Enzyme-Induction Dependent Bioactivation of Troglitazone and

adduct (ML3) but it was substituted in the thiazolidinedione moiety. Pretreatment with DEX ... benzyl]-2,4-thiazolidinedione; Figure 1)1 is the protot...
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Chem. Res. Toxicol. 2001, 14, 965-974

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Enzyme-Induction Dependent Bioactivation of Troglitazone and Troglitazone Quinone In Vivo Justice N. Tettey,†,‡ James L. Maggs,† W. Garth Rapeport,§ Munir Pirmohamed,† and B. Kevin Park*,† Department of Pharmacology and Therapeutics, The University of Liverpool, Liverpool L69 3GE, U.K., and GlaxoWellcome Research and Development, Greenford, Middlesex UB6 0HE, U.K. Received September 6, 2000

Troglitazone (TGZ), a 2,4-thiazolidinedione antidiabetic, causes hepatotoxicity in 1.9% of patients. TGZ is an inducer of, and substrate for, hepatic P450 3A. Microsomal metabolism yields a benzoquinone (TGZQ) and reactive intermediates. Kassahun et al. [Kassahun et al. (2001) Chem. Res. Toxicol. 14, 62-70] have trapped the intermediates as thioester, thioether, and disulfide conjugates of glutathione and found five conjugates in rat bile. The thioether was substituted in the chromane moiety. We have investigated the effect of the P450 3A inducer, dexamethasone (DEX), on metabolism of TGZ and TGZQ in rats and assessed the compounds’ cytotoxicity. TGZ-glucuronide and sulfonate were confirmed as principal biliary metabolites of TGZ (50 mg/kg, iv). Bile from noninduced animals also contained a TGZ-glutathione thioether adduct (ML3) but it was substituted in the thiazolidinedione moiety. Pretreatment with DEX (50 mg/kg/day for 3 days) resulted in a 2-5-fold increase in the biliary concentration of ML3 and a 2-fold increase in the concentration of TGZQ, which was commensurate with the induction of hepatic P450 3A. Three of the known glutathione-conjugated metabolites were also found. TGZQ (50 mg/kg, iv) was metabolized to an analogue of one of the TGZ-glutathione thioesters and a glutathione adduct of TGZQ hydroquinone after DEX pretreatment. TGZ quinol glucuronide was a biliary metabolite of TGZ and TGZQ. Its formation would represent deactivation of TGZQ. TGZ was toxic to rat hepatocytes and Hep-G2 cells at concentrations exceeding 50 and 25 µM, respectively, after 24 h. In contrast, TGZQ was nontoxic to rat hepatocytes and toxic to Hep G2 cells only at concentrations exceeding 100 µM. Our results show that TGZQ as well as TGZ yields reactive metabolites in vivo, and that bioactivation is enhanced by induction of P450 3A. However, hepatotoxicity is unlikely to be due to either TGZQ or its metabolites.

Introduction The 2,4-thiazolidinedione oral hypoglycaemic agents, or glitazones, of which troglitazone (TGZ; Rezulin; (()5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)benzyl]-2,4-thiazolidinedione; Figure 1)1 is the prototype, are insulin-sensitizing compounds which are effective in the management of Type-II or noninsulin-dependent diabetes (1). However, treatment with TGZ has been accompanied by elevation of serum alanine aminotransferase levels (more than 3-fold normal) in 1.9% of patients (2, 3). In a small number of patients, severe hepatotoxicity has been reported (4-6). To date, there have been a few reports of hepatotoxicity associated with rosiglitazone (7) but none with pioglitazone. These differ from TGZ in not possessing the benzoquinone-forming 6-hydroxychromane nucleus. This feature, in view of the established toxicity of quinones (8), has been suggested * To whom correspondence should be addressed. E-mail: bkpark@ liv.ac.uk. † University of Liverpool. ‡ Present address: Department of Pharmaceutical Sciences, Strathclyde Institute for Biomedical Sciences, 27 Taylor Street, Glasgow G4 0NR, U.K. § Glaxo Wellcome Research and Development. 1 Abbreviations: TGZ, troglitazone; TGZQ, troglitazone quinone; DEX, dexamethasone; GSH, reduced glutathione.

as a possible requirement for the hepatotoxicity associated with TGZ (5). The oxidative biotransformation of the 6-hydroxy-5,7,8trimethylchromane moiety of TGZ to give troglitazone quinone (TGZQ; Figure 1) is catalyzed by microsomal P450 3A4 and 2C8 (9). In human and rat hepatocytes, TGZ induces P450 3A activity in a dose-dependent fashion (10), the induction capacity being between that of rifampicin and dexamethasone (DEX). Therefore, it might be expected that TGZ would increase its own turnover to TGZQ (9, 11). Rosiglitazone, which shows a lower tendency for hepatic toxicity than TGZ, also possesses a lower capacity to induce P450 3A (12). It is not known if this difference in toxicity is related to the difference in inducing capacity. TGZQ is essentially a relatively stable compound: the trimethyl-substitution pattern precludes direct attack by nucleophiles. Nevertheless, human liver microsomes can metabolize TGZ to intermediates which bind covalently and selectively to P450 3A4/5 (13). Kassahun et al. (14) have trapped the electrophilic metabolites of TGZ with glutathione, obtaining thioester and disulfide conjugates and one thioether conjugate of TGZ substituted in the 5-methyl group on the chromane ring. The thioesters and disulfides are thought to derive from reactive intermediates generated through an oxidative opening of the

10.1021/tx0001981 CCC: $20.00 © 2001 American Chemical Society Published on Web 07/13/2001

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Tettey et al.

Figure 1. Chemical structures of troglitazone (TGZ) and troglitazone quinone (TGZQ).

thiazolidinedione ring which is mediated principally by P450 3A. Formation of one of the disulfides additionally requires oxidation at the chromane ring to create a quinone function. The precursor of the TGZ conjugate is produced by a distinct pathway, catalyzed by multiple P450 isoforms in human liver microsomes, which entails oxidation of the chromane moiety to a putative o-quinone methide. Five glutathione-conjugated metabolites have been found in the bile of rats administered TGZ (14). Their presence raises the possibility that chemically reactive intermediates could be causally involved in the hepatotoxicity associated with TGZ. Although the metabolism of TGZ is documented (11, 15-17), the effect of P450 3A induction on the formation of TGZQ and the reactive intermediates in vivo has not been established. An increase in hepatic P450 3A in rats would be expected following administration of TGZ in an analogous fashion to the increase demonstrated with rat hepatocytes (10). The induction of hepatic P450 3A in the rat is effectively achieved by administration of DEX (18). In this study, using adult male rats, the nature of the biliary metabolites of TGZ with and without DEX pretreatment has been investigated. Evidence for the metabolism of TGZ to reactive intermediates, as represented by the elimination of a variety of glutathione adducts in bile, is confirmed and augmented. A novel thioether adduct of TGZ and glutathione is also described. For the first time, it is reported that TGZQ administered to rats undergoes bioactivation to intermediates which form glutathione adducts. The enhanced turnover of TGZ to TGZQ has been assessed. The relative cytotoxicity of TGZ and TGZQ to rat hepatocytes and the human hepatoma Hep G2 cell-line has been examined in order to define the effect of an increase in TGZQ concentrations on cell viability.

Materials and Methods Materials. TGZ was a gift from GlaxoWellcome (Stevenage, U.K.). TGZQ was synthesized by the method of Yoshioka et al. (19) and its purity determined by LC-MS (g95%). Collagenase type-IV and modified Chee’s medium were obtained from Gibco BRL (Life Technologies, Paisley, U.K.). CellTiter 96 cell proliferation assay kits were obtained from Promega Corporation (Madison, WI). The Western blotting assay kit with purified goat anti-rabbit IgG alkaline phosphatase conjugate was obtained from Bio-Rad Laboratories (Hercules, CA). Rabbit anti-rat cytochrome P450 3A antibody was obtained from Amersham Life Sciences (Little Chalfont, U.K.) Unless otherwise stated, all enzymes, general and cell-culture reagents were obtained from Sigma-Aldrich Chemical Co. (Poole, U.K.). Solvents were products of Fisher Scientific (Loughborough, U.K.) and were of chromatographic, analytical, or cell-culture grade. Synthesis of TGZ Quinol. TGZQ (1.5 µmol) in pure ethanol (0.4 mL) was reduced to the quinol with sodium dithionite (27.6 µmol) in water (0.3 mL) at room temperature over 100 min. The reaction mixture was concentrated to an aqueous residue under nitrogen, diluted with five volumes of 1/15 M sodium phosphate buffer, pH 7.5, and extracted with ethyl acetate (four volumes × 2). The extracts were reconstituted in methanol. TGZQ and

the quinol were eluted (tR 10.7 and 6.5 min, respectively) from a Columbus 5-µm C-8 column (250 mm × 4.6 mm i.d.; Phenomenex, Macclesfield, U.K.) with acetonitrile (50-70% over 15 min) in 10 mM ammonium acetate buffer, pH 3.8. Electrospray spectra were acquired at a cone voltage of 70 V for TGZQ m/z 475 ([M + NH4]+, 69), 458 (34), 440 (52), 288 (9), 217 (100), 189 (31), 165 (33) and for TGZ quinol m/z 477 (100), 460 (16), 442 (49), 290 (6), 219 (68), 191 (11), 165 (93). Preparation of Rat and Human Microsomes. Rat and human liver microsomes were prepared by differential centrifugation as described previously (20) and the protein content determined using the BCA protein assay (Pierce, Rockford, IL) with bovine serum albumin as standard. The incubation mixture (final volume, 1 mL) contained microsomal protein (1 mg), TGZ (100 µM), MgCl2 (10 mM), phosphate buffer (100 mM, pH 7.4) and NADPH (8.33 mg/mL). Incubations without NADPH or substrate were used as negative controls. After a 30-min incubation at 37 °C, acetonitrile (2 mL) was added to the incubation mixture and centrifuged at 3500g (10 min). Following evaporation of the separated supernatant at 50 °C under a stream of nitrogen, the residue was reconstituted in methanol (100 µL) and analyzed by LC-MS. In Vivo Metabolism. Male Wistar rats (195-250 g) were obtained from a colony maintained by the University of Liverpool and were treated with DEX (50 mg/kg/day, ip, 200 mg/mL in sesame oil, DEX/TGZ) or TGZ (50 mg/kg/day, ip, 200 mg/ mL in sesame oil, TGZ/TGZ) for 3 days. This was followed by a 24-h wash-out period before the animals were used for in vivo metabolism studies. Control animals (-/TGZ) were administered sesame oil (250 µL/kg/day, ip) for 3 days. Animals were anaesthetized with urethane (1.4 mg/mL in isotonic saline; 1 mL/kg, ip). Cannulae were inserted into the jugular vein, trachea and the common bile duct, and the penis ligated. Bile was collected for about 10 min prior to dosing with TGZ (5 mg/kg and 50 mg/kg body weight) or TGZQ (50 mg/kg). The compounds were dissolved in DMSO (100 µL) and administered via the jugular vein. Bile was collected at hourly intervals for 3 h and the concentration of TGZQ formed from TGZ determined by HPLC. Chromatographic peak assignment of the known metabolites of TGZ was facilitated by LC-MS in selected ion monitoring mode. The bile was also searched for known and novel glutathione-conjugated metabolites and metabolites of TGZQ by LC-MS/MS analysis. For the confirmation of glucuronidation and sulfonation, the conjugates in bile were hydrolyzed, after diluting with sodium acetate buffer (0.1 M, pH 5.0), with H-2 β-glucuronidase-arylsulfohydrolase at 37 °C for 4-18 h and subsequently sampled for LC-MS analysis. Determination of Rat Hepatic P450 3A Protein. The protein content of P450 3A in rat hepatic microsomes was visualized using Western immunoblotting. Microsomal protein samples (total protein, 10 µg) from livers of variously pretreated rats (DEX/TGZ, TGZ/TGZ and -/TGZ, n ) 4 per group), as described above, were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes were probed with rabbit anti-rat cytochrome P450 3A antibody and detected with purified goat anti-rabbit IgG alkaline phosphatase conjugate according to the manufacturer’s instructions (Bio-Rad Laboratories). Quantification of Biliary TGZQ. Aliquots (25-50 µL) of bile were eluted from a Luna 3-µm C-18 analytical column (75 mm × 4.6 mm i.d.; Phenomenex) at ambient temperature with linear gradients of acetonitrile (20 to 80% over 30 min) in ammonium acetate buffer (25 mM, pH 3.7) using a Dionex P580

Metabolic Activation of Troglitazone pump (Dionex Corporation, Sunnyvale, CA) at a flow rate of 0.7 mL/min. Chromatograms were monitored at 254 nm with a Dionex UVDI70S detector and the data processed with Chromeleon HPLC software (Dionex Corporation). TGZQ (tR 22.0 min) in bile was quantified by HPLC using authentic TGZQ as external standard. Calibration curves were linear within the working range of 0-200 µg/mL TGZQ (r g 0.99) with a limit of quantification of 9 µg/mL (signal/noise ratio, 3). The above chromatographic system is referred to as system-1. Liquid Chromatography-Mass Spectrometry. Full-scanning and selected ion monitoring data (LC-MS) were obtained with a Quattro II triple quadrupole mass spectrometer fitted with an in-line electrospray source (Micromass, Manchester, U.K.). Eluent was delivered by Jasco PU-980 pumps via an HG980-30 mixing module. Metabolites and standards were eluted from a HyPURITY Elite 5-µm C-18 column (150 mm × 4.6 mm i.d.; Hypersil, Runcorn, U.K.) at room temperature. The analytes were monitored at 254 nm. Eluate split-flow to the mass spectrometer was approximately 50 µL/min. Nitrogen was used as the nebulizing and drying gas. The capillary voltage was 3.9 × 103 V; the source temperature, 70 °C; the standard cone voltage, 30 V (70 V for fragmentation); and the multiplier voltage, 650 V. Full-scanning mass spectra were acquired between m/z 100 and 1050 at 5 s/scan. Daughter spectra were acquired under the following conditions; cone voltage, 30 V; collision energy, 30 eV; collision gas (argon) pressure, ca. 4 × 10-4 mBar; scan range, m/z 100-500 at one scan per 5 s. For selected ion monitoring, eight channels were recorded with a dwell time of 200 ms and an interchannel delay of 20 ms. All data were processed via MassLynx 2.0 software. LC-MS/MS spectra were obtained by data-dependent scanfunction switching (threshold, 20 counts) on a Micromass Q-Tof2 hybrid quadrupole/time-of-flight instrument equipped with a Z-spray ion source. Aliquots (20 µL) of bile were eluted from a Symmetry 300 3.5-µm C-18 column (100 mm × 2.1 mm i.d.; Waters Ltd., Watford, U.K.) at room temperature. Eluent was delivered with a Waters Alliance 2690 chromatograph, and comprised acetonitrile containing 0.1% formic acid (20% for 5 min; 20-50% over 30 min; 50% for 5 min; 50-90% over 1 min) and 0.1% aqueous formic acid. The flow rate was 0.3 mL/min. Source and desolvation temperatures were 120 and 400 °C, respectively. Capillary and cone voltages were 3.1 kV and 35 V, respectively. The collision energy profile consisted of successive 0.5-s MS/MS scans at 10, 15, 20, 25, and 30 eV. Argon was used as collision gas. Calibration files were created with the spectra of [Glu1]-fibrinopeptide B and polyalanine in positiveand negative-ion mode, respectively. Spectra were acquired between m/z 50-999 at 1 s/scan. Mass resolution was 10 × 103 fwhm. Data were processed via MassLynx 3.5 software. Metabolism and Cytotoxicity Assays for Rat Hepatocytes. Hepatocytes were isolated from whole livers of male Wistar rats by a two-step collagenase perfusion technique (21). The viability of the cell suspension was determined by trypanblue exclusion (typically g87%). For metabolism studies, hepatocytes were incubated in suspensions (2 × 106 viable cells/mL) as described previously (22) with TGZ (50 µM) in Krebs-Henseleit buffer (pH 7.4). TGZ was dissolved in DMSO. The final concentration of DMSO in incubations was 0.1% (v/v). Incubations were terminated after 2 h with an equivalent volume of ice-cold acetonitrile and the total incubate extracted with 4 vol of ice-cold acetonitrile. The mixture was centrifuged (3000 rpm, 5 min) to sediment cell debris, and the supernatant evaporated under a stream of nitrogen at 50 °C. The residue was reconstituted in methanol (150 µL) and analyzed by LC-MS. For cytotoxicity assays, hepatocytes isolated from untreated, vehicle-treated and DEX-treated (50 mg/kg/day, ip, 200 mg/mL in sesame oil, for 3 days) rats were suspended in Chee’s medium and plated at a density of 104 cells/100 µL/well into collagencoated (type-1, 25 µg/cm2) 96-well plates. After incubation for 4 h at 37 °C in a humidified atmosphere (5% CO2/air), the medium was aspirated to remove unattached dead cells. The cultures

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Figure 2. Western blotting of rat liver microsomes for rat P450 3A protein. Microsomes (10 µg of total protein) were prepared from rat livers and subjected to Western blot analysis (as detailed in Materials and Methods). Blots were probed with antibodies raised against rat P450 3A. Lane 1, rat P450 3A positive control; 2 and 3, DEX-treated (50 mg/kg/day for 3 days, ip, 200 mg/mL in sesame oil) rats (n ) 4); 4 and 5, TGZ-treated (50 mg/kg/day for 3 days, ip, 200 mg/mL in sesame oil) rats (n ) 4); 6 and 7, sesame oil-treated (negative control, 250 µL/kg/ day for 3 days ip) rats. were then incubated for an additional 16 h in modified serumfree Chee’s medium supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 0.4 mg/mL DEX, before the treatments. Culture of Hep G2 Cells. Hep G2 cells were plated at a density of 104 cells/100 µL/well into 96-well plates. The cultures were then incubated in modified Eagle’s medium, supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% (v/v) foetal calf serum, for 24 h at 37 °C in a humidified atmosphere (5% CO2/air) before treatments. Cytotoxicity assays with TGZ and TGZQ were performed in media free of foetal calf serum. Cytotoxicity Assays. Primary cultures of rat hepatocytes and Hep G2 cells were treated with a series of concentrations of TGZ (0-100 µM) and TGZQ (0-100 µM) for 24 h. TGZ and TGZQ were dissolved in DMSO, the final concentration of which in the culture medium was less than 0.1% (v/v). Cell viability was assessed with the CellTiter 96 assay kit. Briefly, 20 µL of a combined solution of a tetrazolium compound (MTS, 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and phenazine methosulfate was added to each well. After incubation for 1 h at 37 °C in a humidified atmosphere (5% CO2/air), the absorbance at 490 nm was measured on an enzyme-linked immunosorbent assay reader. Statistical Analyses. Unless otherwise specified, data presented are mean ( SE of four separate experiments. Sample comparisons were made using a two-sample nonparametric test (Microsoft Excel 2000) with statistical significance at p < 0.05.

Results Oxidative Metabolism of TGZ by Rat and Human Microsomes. LC-MS analyses of incubations of TGZ with microsomes from human and rat liver identified the presence of TGZQ (retention time, 22.3 min; m/z 475, [M + NH4]+) [acetonitrile (45-65% over 15 min) in 25 mM ammonium acetate, pH 3.8]. The metabolite yielded the same daughter spectrum [m/z 458, 440, 217 (100%), 189, 165] as the authentic standard and its formation was dependent on the presence of NADPH. No other product of oxidative metabolism of TGZ was detected in incubations with either human or rat liver microsomes. Effect of DEX and TGZ Pretreatment on Rat Hepatic P450 3A Protein. Western blot analyses (Figure 2) of the protein content of P450 3A in liver microsomes (total protein, 10 µg) from DEX pretreated (DEX/TGZ) and TGZ pretreated rats showed an increase in expression (2-fold) with the former but no significant change with the latter, relative to controls (-/TGZ). Metabolism in Vivo and Analysis of Rat Biliary Metabolites. TGZQ was not detected in the first 3 h bile collections from animals administered a 5 mg/kg dose (iv) of TGZ. The conjugated metabolites identified by mass spectral (LC-MS) and spectrophotometric comparison of pre- and postdosing bile were TGZ glucuronide (retention

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Figure 3. Determination of TGZQ in bile. The other major metabolites, troglitazone glucuronide (TGZG) and troglitazone sulfonate (TGZS) are shown. The chromatographic conditions were as described in the text for system-1. Table 1. Concentration of TGZQ in Bile of TGZ- and DEX-Treated Rats Concentration (µG/ML) time (h)

-/TGZ

TGZ/TGZ

DEX/TGZ

0-1 1-2 2-3

83.9 ( 15.5 59.4 ( 7.6 37.6 ( 7.2

59.8 ( 23.7 39.8 ( 14.1 24.5 ( 10.4

194.9 ( 82.9a 81.8 ( 6.7 42.7 ( 5.8

a p < 0.05. -/TGZ: no pretreatment + TGZ (50 mg/kg iv on day 5). TGZ/TGZ: pretreatment with TGZ (50 mg/kg ip, 3 days) + TGZ (50 mg/kg iv on day 5). DEX/TGZ: pretreatment with DEX (50 mg/kg ip, 3 days) + TGZ (50 mg/kg iv on day 5).

time, 13 min; m/z 635 for [M + NH4]+) [acetonitrile (2565% over 25 min) in 25 mM ammonium acetate, pH 3.8], TGZ sulfonate (17.5 min; m/z 539, [M + NH4]+), hydroxyTGZ sulfonate/benzoquinone sulfonate (17.5 min; m/z 555, [M + NH4]+), and TGZ benzoquinol sulfonate (17.5 min; m/z 557, [M + NH4]+). The glucuronide and sulfonates underwent diagnostic elimination of dehydroglucuronic acid (176 amu) and SO3, respectively. Enzymatic hydrolysis of the conjugates using β-glucuronidase-arylsulfohydrolase and subsequent LC-MS analysis of the hydrolysate confirmed the presence of TGZ and trace quantities of TGZQ. The administration of a higher dose of TGZ (50 mg/kg iv) was characterized by the presence of TGZQ in bile (Figure 3) from control (-/TGZ) and TGZ and DEX pretreated rats. The concentrations of TGZQ in hourly collections of bile over 3 h are shown in Table 1. Using selected ion monitoring during LC-MS analysis, a peak in the negative-ion mass chromatogram (retention time, 25.0 min) [acetonitrile (15% for 10 min, 15-50% over 15 min) in 0.1% (v/v) formic acid] corresponding to a glutathione adduct of TGZ (ML3; m/z 745 for [M - 1]-) was found after, but not before, the dosing of otherwise untreated rats with TGZ (50 mg/kg). The adduct in bile from DEX pretreated rats was more abundant, and yielded a positive-ion spectrum indicative of a glutathione conjugate: m/z 764 ([M + NH4]+), 440 ([M + NH4-NH3GSH]+), and 308 ([GSH + 1]+). When the ammonium acetate was replaced with formic acid (0.1%), to obtain the eluent used by Kassahun et al. (14), ML3 gave a spectrum containing ions at m/z 747 ([M + H]+), 618 ([M + H-glutamyl]+), 440, 308, and 179 ([308-glutamyl]+). The metabolite peaks in bile from untreated and DEXinduced animals coeluted. Accurate mass measurements of ions in the MS/MS spectrum of m/z 747 were achieved with the Q-Tof2 and confirmed the assignment of a TGZglutathione adduct (Figure 4; Table 2). However, positiveion analysis did not yield fragments from which the regional location of thioether substitution could be de-

Tettey et al.

Figure 4. Positive-ion electrospray mass spectrum of ML3 (tR 21 min) obtained on a Micromass Q-Tof2 by LC-MS/MS analysis of the first hourly collection of bile from a rat pretreated with DEX and dosed with TGZ (50 mg/kg, iv). Table 2. Accurate Mass Measurements of TGZ-Glutathione Adduct ML3 massa

calcd mass

mDa

ppm

formula

747.2342b 618.1987 440.1534 308.0918c 233.0591

747.2370 618.1944 440.1532 308.0916 233.0596

-2.8 +4.3 +0.2 +0.2 -0.5

3.7 6.9 0.4 0.6 2.0

C34H43N4O11S2 C29H36N3O8S2 C24H26NO5S C10H18N3O6S C8H13N2O4S

a Derived from LC-MS/MS spectra acquired on a Micromass Q-Tof2. b [M + 1]+. c [GSH + 1]+.

Figure 5. Negative-ion electrospray mass spectrum of ML3 obtained on a Q-Tof2 by LC-MS/MS analysis of the first hourly collection of bile from a rat pretreated with DEX and dosed with TGZ (50 mg/kg, iv). G represents C10H16N3O6.

duced. This was accomplished by negative-ion analysis. The anion chromatogram for glutathione adducts (m/z 745) contained a single major peak at 25.0 min present only after administration of the drug. At a cone voltage of 70 V, the anion fragmented extensively (m/z 306, 272, 254, 160), the major fragment being m/z 306 ([GS]-). All of the ions were found in the product-ion spectrum of m/z 745 (Figure 5). Peaks at m/z 128 and 143 can be derived from the glutamyl and glutamlycysteinyl moieties, respectively. The peak at m/z 179 can be derived from the chromane ring. The possible sites of glutathione substitution in ML3 were limited by the presence of m/z 254. This fragment, it was concluded, was formed by cleavage of the C-S bond within the glutathione moiety and loss of the chromane ring system; and therefore consisted of the phenoxy and thiazolidinedione rings and a sulfur atom. Subsequent elimination of the phenoxy moiety, producing

Metabolic Activation of Troglitazone

Figure 6. Positive-ion electrospray mass spectrum of TGZ quinol glucuronide obtained on a Quattro II by LC-MS analysis of the first hourly collection of bile from a rat pretreated with DEX and dosed with TGZQ (50 mg/kg, iv). This metabolite was also present in bile following administration of TGZ (50 mg/kg) and in incubations of rat hepatocytes with TGZ (50 µM).

the ion at m/z 160, restricted the thioether linkage to either the thiazolidinedione ring or the adjacent methylene bridge. TGZ quinol glucuronide (m/z 653, [M + NH4]+), a metabolite of TGZ, was found in bile by LC-MS analysis (retention time, 20 min) [acetonitrile (15% for 10 min, 15-50% over 15 min) in 25 mM ammonium acetate, pH 3.8]. The formation of the quinol glucuronide from TGZQ was confirmed by the iv administration of TGZQ (50 mg/ kg, iv). This metabolite yielded diagnostic fragments (Figure 6) at m/z 442 ([M + NH4-NH3-dehydroglucuronic acid-H2O]+), 424 ([442-H2O]+), 219 and 165; all of which were found in the product-ion spectrum of [M + 1]+ (m/z 636) obtained on the Q-Tof2 (data not shown). Formation of m/z 219, a ring-opened chromane fragment, was ascribed to scission of the O-CH2 linkage following elimination of dehydroglucuronic acid and subsequent dehydration of a hypothetical ring-opened hydroxychromane intermediate. The ion at m/z 165 can be derived via elimination of a C-4 fragment from the alkyl moiety of m/z 219. It is common to the mass spectra of TGZ, TGZQ and a number of R-tocopherol derivatives with oxygenated chromane rings (23). Enzymatic hydrolysis of the conjugates in bile from TGZQ-treated rats using β-glucuronidase resulted in the recovery of large quantities of TGZQ, formed presumably by oxidation of the corresponding quinol. This was not prevented by coincubation with ascorbic acid (1.0 mM). The susceptibility of TGZ quinol to autoxidation was demonstrated by incubating synthetic quinol (approximately 1.5 mM) in sodium acetate (100 mM, pH 5.0) at 37 °C for 16 h, when complete conversion to TGZQ occurred. Metabolism of TGZ and TGZQ in Rats Pretreated with TGZ or DEX. Pretreatment of rats with TGZ (50 mg/kg/day for 3 days) did not result in an increase in the biliary concentration of the TGZ-glutathione adduct (estimated from peak areas for m/z 745) nor of TGZQ over the 3-h collection period (Table 1). The metabolite profile was essentially similar to that of rats without pretreatment. This result was consistent with the amount of immunoreactive P450 3A protein, which did not show a difference between TGZ/TGZ and -/TGZ. DEX pretreatment resulted in a 2-5-fold (n ) 4) increase in the concentration of the TGZ-glutathione adduct relative to vehicle-treated and TGZ pretreated rats. The magnitude of the change in concentration of the TGZ-glutathione adduct was determined by the LC-MS analysis of all bile samples on the same day. The concentration of the glutathione conjugate in bile declined by about 80% from

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Figure 7. Positive-ion electrospray mass spectrum of ML2 metabolite of TGZQ (50 mg/kg, iv) obtained on a Q-Tof2 by LCMS/MS analysis of the first hourly collection of bile from a rat pretreated with DEX. This conjugate was characterized as the quinone analogue of the thioester metabolite designated M2 by Kassahun et al. (14).

the first to the second hourly collection. Also, the pretreatment of animals with DEX resulted in a 2-fold increase in the concentration of TGZQ in the first hourly bile collections (Table 1). Three of the glutathione-conjugated biliary metabolites of TGZ described by Kassahun et al. (14) were found through LC-MS/MS analysis (Q-Tof2) of bile (0-1 h collection) from rats pretreated with DEX, namely the two isomers of the thioester M2 (retention times, 8.0 and 9.0 min) and the thioester M3 (16.0 min). The [M + 1]+ ions of the M2 isomers at m/z 781 yielded identical spectra: m/z 763 ([M + 1-H2O]+), 706 ([M + 1-glycine]+), 688 ([706-H2O]+), 652 ([M + 1-glutamyl]+), 634 ([652H2O]+), 616, 549, 531, 456 ([M + 1-H2O-GSH]+), 411 (100), 394, 393, 308 ([GSH + 1]+), 265, 233, 191, 179, 145. The [M + 1]+ ion of M3 at m/z 763 exhibited an analogous spectrum: m/z 745 ([M + 1-H2O]+), 688 ([M + 1-glycine]+), 634 ([M + 1-glutamyl]+), 616 ([634-H2O]+), 531, 522, 513, 470, 438 ([745-GSH]+), 393, 372, 308 ([GSH + 1]+), 215, 189, 145. A fourth metabolite, the carboxyl derivative of M1 ([M + 1]+ at m/z 722), was detected but at a concentration too low to actuate MS-to-MS/MS switching. A novel glutathione-conjugated metabolite (ML2; retention time, 3.4 min), exhibiting an [M + 1]+ at m/z 797, was located by LC-MS/MS in the bile (0-1 h collection) of rats pretreated with DEX and administered TGZQ (50 mg/kg). It yielded fragments (Figure 7) which included a series of ions 16 mass units heavier than ions obtained from M2: m/z 722 ([M + 1-glycine]+), 668 ([M + 1-glutamyl]+), 650, 632, 614, 565, 547, 308 ([GSH+1]+), 249. From this, the metabolite was identified tentatively as the quinone derivative of M2. A glutathione adduct of the quinol (ML1; [M + NH4]+ at m/z 782, retention time, 19 min; [M - 1]- at m/z 763, 21 min; Figure 8) was only found, and apparently in trace amounts, in the bile of rats predosed with DEX and administered TGZQ (50 mg/kg). Its negative-ion spectrum was similar to that of the TGZ adduct (Figure 5). The ion at m/z 254 which was taken to be indicative of thioether substitution at either the phenoxy or thiazolidinedione ring was found although the fragment at m/z 160 comprised of the thiazolidinedione nucleus, benzylic carbon, and attached sulfur could not be distinguished. The fragment of the glutathione moiety at m/z 272 was much more abundant in this spectrum. The glutathione

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Toxicity of TGZ to Hepatocytes from DEX-Treated Rats. The IC50 of TGZ in hepatocytes isolated from vehicle and DEX-treated rats was 33.5 ( 10.2 µM and 22.3 ( 1.3 µM (mean ( SD, n ) 4), respectively. A twosample t-test gave a value of 0.058, indicating no more than a marginal effect of pretreatment with DEX on the susceptibility of the cells to TGZ’s cytotoxicity.

Discussion

Figure 8. Negative-ion electrospray mass spectrum of ML1 obtained on a Quattro II by LC-MS analysis of the first hourly collection of bile from a rat pretreated with DEX and dosed with TGZQ (50 mg/kg, iv). G represents C10H16N3O6.

adduct and the parent quinone were detected only in the first hourly collection. Metabolism by Rat Hepatocytes. TGZ (50 µM) was incubated with rat hepatocytes for 2 h. Ion-current peaks ([M + NH4]+) of m/z 475 (TGZQ), m/z 635 (TGZ glucuronide), m/z 539 (TGZ sulfonate), m/z 555 (hydroxy TGZ sulfonate/benzoquinone sulfonate), m/z 557 (TGZ quinol sulfonate), and m/z 653 (TGZ quinol glucuronide) corresponding to those found in the bile of rats administered TGZ were obtained by LC-MS analysis. Toxicity of TGZ and TGZQ to Rat Hepatocytes and Human-Derived Hep G2 Cells. The loss of cell viability, as determined by a decrease in the reduction of MTS by dehydrogenase activity in rat hepatocytes exposed to 2.5-100 µM TGZ for 24 h, was significantly different from that of control cells at 50 µM and the IC50 was 35 µM (Figure 9a). TGZ was more toxic than TGZQ in a dose-dependent manner and this difference was significant, relative to control cells, at 50 µM. TGZQ was nontoxic to rat hepatocytes at 100 µM. Incubation with the human-derived Hep G2 cells showed a similar toxicity profile (Figure 9b) to that obtained with primary cultures of hepatocytes. The difference in viability between TGZ-treated cells and untreated controls was significant at 25 µM with an IC50 of 20 µM. The toxicity of TGZQ was only significant at 100 µM (p < 0.05).

In this study, the evidence for hepatic bioactivation of TGZ, represented by the occurrence of several glutathione-conjugated metabolites in the bile of rats given a large dose of the drug, has been confirmed and expanded. For the first time it has been demonstrated that induction of P450 3A, which is the predominant catalyst of the activation of TGZ by human liver microsomes (13, 14), might be associated with an increased formation of certain of these conjugates in vivo. TGZ was first reported to be bioactivated by human liver microsomes (13) and human primary hepatocytes (24). The reactive species underwent extensive and selective covalent binding to P460 3A isoforms. Kassahun et al. (14) have trapped these intermediates in microsomal incubations with GSH, and from the structures of the resulting conjugates proposed two pathways of bioactivation. First, S-oxidation, yielding a sulfoxide intermediate, initiates opening of the thiazolidinedione ring to generate an electrophilic R-keto isocyanate derivative (Figure 10) which is ultimately trapped as two thioesters (M2 and M3; M2 being formed as an isomeric pair) and a disulfide (M1). Second, one-electron oxidation of the chromane moiety creates a phenoxy radical which yields, by one route, TGZQ, and by another, an o-quinone methide which is trapped by GSH as a thioether adduct of TGZ (M5). Oxidation of TGZQ at the thiazolidinedione ring leads to another disulfide (M4).The formation of TGZQ and the glutathione adducts derived by activation of the thiazolidinedione ring is catalyzed selectively by P460 3A in human liver microsomes, whereas the formation of M5 appears to be mediated by multiple P450 isoforms (14). When unlabeled TGZ (100 mg/kg) in DMSO was administered ip or po to a fasted male Sprague-Dawley rat, M2, M3, M5, and a derivative of M1 were found in bile (14). The excretion of M2 and M3 was confirmed

Figure 9. Effect of TGZ and TGZQ on the viability of (a) cultured rat hepatocytes and (b) human hepatoma Hep G2 cells. Cells were treated with different concentrations of TGZ and TGZQ for 24 h. Cell viability was determined by the MTS assay as described. The percentage of cell viability was calculated as the ratio of A490 of treated cells and control cells (treated with DMSO, 0.1% v/v in Chee’s medium). Data presented are mean ( SEM (n ) 4). Statistical significance [(*) p < 0.05] was calculated relative to control incubations.

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Figure 10. Proposed pathways for metabolic activation of the 2,4-thiazolidinedione ring of glitazones and formation of the glutathioneconjugated metabolites found in the bile of Wistar rats administered either TGZ (ML3, M2, and M3) or TGZQ (ML1 and ML2). ML1-ML3 are novel metabolites. The pathways leading to M2-M3 and M5 were proposed by Kassahun et al. (14); who found M5 in bile rather than ML3.

during the present investigations, in the case of male Wistar rats pretreated with DEX, and apparently trace amounts of the M1 derivative were also detected, but the only glutathione adduct of TGZ located in the bile of either uninduced or DEX-treated rats (ML3) did not correspond to M5. Although the two metabolites yielded very similar positive-ion spectra, dominated by peaks representing protonated GSH and loss of a glutamyl residue, the negative-ion spectrum of ML3 was consistent with substitution on the thiazolidinedione ring, not the chromane C-5 methyl of M5 (14). The proposed mechanism of formation of ML3 involves two-electron Soxidation, suggested previously (14), or one-electron oxidation at C-5 on the thiazolidinedione ring, which in this scheme leads to substitution at C-5 of the closed ring rather than ring opening. Differences due to animal strain (including expression of P450 3A4), dose and route of administration might all be invoked to rationalize this apparent divergence in the routes of bioactivation. While absolute quantitation of ML3 was not possible, relative measurements by LC-MS demonstrated a 2-5fold increase in the concentration of adduct in DEXtreated rats compared to either vehicle-pretreated or TGZ-pretreated animals. This corresponded to an induction of hepatic P450 3A in the rat. The effect becomes even more pronounced when considering the increase in bile flow from 0.7 mL/h/200 g body weight in noninduced rats to 1.2 mL/h/200 g in DEX-treated animals during the hour after administration of TGZ. DEX is a potent inducer of hepatic P450 3A in the rat (18). Bioactivation of TGZ by P450 3A has particular toxicological implications because TGZ induces P450 3A in human and rat

hepatocytes (10, 24). Autoinduction might lead to accelerated formation of reactive metabolites and hence localized toxicity (14, 25). Although in this study, the induction capacity of TGZ is apparently less than that of DEX over a 3-day treatment period, the therapeutic use of TGZ involved long-term administration of a daily regimen. Rosiglitazone, in contrast, although it also is activated by human liver microsomes (24), does not form an intermediate which binds preferentially to P450 3A and is an extremely weak inducer of P450 3A (12, 24). If bioactivation by P450 3A is a prerequisite for the hepatotoxicity of glitazones, the difference between TGZ and rosiglitazone in terms of enzyme selectivity and P450 induction may be an explanation for the former’s particular toxicity. The 5,7,8-trimethyl substitution pattern of TGZQ, by precluding direct nucleophilic attack on the p-benzoquinone function, would be expected to favor either reduction to the semiquinone or hydroquinonesas demonstrated with a structural paradigm, 2,3,5,6-tetramethylbenzoquinone (26, 27)sor activation of the thiazolidinedione ring. Both of the novel glutathione-conjugated metabolites of TGZQ (ML1 and ML2) found in bile and the quinone metabolite of TGZ described previously (14) appear to be products of the latter process but ML1 had also undergone reduction to the hydroquinone. The metabolism of R-tocopherol is particularly relevant to that of TGZ due to the presence of a trimethyl-6hydroxychromane nucleus in both compounds. R-Tocopherol is oxidized to the benzoquinone in rats (28) and man (29). The quinone is thought to be partly reduced to the quinol (30, 31), which undergoes glucuronidation

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Scheme 1. Proposed Scheme for the Pathways of Bioactivation and Bioinactivation of TGZ and TGZQ

and/or sulfonation (28). TGZQ was found to be metabolized to a novel quinol glucuronide in vivo and by hepatocytes. That the quinol was not detected could be attributed to spontaneous autoxidation to the quinone, a phenomenon previously described for R-tocopherol hydroquinone (31). Reduction and glucuronidation represents an additional pathway, together with sulfonation (11), for the elimination of TGZQ. The metabolism of TGZ to TGZQ, by analogy with the established involvement of quinones in cytotoxicity (8), immediately suggests the possibility of an inducible, oxidation-dependent contribution to the drug’s hepatotoxicity. Pretreatment with DEX produced a significant increase in the biliary concentrations of TGZQ only 1 h after dosing with TGZ, compared with that observed for TGZ and vehicle pretreatment. This is consistent with the lower P450 3A inducing activity of TGZ in rats (10). The selection of rat hepatocytes as a model to study TGZQ toxicity was based on their ability to reproduce the metabolism of TGZ in vivo. They effected sulfonation and glucuronidation, oxidation to TGZQ, and reduction of TGZQ to TGZ quinol followed by sulfonation and glucuronidation. TGZ was toxic to cultured rat hepatocytes at concentrations exceeding 25 µM. The effect was clearly dose dependent, and the response after 24 h was identical to the toxicity profile observed at 4 h (data not shown). TGZQ was not toxic to cells at the concentrations which elicited toxicity from TGZ and there was no significant reduction in the number of viable hepatocytes after 24 h at 100 µM (equivalent to biliary concentrations in rats). Concentrations of TGZQ of >100 µM could not be assessed owing to its limited solubility in the medium. None of the TGZQ concentrations used in this study depleted hepatocyte GSH significantly over 24 h relative to vehicle-treated cells (data not shown). This conforms with a report (16) that the toxicity of TGZ to human and porcine hepatocytes is not a function of the metabolic formation of quinone. The authors suggested that either the parent compound or an unknown metabolite was responsible for the toxicity. The absence of an unambiguous increase in the sensitivity of isolated rat hepatocytes to TGZ associated with DEX treatment in vivo, anticipated because DEX induces the P450 which metabolizes TGZ to a reactive species (13, 14, 24), might be attributable to the circumstances of the toxicity assessment. Modified Chee’s medium, used here, is regarded as the most suitable for maintaining P450 levels in rat hepatocytes (32). However, this depends on additives which include DEX (33). (Modified Chee’s medium contains 1 µM DEX.) Induction of P450 3A in cultured hepatocytes from the vehicle-treated rats could have effectively nullified any measurable difference between those cells and hepatocytes from DEX-treated animals. In general, any substitution which blocks the alkylation of a benzoquinone, and thereby shifts the compound’s

mechanism of toxicity from alkylation to oxidative stress, will render that compound less cytotoxic (34). Furthermore, the antioxidant property of TGZ, analogous to R-tocopherol (35) would be expected to enhance cellular antioxidant defenses. This is illustrated by R-tocopherol benzoquinone which, unlike its mono- and dimethylated homologues, cannot act as an alkylating electrophile and is not toxic toward leukaemia cells (36). Also, 2,3,5,6tetramethylbenzoquinone (duroquinone) is nontoxic to rat hepatocytes at a concentration of 600 µM, and only causes significant depletion of GSH followed by cytotoxicity at 100 µM after inactivation of the cell’s catalase and/or glutathione reductases (37). Although the thiazolidinedione moiety in TGZQ may be implicated in conjugation with GSH as shown in this study, the absence of toxicity and/or significant depletion of cellular GSH might point to a low level of chemical stress which can be efficiently contained by the hepatocytes. In the mouse-model of metabolism-induced chemical stress (38), transcripts of the immediate early genes c-fos and c-jun and γ-glutamylcysteine synthetase are upregulated in response to depletion of cellular GSH by known hepatotoxicants such as acetaminophen and carbon tetrachloride. Exposure to TGZ did not result in an upregulation of these genes (data not shown). This indicates that any GSH depletion caused by formation of the glutathione-conjugated metabolites does not represent a danger signal in competent rodent hepatocytes. By analogy with the formation of R-tocopherol quinone (35), the formation of TGZQ should involve potentially toxic intermediates such as the pro-oxidant phenoxy radical (14). If so, it would be anticipated that cells effecting a high turnover are more susceptible to TGZ’s toxicity. Generally, Hep G2 cells possess less P450 activity than hepatocytes (39), and this was expressed in their lower turnover of TGZ to TGZQ relative to rat hepatocytes (data not shown). In Hep G2 cells, as in rat hepatocytes, TGZQ was significantly less toxic than TGZ. Nevertheless, TGZ was more toxic to Hep G2 cells than rat hepatocytes. Consequently, there is no compelling evidence for cytotoxic intermediates in the conversion of TGZ to TGZQ. Considering TGZQ is a minor human metabolite (ca. 10% of initial dose), its toxicity to hepatocytes might require cellular accumulation. However, our and other (11) in vivo studies indicate that the elimination of TGZQ is fairly rapid and this in combination with metabolic clearance and the lack of cytotoxicity (16) at and above therapeutic concentrations might exclude TGZQ from a role in TGZ-induced hepatotoxicity. In conclusion, our findings indicate that Wistar rats can metabolize TGZ and TGZQ inter alia to novel glutathione conjugates (ML1 and ML3) via an inducible pathway mediated by hepatic P450 3A (Scheme 1). The conjugates remain to be fully characterized but appear to be thioethers substituted in the ring-closed 2,4thiazolidinedione moiety, which implies the operation of

Metabolic Activation of Troglitazone

a hitherto unreported mechanism of bioactivation of this ring system. However, TGZQ is not toxic to isolated hepatocytes at high concentrations and is certainly less toxic than TGZ. The newly described pathway of reduction and glucuronidation, together with sulfonation, would constitute an effective mechanism for deactivating TGZQ. Notably, the glutathione conjugate of TGZQ (ML1) unlike that of TGZ was found in bile only after induction of P450 3A. Reactive intermediates other than those on the pathway to TGZQ are potential candidates for hepatotoxic metabolites. Because the 2,4-thiazolidinedione ring is the generic feature of glitazones, further work should establish the generality and toxicological implications of bioactivation of this moiety.

Acknowledgment. J.N.T. was a GlaxoWellcome Research Fellow. The Quattro II mass spectrometer was purchased and maintained by grants from the Wellcome Trust. B.K.P. is a Wellcome Principal Research Fellow. We are indebted to Dr. Kirsten Hobby, of Micromass U.K., for obtaining mass specta on the Q-Tof2. The contributions of Drs Paul Stocks and Anne Stalford to the synthesis of troglitazone quinone are gratefully acknowledged.

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