Biotransformation of the Naturally Occurring Isothiocyanate

activities in laboratory animals. The present studies were carried out to elucidate the metabolic fate of SFN in the rat. Particular emphasis was plac...
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Chem. Res. Toxicol. 1997, 10, 1228-1233

Biotransformation of the Naturally Occurring Isothiocyanate Sulforaphane in the Rat: Identification of Phase I Metabolites and Glutathione Conjugates Kelem Kassahun,*,† Margaret Davis,† Pei Hu, Bryan Martin, and Thomas Baillie† Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195 Received May 9, 1997X

Sulforaphane (SFN) is a naturally occurring isothiocyanate present in cruciferous vegetables, such as broccoli, that has been identified as a potent inducer of glutathione S-transferase activities in laboratory animals. The present studies were carried out to elucidate the metabolic fate of SFN in the rat. Particular emphasis was placed on glutathione (GSH)-dependent pathways because conjugation with GSH is a major route by which many isothiocyanates are eliminated in mammals. Male Sprague-Dawley rats were administered a single dose of SFN (50 mg kg-1 ip), and bile and urine were collected over ascorbic acid. Analysis of biological fluids was carried out by ionspray LC-MS/MS using the neutral loss (129 Da) and precursor ion (m/z 164) scan modes to detect GSH and N-acetylcysteine (NAC) conjugates, respectively. In bile, five thiol conjugates (designated M1-M5) were detected. Metabolites M2 and M4 were identified as the GSH conjugates of SFN and erucin (ERN, the sulfide analog of SFN), respectively, by comparing their LC-MS/MS properties with those of standards obtained by synthesis. M1 was characterized as the GSH conjugate of a desaturated metabolite of SFN (tentatively assigned the structure of ∆1-SFN), suggesting that the parent compound also undergoes oxidative metabolism. Metabolites M3 and M5 were identified as the NAC conjugates of SFN and ERN, respectively, and together with the NAC conjugate of ∆1-SFN, these species also were detected in urine. Quantitative determination of the former two mercapturates in urine indicated that ∼60% and ∼12% of a single dose of SFN is eliminated in 24 h as the NAC conjugates of SFN and ERN, respectively. The corresponding figures in rats dosed with ERN were ∼67% and ∼29%. When the GSH conjugate of SFN was incubated with phosphate buffer (pH 7.4, 37 °C), 70% conversion) when incubated in the presence of excess cysteine, thereby acting as an effective carbamoylating agent. It is concluded that SFN undergoes metabolism by S-oxide reduction and dehydrogenation and that GSH conjugation is the major pathway by which the parent compound and its phase I metabolites are eliminated in the rat.

Introduction Consumption of cruciferous vegetables, such as broccoli, cabbage, and cauliflower, is known to be associated with a lower risk of developing cancer (1). Compounds bearing the reactive isothiocyanate moiety represent one group of natural products that occurs widely in these vegetables. Certain isothiocyanates appear to have cancer chemoprotective properties which may stem from their effects on enzyme systems involved in the metabolism of carcinogens. For example, phenethyl isothiocyanate has been shown to be an inhibitor of cytochrome P450 2E1 (2), while many other isothiocyanates are inducers of phase II enzymes such as glutathione Stransferases (3, 4). The alkyl sulfoxide isothiocyanate derivative sulforaphane (SFN;1 Chart 1) is present in broccoli as a * Send correspondence to this author at Department of Drug Metabolism, Merck Research Laboratories, WP26A-2044, West Point, PA 19486. † Present address: Merck Research Laboratories, West Point, PA 19486. X Abstract published in Advance ACS Abstracts, October 1, 1997. 1 Abbreviations: SFN, sulforaphane; GSH, glutathione; NAC, Nacetylcysteine; ERN, erucin; Cys, cysteine; CID, collision-induced dissociation.

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Chart 1. Structures of SFN, Its Deuterated and Desaturated Derivatives, and ERN

glucosinolate and was identified recently as a major and very potent inducer of glutathione S-transferase and NADPH (quinone-acceptor) oxidoreductase activities (3, 5). Because no information is available on the biological fate of SFN in mammals, the present studies were carried out to elucidate the metabolism of SFN in the rat. Studies on the metabolic fate of several isothiocyanates in humans and animals indicate that a major pathway is the formation of NAC conjugates (6-11). Therefore, it was of interest to know if, and to what extent, SFN served as a substrate for glutathione S-transferases in vivo. The in vitro dissociation and exchange properties © 1997 American Chemical Society

Metabolism of Sulforaphane in Rats

of the GSH conjugate of SFN also were studied, since GSH conjugates of several isocyanates and isothiocyanates are known to be formed reversibly, thereby acting as “transport forms” of the parent compound (12, 13).

Experimental Procedures Syntheses. SFN and ERN. The amine precursors, 4-(methylsulfinyl)-n-butylamine and 4-(methylthio)-n-butylamine, were synthesized according to Zhang et al. (3). These amines were converted to the corresponding isothiocyanates by reaction with equimolar amounts of di-2-pyridyl thionocarbonate in CH2Cl2 at room temperature (14). The structures of the products, SFN and ERN, were confirmed by 1H-NMR and ionspray MS/MS analysis. 1-(Isothiocyanato)-4-(methylsulfinyl)-1,1-dideuteriobutane ([1,1-2H2]SFN). A solution of methyl 4-(methylthio)butyrate in diethyl ether (80 mL, 80 mmol) was added to a chilled slurry of lithium aluminum deuteride in diethyl ether (2.4 g, 57 mmol) at a rate that maintained the reaction temperature below 5 °C. The cooling bath was removed, and the reaction mixture was stirred under N2 for 2 h. The mixture was cooled to 0 °C and hydrolyzed by the addition of water (5 mL) and 10% HCl (90 mL). The organic layer was separated, and the aqueous phase was extracted with diethyl ether (4 × 500 mL). The combined extracts were dried (MgSO4), and the solvent was removed under reduced pressure. The resulting alcohol was converted to the corresponding amine essentially according to the general method of Fabiano et al. (15). 1HNMR: δ 1.49-1.68 (m, 4H, -CH2CH2CH2CD2-), 2.09 (s, 3H, CH3S-), 2.48 (t, 2H, -S-CH2-). As expected, the triplet at δ 2.65 due to -CH2-NH2 was missing in the spectrum of this deuterated analog. The amine then was converted to the corresponding sulfoxide and isothiocyanate as described above for SFN. The final product was isolated by preparative silica gel TLC using CH3CN as the developing solvent. Upon electrospray MS analysis, the isolated material yielded an MH+ ion of m/z 180. GSH Adduct of SFN. GSH (123 mg, 0.4 mmol) was dissolved in aqueous ethanol (50%, 6 mL), and the pH of the resulting solution was adjusted to ∼7.8 using 1 N NaOH. SFN (36 mg, 0.2 mmol) dissolved in ethanol (3 mL) was added to the GSH solution and the mixture was stirred at ambient temperature under N2 for 3 h. After the solvent was evaporated, the crude product was purified by reverse phase HPLC using a gradient of 0.05% TFA in CH3CN. The product gave the following NMR and MS data. 1H-NMR: δ 1.75-1.90 (m, 4H, -CH2-CH2-CH2-CH2-), 2.18-2.30 (m, 2H, Gluβ), 2.52-2.61 (m, 2H, Gluγ), 2.68 (s, 3H, CH3-SO-), 2.89-2.98 (m, 2H, -SO-CH2-), 3.52-3.63 (m, 1H, Cysβ), 3.72-3.79 (m, 2H, -CH2-NH-CS-), 3.83-3.92 (m, 1H, Cysβ′), 4.02 (s, 2H, GlyR), 4.10 (t, J ) 7.3 Hz, 1H, GluR), and 4.78 (dd, J ) 5.2, 8.4 Hz, 1H, CysR). MS: m/z 485 (MH+). GSH Adduct of ERN. This conjugate was prepared as described above for the corresponding conjugate of SFN and gave the following NMR and MS data. 1H-NMR: δ 1.59-1.80 (m, 4H, -CH2-CH2-CH2-CH2-), 2.05 (s, 3H, CH3-S-), 2.15-2.29 (m, 2H, Gluβ), 2.50-2.60 (m, 2H, Gluγ), 2.93-2.96 (m, 2H, -SCH2-), 3.49-3.60 (m, 1H, Cysβ), 3.70 (t, J ) 5.0 Hz, 2H, -CH2NH-CS-), 3.79-3.87 (m, 1H, Cysβ′), 4.00 (s, 2H, GlyR), 4.05 (t, J ) 6.8 Hz, 1H, GluR), 4.65 (dd, J ) 5.3, 8.9 Hz, 1H, CysR). MS: m/z 469 (MH+). NAC and Cys Adducts of SFN and ERN. These conjugates were synthesized according to the procedure described above but using NAC or cysteine (Cys) in place of GSH. NAC conjugate of SFN: 1H-NMR δ 1.80-1.82 (m, 4H, -CH2-CH2-CH2CH2-), 1.96 (s, 3H, CH3-CO-), 2.64 (s, 3H, CH3-SO-), 2.81-2.89 (m, 2H, -SO-CH2-), 3.54 (dd, J ) 13.9, 8.0 Hz, 1H, Cysβ), 3.723.74 (m, 2H, -CH2-NH-), 3.88 (dd, J ) 13.9, 4.3 Hz, 1H, Cysβ′), 4.54 (dd, J ) 7.4, 4.7 Hz, 1H, CysR); MS m/z 341 (MH+). NAC conjugate of ERN: 1H-NMR δ 1.59-1.77 (m, 4H, -CH2-CH2-CH2CH2-), 1.95 (s, 3H, CH3-CO-), 2.06 (s, 3H, CH3-S-), 2.49-2.53 (m, 2H, -S-CH2-), 3.51 (dd, J ) 14.1, 8.7 Hz, 1H, Cysβ), 3.663.71 (m, 2H, -CH2-NH-), 3.96 (dd, J ) 14.1, 4.7 Hz, 1H, Cysβ′), 4.65 (dd, J ) 8.6, 4.6 Hz, 1H, CysR); MS m/z 325 (MH+). Cys conjugate of SFN: upon LC-MS analysis, this conjugate yielded an MH+ ion of m/z 299.

Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1229 In Vivo Studies. Male Sprague-Dawley rats were obtained from Charles River Laboratories (Willmington, MA). Animals were allowed free access to food and water before use. Groups of four animals, each weighing ∼180-220 g, were given an ip dose (50 mg kg-1) of a solution of SFN or ERN in corn oil. Urine was collected over aqueous ascorbic acid (10 mL, 50 mg mL-1) for 24 h from conscious animals housed in metabolic cages. Bile (0-4 h) was collected, under urethane anesthesia (1 g kg-1), from another group of animals that was dosed similarly with either SFN or [1,1-2H2]SFN. Dissociation and Exchange Reactions of the Cys and GSH Adducts of SFN. The dissociation of the GSH and Cys conjugates to free SFN was studied by incubating, at 37 °C, a solution of each conjugate (40 mM) in potassium phosphate buffer (0.05 M, pH 7.4). Samples (60 µL) were withdrawn at 0.5, 1, 2, and 4 h after the start of incubation. The samples were acidified immediately with 10% TFA (10 µL) and stored on ice until analyzed by HPLC. The reaction of the GSH adduct of SFN with Cys was studied by incubating solutions of the adduct (750 µL, 1 mM) and Cys (750 µL, 5 mM) in phosphate buffer (pH 7.4), at 37 °C. Aliquots (60 µL) of the incubation mixture were taken at specified times for quantitation of the GSH and Cys conjugates by HPLC. Similarly, the reaction of the Cys adduct of SFN with GSH was studied by incubating the adduct (1 mM) with GSH (5 mM). Instrumentation and Analytical Methods. Proton NMR spectra of S-linked conjugates were recorded on a Varian VXR300 spectrometer operating at 300 MHz. Samples were dissolved in D2O or D2O/CDCl3 (75/25). Chemical shifts are reported relative to residual H2O (δ 4.67). The NMR spectra of other compounds were recorded in CDCl3, and chemical shifts were calculated relative to residual CHCl3 (δ 7.25). Ionspray mass spectrometry was carried out on a Sciex API III triplequadrupole instrument using the ionspray interface. Full scan mass spectra of synthetic compounds and product ion spectra, obtained by collision-induced dissociation (CID) of the corresponding MH+ precursor ions, were recorded by direct infusion of samples into the ion source. This was performed by dissolving ∼6 µg of sample in MeOH/1% aqueous HCOOH (1:1, 1 mL) and infusing the solutions into the instrument at a flow rate of 5 µL min-1. LC-MS/MS was performed by injecting filtered samples of bile or urine (50 µL) onto a narrow-bore C18 HPLC column (2 × 150 mm, 5 µm; Beckman Instruments, San Ramon, CA) coupled to the mass spectrometer via a splitting tee. The initial solvent was 0.06% aqueous TFA, and after 5 min, 0.06% TFA in CH3CN was added to the mobile phase at a rate of 1% min-1. The mobile phase (flow rate 1 mL min-1) was delivered by an Applied Biosystems Model 140B pump, and the column effluent was split such that the flow to the ion source was 50 µL min-1. The concentrations of NAC conjugates in urine were determined by MS/MS using the technique of selected reaction monitoring. Crude urine specimens, which had been filtered and diluted (50-fold) with MeOH/1% HCOOH, were infused directly into the mass spectrometer, and the transitions m/z 341 to 178 (NAC conjugate of SFN), 325 to 164 (NAC conjugate of ERN), and 249 to 164 (internal standard, NAC conjugate of propyl isocyanate) were monitored. The ratios of the ion current signals for analyte to internal standard then were related to concentrations of analytes using standard curves prepared by adding varying amounts of the synthetic conjugates and a fixed amount of the internal standard to specimen of control urine. The SFN liberated from S-linked conjugates was quantified using HPLC analysis with UV detection (214 nm). The separation of SFN was achieved on a Hypersil ODS C18 column (4.6 × 150 mm, 5 µm; Beckman Instruments, San Ramon, CA) with the solvent system 0.06% aqueous TFA and 0.06% TFA in CH3CN. The pump (Shimadzu LC 600) was programmed to increase the organic content from 0% to 40% over 30 min (flow rate 1 mL min-1). Under these conditions, SFN eluted at 22.5 min. Standards of SFN (0.25-3.25 mM) in phosphate buffer (0.05 M, pH 7.4) were analyzed, and a standard curve was constructed using the ratio of the area of SFN to that of the phosphate contained in the solvent (retention time 5.5 min). Similarly, standard curves were constructed over the concentration range

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Figure 1. Product ion spectrum obtained by collision-induced dissociation of the (M + H)+ ion (m/z 485) of the glutathione conjugate of SFN.

Figure 2. Product ion spectrum obtained by collision-induced dissociation of the (M + H)+ ion (m/z 469) of the glutathione conjugate of ERN. of 0.05 to 1 mM to quantify the GSH and Cys conjugates of SFN formed during thiol exchange reactions.

Results Analysis of bile and urine from rats dosed with SFN and ERN was carried out by LC-MS/MS using the neutral loss (129 Da) and precursor ion (m/z 164) scan modes to detect GSH and NAC conjugates, respectively. Neutral loss scan analysis of bile from rats given SFN resulted in the detection of five candidate conjugates (designated M1-M5 on the basis of their HPLC elution order) which were absent in bile from control animals. Metabolites M2 and M4 exhibited an MH+ ion at m/z 485 and 469, suggesting that these metabolites may be

the GSH adducts of SFN and ERN (sulfide metabolite of SFN), respectively. This was indeed the case as the isolated metabolites yielded identical retention times and product ion spectra (Figures 1 and 2) to those of the corresponding synthetic standards. Metabolite M1 exhibited an MH+ species at m/z 483 which indicated that this metabolite may be the GSH adduct of a desaturated metabolite of SFN. It was suspected that the site of desaturation was the carbon R to the isothiocyanate functional group. To elucidate the structure of this conjugate, [1,1-2H2]SFN was synthesized and administered to rats. This approach was taken because the deuterated analog was more accessible

Metabolism of Sulforaphane in Rats

Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1231

Figure 3. Product ion spectrum obtained by collision-induced dissociation of the (M + H)+ ion (m/z 341) of the N-acetylcysteine conjugate of ERN.

Figure 4. Product ion spectrum obtained by collision-induced dissociation of the (M + H)+ ion (m/z 325) of the N-acetylcysteine conjugate of ERN.

synthetically than the R-desaturated derivative. Analysis (neutral loss of 129 Da) of bile from rats dosed with [1,1-2H2]SFN revealed metabolites with MH+ ions at m/z 484, 487, and 471. The MH+ ion at m/z 484 was consistent with an R-desaturated metabolite of SFN, since one of the two deuterium atoms on the carbon on position 1 of the molecule (Chart 1) would be lost when the desaturated metabolite is formed. CID of m/z 484 resulted in diagnostic ions at m/z 308 (GSH2)+ and 355 (MH - 129)+. Thus, M1 was tentatively identified as the GHS conjugate of ∆1-SFN. As expected, the MH+ ions of the conjugates of SFN and ERN shifted by 2 Da (m/z 485 vs 487 and m/z 469 vs 471) when rats were dosed with [1,1-2H2]SFN in place of SFN.

Biliary metabolites M3 and M5 yielded MH+ ions at m/z 341 and 325, respectively. Based on their MH+ ions, these conjugates appeared to be respectively the NAC adducts of SFN and ERN. This identification was confirmed when the retention times and MS/MS spectra (Figures 3 and 4) of the isolated metabolites were found to be identical to those obtained from the authentic standards. Analysis of urine from SFN-dosed rats using the technique of precursor ion scanning (m/z 164) resulted in the detection of metabolites having MH+ ions at m/z 341, 325, and 339. The former two species were identified as the NAC conjugates of SFN and ERN, which also were identified as biliary metabolites of SFN. The

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Table 1. Amount (%) of NAC Conjugates Excreted in the Urine (24 h) of Rats after an ip Dose of 50 mg kg-1 SFN or ERN compound administered SFN ERN a

fraction of dose excreted as NAC conjugate of SFN ERN 12.3a

60.2 ( 66.7 ( 3.1

11.6 ( 2.2 28.5 ( 3.0

total 71.8 95.2

exchange in the presence of a suitable sulfhydryl reagent. With the Cys conjugate, by the first sampling time the concentration of the GSH conjugate reached 20% of total conjugates and by 4 h the Cys conjugate had decreased to less than 15% of the original concentration (Figure 5). In the case of the GSH conjugate, equilibrium was reached before 2 h and at 2 h the concentration of the Cys conjugate exceeded that of the GSH conjugate.

Data represent mean ( SD (N ) 4).

Discussion

Figure 5. Concentrations of the cysteine (SFN-Cys) and glutathione (SFN-GSH) conjugates of SFN following incubation of SFN-Cys (1 mM) with GSH (5 mM).

Figure 6. Concentrations of the glutathione (SFN-GSH) and cysteine (SFN-Cys) conjugates of SFN following incubation of SFN-GSH (1 mM) with Cys (5 mM).

metabolite giving rise to an MH+ ion at m/z 339 most likely is the NAC conjugate of ∆1-SFN, although this structural assignment could not be confirmed due to the absence of a standard of this compound. Quantitative determination of the NAC adducts of SFN and ERN excreted in urine after an ip dose of SFN resulted in the data shown in Table 1, which indicates that the majority of a single dose of the parent isothiocyanate is eliminated as the corresponding NAC adduct. A small fraction of the dose also appeared in urine as the NAC adduct of ERN. In rats administered ERN, some 95% of the dose was excreted as mercapturates, with the majority being the NAC conjugate of SFN. When the GSH adduct of SFN was incubated in aqueous phosphate buffer in the absence of a thiol, a maximum of only 0.31% of the conjugate dissociated over a 4 h period. The corresponding figure for the Cys adduct was 1.2%. On the other hand, incubation of each conjugate in the presence of either excess Cys or GSH resulted in the data shown in Figures 5 and 6, which indicate that these conjugates are reactive toward thiol

In this study of the metabolic fate of SFN and ERN, particular emphasis was placed on GSH-dependent pathways since it has been reported that conjugation with GSH is the major route by which many isothiocyanates are eliminated from the body (6-11). The MS/MS technique of neutral loss scanning proved to be extremely valuable in detecting conjugated SFN metabolites in bile, in that neutral loss scan of 129 Da revealed the presence of not only GSH adducts (16) but also NAC conjugates. The neutral lost from the latter conjugates probably is CH2dC-(NHCOCH3)-COOH. In bile of animals dosed with SFN, three GSH conjugates were detected which were identified as those of SFN, ERN, and ∆1-SFN. The corresponding NAC conjugates were identified in both bile and urine, indicating that the enzymes that convert these GSH adducts to mercapturates are present in both the liver and kidney (17). The NAC conjugates of SFN and ERN showed marked differences in the product ion spectra generated by CID of the respective MH+ ions. In the spectrum of the NAC conjugate of SFN, the most abundant product ion was m/z 178 ([MH - NAC]+). In contrast, the corresponding ion (m/z 162) was absent from the spectrum of the NAC conjugate of ERN, which was dominated by the fragment ion at m/z 164 (protonated NAC) which has been reported for NAC conjugates of isocyanates (18). By monitoring the respective transitions, a sensitive and highly selective assay was developed for the quantitative detemination of these conjugates in rat urine. Using this assay, it was shown that ∼72% of the dose of SFN administered to rats was excreted as the NAC adducts of SFN and ERN over 24 h. Most of the remaining dose probably is accounted for by the GSH and NAC conjugates excreted in the bile. Rats also were dosed with ERN to assess the reversibility of the oxidation-reduction biotransformation of the sulfur atom in ERN and SFN. The results demonstrated that almost all of the administered ERN was excreted as NAC conjugates of ERN and SFN, and it was found that oxidation of the sulfide in ERN was a more favored metabolic reaction than the reduction of the sulfoxide in SFN (Table 1). Thus, the metabolic fate of SFN in the rat was similar to that reported for other isothiocyanates in as much as the majority of the dose was cleared via the mercapturic acid pathway. However, SFN also underwent oxidative metabolism to a desaturated derivative (most likely ∆1SFN) which appeared to contribute appreciably to the clearance of the parent compound. The identification of ∆1-SFN (∆3-SFN occurs naturally) as an in vivo metabolite of SFN is of particular interest since one of the mechanisms by which isothiocyanates exert chemoprotective effects is believed to be through inhibition of cytochrome P450 enzymes (2, 19). Although we have not yet studied the genesis of this particular metabolite, cytochrome P450 enzymes are known to catalyze the desaturation of many substrates with aliphatic function-

Metabolism of Sulforaphane in Rats

alities (20-22), and it is possible that ∆1-SFN could be a product of oxidation of SFN by cytochrome P450 enzymes. This metabolic pathway could represent one way by which SFN interacts with cytochrome P450 enzymes. Additionally, SFN could undergo cytochrome P450-mediated activation to the more reactive isocyanate derivative, as has been shown for other isothiocyanates (23, 24), although such a metabolite was not detected in the present in vivo study. The reversibility studies conducted with the synthetic conjugates indicate that both the GSH and Cys adducts of SFN were stable in aqueous media at physiological pH and temperature. At the end of a 4 h incubation period,