figure 8). Increasing the TOLB concentration to 2.0 mM produced no effect on yeast GR activity, and similar negative results were obtained with the form of the enzyme from bovine intestinal mucosa (data not shown). In contrast, SBuG proved to be an effective inhibitor of both bovine intestinal mucosa GR (Figure 4A) and yeast GR (Figure 8) at concentrations of 0.75 and 0.8 mM, respectively.

Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1141

Figure 4. (A) Inhibition of GR from bovine intestinal mucosa by various concentrations of SBuG: (4) 0.25, ([) 0.5, (b) 0.75, and (O) 1 mM. The enzyme activity was determined at the indicated time points, and the results are expressed as the natural log of GR activity (relative to matched control samples incubated in the absence of SBuG) as a function of time. The control activity was 13.5 ( 1.3 nmol of GSH formed mL-1 min-1 (n ) 4). (B) A plot of the reciprocals of the slopes (1/kapp) of the inhibition curves of panel A vs the reciprocals of the corresponding inhibitor concentrations, from which the kinetic constants Ki and kinact were calculated.

Figure 5. Inhibition of GR from bovine intestinal mucosa by SBuG (A) and n-butyl isocyanate (B). GR was incubated with the inhibitor (0.25 mM) for 30 min at 25 °C as described in Experimental Procedures. The results are expressed as the percent activity of matched control samples which were incubated in the absence of the inhibitor, and represent means ( SE (n ) 3). The control activity was 18.2 ( 2.1 nmol of GSH formed mL-1 min-1 (n ) 3).

Discussion The study presented here has provided evidence for the operation of a novel pathway of metabolism of TOLB in the rat, namely, formation of n-butyl isocyanate, an electrophilic intermediate which gives rise to the S-linked conjugate SBuG by reaction with GSH. Although this route of metabolism appears to be a minor one based on the biliary excretion of SBuG (0.2% of the TOLB dose over the course of 5 h), it is nevertheless toxicologically significant in that carbamate thioester derivatives of GSH analogous to SBuG are known to act as carbamoy-

lating agents under physiological conditions, when they donate the elements of the parent isocyanate to biological macromolecules (23-25). In agreement with this chemical reactivity, SBuG is found to readily carbamoylate thiol groups (-SH) (19), which are abundant in vivo. The relevance of these findings with respect to the origin of the teratogenic effects of TOLB is that they provide a potential explanation for the reported inhibitory properties of TOLB on GR in vivo, since the structurally related methyl and 2-chloroethyl isocyanates, together

1142

Chem. Res. Toxicol., Vol. 12, No. 12, 1999

Figure 6. Protective effect of GSSG on the inhibition of GR by SBuG. The enzyme was incubated for 10 min with SBuG (0.25-1.0 mM) in the presence of various concentrations of GSSG [([) 5, (b) 30, and (O) 50 µM] and a fixed concentration (0.2 mM) of NADPH at 25 °C. Results are expressed as the percent activity of matched controls which were incubated in the absence of SBuG, and represent means ( SE (n ) 3). The control activity was 13.5 ( 1.3 nmol of GSH formed mL-1 min-1 (n ) 4).

Figure 7. Effects of NADPH on the inhibition of GR by SBuG. The enzyme was incubated with SBuG (0.25 mM) in the absence ([) or presence (O) of NADPH (0.2 mM), as described in Experimental Procedures. The enzyme activity was determined at the indicated time points, and the results are expressed as the percent activity of matched controls which were incubated in the absence of the inhibitor. Values represent means ( SE (n ) 3). The control activity was 13.5 ( 1.3 nmol of GSH formed mL-1 min-1 (n ) 4).

with their respective GSH conjugates, serve as active sitedirected inhibitors of this enzyme (16-18). Indeed, when SBuG was incubated with GR from either yeast or bovine intestinal mucosa, the enzyme was inhibited in a timeand concentration-dependent manner. Moreover, the inhibition could be blocked by the natural substrate for GR, viz. GSSG, and was dependent on prior reduction of the enzyme by NADPH. As discussed previously (17), these findings are indicative of carbamoylation by the inhibitor of a key functional group(s) at the active site of GR. Since alkyl isocyanates generated from nitrosoureas have been suggested to be the species inhibiting GR (14), we investigated whether n-butyl isocyanate was a GR inhibitor and, if it was, whether GR inhibition by SBuG occurred through interaction of the enzyme with n-butyl isocyanate generated from SBuG rather than with SBuG itself. Our data indicated that n-butyl isocyanate was a much more potent GR inhibitor than SBuG (Figure 5). However, it appears unlikely that inhibition of GR by SBuG was through n-butyl isocyanate since the decomposition rate constant (7.47 × 10-4 min-1, unpublished results) of SBuG in 0.1 M phosphate buffer (pH 7.4) was much lower than the enzyme inactivation rate constant kinact (0.27 min-1) for SBuG. Therefore, the GR inactiva-

Guan et al.

Figure 8. Comparison of the effects of equimolar concentrations (0.8 mM) of SBuG (O) and TOLB ([) on the activity of yeast GR. The results are expressed as the percent activity of matched control samples which were incubated in the absence of the inhibitor, and represent means ( SE (n ) 3). The control activity was 13.5 ( 1.3 nmol of GSH formed mL-1 min-1 (n ) 4).

tion is primarily produced by SBuG directly not via n-butyl isocyanate. Reactive oxygen intermediates, which form as byproducts of various normal biochemical processes and xenobiotic metabolism, and electrophilic toxicants are known to be associated with embryotoxicity and teratogenicity (26, 27). Detoxification of reactive oxygen intermediates and their resulting products such as peroxides is mainly through reduction of these species coupled with oxidation of GSH to GSSG either nonenzymaticaly or enzymaticaly by glutathione peroxidase (6). Similarly, removal of electrophilic toxicants is accomplished by either nonenzymatic or enzymatic (glutathione S-transferase) reactions with GSH in forming glutathione conjugates (6). The presence in mammalian embryos at an early stage of development of GSH-related enzymes (GR, glutathione peroxidase, and glutathione S-transferase) points to the importance of these protective systems during organogenesis (28). As noted earlier, a certain basal level of GSH is essential for normal development of mammalian embryos, and depletion of GSH can cause death and malformation of embryos in culture. The critical role of GSH is even more significant during the preimplantation stage of embryo development (11, 12), and GR plays a major protective role at this stage by recovering GSH via reduction of GSSG. Therefore, it seems reasonable to expect that inhibition of GR by SBuG would contribute to the teratogenic and embryotoxic effects of TOLB. In support of this hypothesis, S-(N-methylcarbamoyl)glutathione (SMG, Figure 1), the GSH conjugate of methyl isocyanate and an effective inhibitor of GR (17), was found to produce concentration-dependent decreases in the rates of growth and development of mouse embryos explanted on day 8 of gestation and cultured in rat serum for 42 h (29). Interestingly, the embryotoxicity of SMG in these studies was blocked by the addition of GSH to culture media. In view of the close structural similarity of SBuG and SMG (Figure 1), it would be predicted that SBuG will produce similar toxic effects in developing embryos. A recent report also reveals that depletion of GSH by BSO produced signs of developmental delay in rat embryos cultured in diabetic serum, which further supports our hypothesis (30). It may be argued whether the amount of SBuG formed as a metabolite of TOLB is significant enough to produce any biological effects. As indicated earlier, SBuG is a carbamoylating agent and can react with thiol groups, which are abundant in vivo.

Metabolic Activation of Tolbutamide

The amount of SBuG collected in bile of the TOLBtreated rats represented only the unreacted portion of the formed SBuG. Moreover, a stability study revealed that incubation of SBuG (0.1 mM) in drug-free rat bile at 0 °C caused a 21 ( 3.2% (n ) 3) loss of the compound over the course of 5 h (unpublished results). In addition to chemical instability, SBuG is likely to undergo in vivo enzymatic degradation to produce the corresponding mercapturic acid conjugate, a known metabolic pathway of glutathione conjugates (31). Therefore, the amount of SBuG recovered in bile should be considered as a lower limit in terms of the quantity of n-butyl isocyanate actually released from TOLB during metabolism. Further, although the amount of n-butyl isocyanate and its related metabolite SBuG generated from therapeutic doses of TOLB may not cause embryotoxicity under normal conditions, the same amount may be significant enough to cause toxicity when an oxidative stress condition exists. In conclusion, TOLB has been found to undergo metabolism in the rat to a reactive GSH conjugate, SBuG, which inhibits GR in vitro. Since TOLB itself did not inhibit GR under these conditions, it is possible that SBuG and/or the n-butyl isocyanate from which it is derived is responsible for inhibiting this enzyme in vivo. Although further studies will be required to establish the contributions of these reactive carbamoylating metabolites of TOLB to the teratogenicity and embryotoxicity of the parent drug, the investigation presented here has provided new insight into the possible underlying biochemical mechanism of the serious adverse effects of the widely used antidiabetic agent.

Acknowledgment. We thank Dr. David G. Kaiser (Upjohn Co.) for a gift of tolbutamide. This research was supported by a grant from the National Institutes of Health (ES05500), which is gratefully acknowledged.

References (1) Schiff, D., Aranda, J. V., and Stern, L. (1970) Neonatal thrombocytopenia and congenital malformations associated with administration of tolbutamide to the mother. J. Pediatr. 77, 457458. (2) Saili, A., and Sarna, M. S. (1991) Tolbutamide: teratogenic effects. Indian Pediatr. 28, 936-940. (3) Smoak, I. W. (1992) Teratogenic effects of tolbutamide on earlysomite mouse embryos in vitro. Diabetes Res. Clin. Pract. 17, 161167. (4) Ziegler, M. H., Grafton, T. F., and Hansen, D. K. (1993) The effect of tolbutamide on rat embryonic development in vitro. Teratology 48, 45-51. (5) Ziegler, M. H., and Medon, P. J. (1990) Effect of tolbutamide on both BCNU-mediated inhibition of insulin release from isolated pancreatic islets and the activity of yeast glutathione reductase in vitro. FASEB J. 4, A401. (6) Dolphin, D., Poulson, R., and Avramovic, O., Eds. (1989) Glutathione, Chemical, Biochemical, and Medical Aspects, Vol. III, Parts A and B, Wiley, New York. (7) Slott, V. L., and Hales, B. F. (1987) Enhancement of the embryotoxicity of acrolein, but not phosphoramide mustard, by glutathione depletion in rat embryos in vitro. Biochem. Pharmacol. 36, 2019-2025. (8) Slott, V. L., and Hales, B. F. (1987) Effect of glutathione depletion by buthionine sulfoximine on rat embryonic development in vitro. Biochem. Pharmacol. 36, 683-688. (9) Stark, K. L., Harris, C., and Juchau, M. R. (1989) Influence of electrophilic character and glutathione depletion on chemical dysmorphogenesis in cultured rat embryos. Biochem. Pharmacol. 38, 2685-2692. (10) Hales, B. F., and Brown, H. (1991) The effect of in vivo glutathione depletion with buthionine sulfoximine on rat embryo development. Teratology 44, 251-257.

Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1143 (11) Gardiner, C. S., and Reed, D. J. (1995) Synthesis of glutathione in the preimplantation mouse embryo. Arch. Biochem. Biophys. 318, 30-36. (12) Gardiner, C. S., Salmen, J. J., Brandt, C. J., and Stover, S. K. (1998) Glutathione is present in reproductive tract secretions and improves development of mouse embryos after chemically induced glutathione depletion. Biol. Reprod. 59, 431-436. (13) Gardiner, C. S., and Reed, D. J. (1995) Glutathione redox cycledriven recovery of reduced glutathione after oxidation by tertiarybutyl hydroperoxide in preimplantation mouse embryos. Arch. Biochem. Biophys. 321, 6-12. (14) Babson, J. R., and Reed, D. J. (1978) Inactivation of glutathione reductase by 2-chloroethyl nitrosourea-derived isocyanate. Biochem. Biophys. Res. Commun. 83, 754-762. (15) Karplus, P. A., Krauth-Siegel, R. L., Schrimer, R. H., and Schulz, G. E. (1988) Inhibition of human glutathione reductase by the nitrosourea drugs 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea. Eur. J. Biochem. 171, 193-198. (16) Davis, M. R., Kassahun, K., Jochheim, C. M., Brandt, K. M., and Baillie, T. A. (1993) Glutathione and N-acetylcysteine conjugates of 2-chloroethyl isocyanate. Identification as metabolites of N,N′bis(2-chloroethyl)-N-nitrosourea in the rat and inhibitory properties toward glutathione reductase in vitro. Chem. Res. Toxicol. 6, 376-383. (17) Jochheim, C. M., and Baillie, T. A. (1994) Selective and irreversible inhibition of glutathione reductase in vitro by carbamate thioester conjugates of methyl isocyanate. Biochem. Pharmacol. 47, 1197-1206. (18) Kassahun, K., Jochheim, C. M., and Baillie, T. A. (1994) Effect of carbamate thioester derivatives of methyl and 2-chloroethyl isocyanate on glutathione levels and glutathione reductase activity in isolated rat hepatocytes. Biochem. Pharmcol. 48, 587-594. (19) Guan, X., Davis, M. R., Jin, L., and Baillie, T. A. (1994) Identification of S-(n-butylcarbamoyl)glutathione, a reactive carbamoylating agent as a biliary metabolite of benomyl in the rat. J. Agric. Food Chem. 42, 2953-2957. (20) Han, D.-H., Pearson, P. G., Baillie, T. A., Dayal, R., Tsang, L. H., and Gescher, A. (1990) Chemical synthesis and cytotoxic properties of N-alkylcarbamic acid thioesters, metabolites of hepatotoxic formamides. Chem. Res. Toxicol. 3, 118-124. (21) Baillie, T. A., and Davis, M. R. (1993) Mass spectrometry in the analysis of glutathione conjugates. Biol. Mass Spectrom. 22, 319325. (22) Kitz, R., and Wilson, I. B. (1962) Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. J. Biol. Chem. 237, 3245-3249. (23) Baillie, T. A., and Slatter, J. G. (1991) Glutathione: a vehicle for the transport of chemically reactive metabolites in vivo. Acc. Chem. Res. 24, 264-270. (24) Pearson, P. G., Slatter, J. G., Rashed, M. S., Han, D.-H., and Baillie, T. A. (1991) Carbamoylation of peptides and proteins in vitro by S-(N-methylcarbamoyl)glutathione and S-(N-methylcarbamoyl)cysteine, two electrophilic S-linked conjugates of methyl isocyanate. Chem. Res. Toxicol. 4, 436-444. (25) Baillie, T. A., and Kassahun, K. (1994) Reversibility in glutathione conjugate formation. Adv. Pharmacol. 27, 163-181. (26) Wells, P. G., Kim, P. M., Laposa, R. R., Nicol, C. J., Parman, T., and Winn, L. M. (1997) Oxidative damage in chemical teratogenesis. Mutat. Res. 396, 65-78. (27) Parman, T., Wiley, M. J., and Wells, P. G. (1999) Free radicalmediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. Nat. Med. 5, 582-585. (28) Serafini, M. T., Arola, L., and Romeu, A. (1991) Glutathione and related enzyme activity in the 11-day rat embryo, placenta and perinatal rat liver. Biol. Neonate 60, 236-242. (29) Guest, I., Baillie, T. A., and Varma, D. R. (1992) Toxicity of the methyl isocyanate metabolite S-(N-methylcarbamoyl)GSH on mouse embryos in culture. Teratology 46, 61-67. (30) Menegola, E., Broccia, M. L., Prati, M., and Giavini, E. (1999) Development of rat embryos cultured in serum from diabetic rats. Biol. Neonate 75, 65-72. (31) Williams, D. (1995) Drug Metabolism. In Principles of Medicinal Chemistry (Foye, W. O., Lemke, T. L., and Williams, D. A., Eds.) pp 83-140, Williams & Wilkins, Baltimore.

TX990086D