Abstract
Methapyrilene (MP), a 2-thiophene H1-receptor antagonist, is a model toxicant in the genomic and proteomic analyses of hepatotoxicity. In rats, it causes an unusual periportal necrosis that is hypothetically attributed to chemically reactive and cytotoxic metabolites. We have characterized the bioactivation of MP by hepatic microsomes and primary rat hepatocytes, and we established a possible causal linkage with cytotoxicity. Methapyrilene tritiated at C-2 of the diaminoethane moiety ([3H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat > mouse ≈ human). This binding was attenuated by the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole and thiols but not by trapping agents for iminium ions and aldehydes. Reactive intermediates were trapped as thioether adducts of monooxygenated MP. Mass spectrometric and hydrogen/deuterium exchange analysis of the glutathione adduct produced by rat liver microsomes indicated that the metabolite was most probably a thioether of MP S-oxide substituted in the thiophene ring. The glutathione adduct was formed by rat hepatocytes and eliminated in bile by rats administered [3H]MP intravenously. MP produced concentration- and time-dependent cytotoxicity, depleted glutathione, and underwent irreversible binding to the hepatocytes before a significant increase in cell damage was observed. P450 inhibitors reduced turnover of the drug, production of the glutathione adduct, irreversible binding, and cytotoxicity but inhibited glutathione depletion selectively. MP underwent lesser turnover and bioactivation in mouse hepatocytes and was not cytotoxic. Analogs with phenyl and p-methoxyphenyl rings were much less hepatocytotoxic than MP. Hepatotoxicity in rats was diminished by predosing with 1-aminobenzotriazole. For the first time, a thiophene ring substituent is identified as a bioactivation-dependent toxicophore in hepatocytes.
Methapyrilene [N,N-dimethyl-N′-(2-pyridyl)-N′-(2-thienylmethyl)-1,2-ethanediamine (MP); Fig. 1], a 2-thiophene H1-receptor antagonist, was not associated with hepatotoxicity in humans, but it causes unusual dose-dependent periportal damage in adult male rats (Graichen et al., 1985; Ratra et al., 1998a; Ratra et al., 2000). Typical hepatotoxic regimens (150–300 mg/kg/day × 3) produce a mild to moderate injury characterized by hepatocellular necrosis, bile duct proliferation, and inflammatory cell infiltration. MP is also toxic to isolated rat hepatocytes (McQueen and Williams, 1982; Ratra et al., 1998b), which are reported to be more susceptible than mouse hepatocytes (Kelly et al., 1992).
MP has come to be regarded as a model investigatory compound among drug hepatotoxicants, and it is used frequently in differential gene (Huang et al., 2004; Beekman et al., 2006) and protein (Craig et al., 2006) expression analyses of acute liver injury and hepatocytotoxicity. However, although the cytotoxicity has been associated with metabolic activation, oxidative stress, mitochondrial damage (Ratra et al., 1998a; Ratra et al., 1998b), protein modification, and altered protein expression (Craig et al., 2006), its mechanism is still obscure and the toxicophore is unidentified.
The drug-induced hepatotoxicity is considered to be metabolism-dependent because it is diminished by agents that inhibit cytochrome P450 (P450): cobalt protoporphyrin in vivo (Ratra et al., 1998a) and metyrapone in rat hepatocytes (Ratra et al., 1998b). The major metabolites (Fig. 1) commonly described for the rat are desmethyl MP, MP N-oxide, (5-hydroxypyridyl)-MP O-glucuronide, and products arising from oxidative N′-dealkylation of the thienylmethyl moiety (Singer et al., 1987; Kammerer et al., 1988; Ratra et al., 2000). Male-specific CYP2C11 has been tentatively implicated in the biotransformation and hepatocytotoxicity of MP, but the reaction catalyzed by this isoform remains uncharacterized (Ratra et al., 1998a,b).
MP has several structural alerts (Kalgutkar et al., 2005) for metabolic activation of potential toxicophores via N-oxidation, heteroarene oxidation (S-oxidation and thiophene ring epoxidation), and arene oxidation (Fig. 1). This suggests that the metabolism dependence of the hepatotoxicity of the drug might derive from the actions of one or more reactive intermediates of oxidative biotransformations. Evidence for bioactivation has been obtained from irreversible binding of radiolabeled MP to macromolecules in vivo and in vitro. The radiolabeling of liver protein in rats after administration of [3H(G)]MP was much greater than that of hepatic nucleic acids and kidney protein (Lijinsky and Muschik, 1982). Irreversible binding of [thienyl methylene-14C]MP to DNA coincubated with rat liver microsomes was NADPH-dependent and reduced by P450 inhibitors and thiols (Lampe and Kammerer, 1987, 1990). Coincidental binding to the microsomal protein was greater than the binding to DNA.
Iminium ions, iminoquinones, epoxides, and aldehyde species have all been suggested as reactive metabolites of MP (Ziegler et al., 1981; Singer et al., 1987; Lampe and Kammerer, 1990; Kelly et al., 1992). Two iminium ions generated by rabbit liver microsomes were trapped with cyanide, and one stable adduct, N-(cyanomethyl)normethapyrilene, was identified (Ziegler et al., 1981). However, they have not been trapped in rat liver microsomes, and they have not been connected with hepatocytotoxicity. Note that although several compounds are known to undergo metabolic activation at a thiophene ring (Kalgutkar et al., 2005), no metabolites of MP arising from oxidation of this moiety have been described previously (Kammerer and Schmitz, 1986; Singer et al., 1987). Differences between the metabolism of MP in rats and mice are both qualitative and quantitative (Lampe and Kammerer, 1990), but they have not provided a rigorously tested metabolic explanation of the greater susceptibility of rat hepatocytes (Kelly et al., 1992).
Although metabolites of MP have been postulated as causal to the hepatotoxicity of the drug (Singer et al., 1987; Kelly et al., 1992; Ratra et al., 1998b), this toxicity has not been linked experimentally with either metabolic activation, as represented by irreversible drug binding, or any stable metabolites of MP derived from reactive intermediates. We have re-examined the metabolism and cytotoxicity of radio-labeled MP in rodent hepatocytes to elucidate metabolic mechanisms of hepatotoxicity. Cell injury was preceded by depletion of total glutathione (tGSH) and irreversible binding of radiolabel to cellular material. All three effects were attenuated by P450 inhibitors. Contrary to the findings of early studies on bioactivation of MP (Ziegler et al., 1981; Singer et al., 1987), the reactive intermediate of MP could not be trapped with cyanide but was stabilized as thioether conjugates, the preliminary structures of which suggested the formation of the S-oxide or an isomeric thiophene epoxide metabolite. For the first time, a thiophene ring is identified as a bioactivation-dependent toxicophore in hepatocytes.
Materials and Methods
Materials. MP hydrochloride, pyrilamine (mepyramine; N-[(4-methoxyphenyl)methyl]-N′,N′-dimethyl-N-pyridin-2-ylethane-1, 2-diamine), tripelennamine [pyribenzamine; N′,N′-dimethyl-N-(phenylmethyl)-N-pyridin-2-ylethane-1,2-diamine], D2O (99.9 atom percentage of D), and monodeuteromethanol (MeOD; 99.5 atom percentage of D) were purchased from Sigma-Aldrich (Poole, Dorset, UK). Nondeuterated methanol was high-performance liquid chromatography (HPLC) grade from Fisher Scientific UK (Loughborough, Leicestershire, UK). Methapyrilene tritiated at C-2 of the diaminoethane moiety ([3H]MP) was prepared from MP by direct tritium-hydrogen exchange in dichloromethane using Crabtree's catalyst (Bushby and Killick, 2007), and it was supplied by Isotope Chemistry (AstraZeneca, Alderley Park, Cheshire, UK; specific activity 27.8 Ci/mmol, radiochemical purity >95% by HPLC) and RC Tritec AG (Teufen, Switzerland; specific activity 56.0 Ci/mmol, radiochemical purity >99% by HPLC). The position of the tritium labels on C-2 of the 1,2-diaminoethane moiety (Fig. 1) was established by 3H NMR. Collagenase A was obtained from Roche Diagnostics (Lewes, Sussex, UK). Bradford reagent was obtained from Bio-Rad (Munich, Germany). CellTiter 96 AQueous One Solution Cell Proliferation Assay kits were purchased from Promega (Southampton, UK). cDNA-expressed rat P450 preparations (BD Supersomes) containing P450 reductase and cytochrome b5 were obtained from BD Gentest via Biotrace Fredbaker Ltd. (Runcorn, Cheshire, UK). Unless otherwise stated, all other reagents were obtained from Sigma-Aldrich. Organic solvents were products obtained from VWR (Lutterworth, Leicestershire, UK) and were of chromatographic, analytical, or cell-culture grade.
Animals. Adult male Wistar rats and CD-1 mice were obtained from Charles River Laboratories (Margate, Kent, UK). All experiments with live animals were undertaken in accordance with criteria outlined in a license granted under the Animals (Scientific Procedures) Act 1986 and approved by the Animal Ethics Committee of the University of Liverpool.
Hepatocytes. Rat and mouse hepatocytes were isolated using a two-step collagenase procedure. The rats (150–300 g) and mice (20–30 g) were anesthetized with sodium pentobarbital (Euthanal, 60 mg/ml; 1 μl/g i.p.). The liver was first perfused in situ via the hepatic portal vein for 9 min (7 min for mouse) with a wash buffer consisting of Hanks' balanced salt solution containing 5.8 mM HEPES and 4.5 mM NaHCO3 but neither CaCl2, MgCl2, MgSO2, nor phenol red (Invitrogen, Paisley, UK). This was followed by a perfusion with wash buffer supplemented with 5 mM CaCl2 and 0.5 mg/ml collagenase until the liver interstitium had been digested. The flow rate was maintained at 40 ml/min (12 ml/min for mouse), and all solutions were kept at 37°C. After digestion, the liver was excised and the liver capsule was broken. The cells were combed and stirred out of the capsule, suspended in 100 ml of wash buffer supplemented with DNase I (0.1 mg/ml), and filtered through a 125-μm mesh (Lockertex, Warrington, Cheshire, UK). They were allowed to settle for 10 min at 4°C, and then they were centrifuged at 50g for 2 min. The supernatant was removed, and the cells were resuspended in wash buffer containing DNase I. This procedure was repeated twice with unsupplemented wash buffer, with final resuspension in wash buffer supplemented with 1 mM MgSO47H2O (incubation buffer). Viability was assessed by trypan blue exclusion (20 μl of trypan blue solution (0.1% w/v) combined with 100 μl of cell suspension). Typically, viability was 85 to 95% and hepatocytes were only used when viability was greater than 75%. In our experience, drug turnover by freshly isolated rat hepatocytes is only compromised when the cell viability rated by trypan blue exclusion is less than 75%.
Rat and mouse hepatocytes from three individual isolations were incubated as separate suspensions (2 × 106 cells/ml, final volume 6 ml) in HEPES incubation buffer with either 20 to 500 μM unlabeled drug (MP, pyrilamine, or tripelennamine) or 200 μM[3H]MP (1 μCi). Unlabeled drug or [3H]MP was added as an ethanol solution (final concentration of ethanol, ≤0.1% v/v), and control incubations contained appropriate amounts of ethanol. Incubations (0.5–6 h) were performed in 20-ml glass vials, in an orbital shaker at 37°C, with gentle stirring throughout the incubation to ensure that cells did not settle. Incubations of rat hepatocytes were also carried out with the addition of 1 mM glutathione (GSH), glutathione O-ethyl ester (GSH-EE), or N-acetylcysteine (NAC) at the same time as the MP or [3H]MP. Coincubations with nonspecific P450 inhibitors [1 mM 1-aminobenzotriazole (ABT) or 50 μM SKF-525A] had a preincubation of 30 min before the addition of MP or [3H]MP (final concentration, 200 μM, 1 μCi). The thiols and P450 inhibitors were dissolved in incubation buffer for addition. Drug was omitted from control incubations. Unlabeled MP was used in incubations where only tGSH contents, i.e., GSH plus glutathione disulfide, were measured.
After incubation of the rat and mouse hepatocytes, an aliquot of the cell suspension (100 μl) was used to assess trypan blue exclusion. Cell suspension was also added to the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution (ratio, 5:1 v/v) from a CellTiter 96 AQueous One Solution Cell Proliferation Assay kit, incubated at 37°C for 1 h, and centrifuged at 50g for 2 min. Supernatant (80 μl) was analyzed at 490 nm on a UV-visible absorbance MRX microtiter plate reader (Dynex Technologies Ltd., Worthing, West Sussex, UK). Determination of the tGSH content of rat hepatocytes was carried out on 1 × 106 cells taken from an incubation and centrifuged at 50g for 2 min, discarding the supernatant. HCl (10 mM, 250 μl) was added to lyse the cells. Lysed hepatocytes (50 μl) were removed for protein determination (Bradford, 1976). This aliquot was replaced with 6.5% (w/v) 5-sulfosalicyclic acid solution, and the whole suspension was kept on ice for 10 min before centrifugation at 18,400g for 5 min. Supernatants were stored at –80°C until analyzed. tGSH was measured spectrometrically (412 nm) by a glutathione reductase-dithio-bis(2-nitrobenzoic acid) recycling assay (Vandeputte et al., 1994). The results were compared with GSH standards (0–40 μM).
To the remaining suspension of rat or mouse hepatocytes, an equal volume of acetonitrile was added, and the mixture was centrifuged at 870g for 10 min. The supernatant was removed and evaporated under nitrogen. The dry residue was reconstituted in 0.1 M ammonium acetate buffer, pH 8.33 (200 μl), centrifuged at 18,400g for 5 min, and the supernatant was analyzed by liquid chromatography-mass spectrometry (LC-MS). Irreversible binding of [3H]MP to the hepatocytes was assessed by exhaustive solvent extraction of the pelleted cells obtained after addition of acetonitrile. Methanol (5 ml) was added, and, after vigorous vortexing, the cellular suspension was centrifuged at 870g for 10 min. The supernatant was discarded. Another two washes of the pellet were performed with 70% methanol (5 ml). The extracted pellet was dried at 60°C and dissolved in 1 M NaOH (1 ml) at 60°C. Samples (100 μl) of the solution were taken for determination of radioactivity by liquid scintillation counting and measurement of the protein content (Bradford, 1976).
Microsomes. Wistar rat (RLM), CD-1 mouse (MLM), and human (HLM) liver microsomes were prepared from tissue minced in ice-cold 67 mM phosphate buffer, pH 7.5, containing KCl (1.15%, w/v). Homogenates were produced with a motor-driven homogenizer and centrifuged (10,000g for 20 min at 4°C) to obtain the supernatant from which microsomes were sedimented (105,000g for 60 min at 4°C). The microsomes were resuspended in KCl phosphate buffer, centrifuged (105,000g for 60 min at 4°C), and reconstituted in 67 mM phosphate buffer, pH 7.5. HLM from two female and two male donors aged 10 to 41 years were pooled. P450 content was determined by reduced-carbon monoxide difference spectroscopy as described previously (Ratra et al., 1998a), and protein content was measured by the Bradford assay (Bradford, 1976). Incubations were carried out in a final volume of 1 ml of Tris-HCl buffer (0.2 M), pH 7.4, containing 1 mg/ml microsomal protein, 20 μM [3H]MP (0.25 μCi), and a NADPH-regenerating system (0.4 mM NADP, 7.5 mM glucose 6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, and 5.0 mM MgCl2). [3H]MP was added as an ethanol solution (final concentration of ethanol, ≤0.6% v/v). The regenerating system was omitted from control incubations. After a 60-min incubation at 37°C, the reaction was terminated by the addition of an equal volume of ice-cold acetonitrile. After overnight precipitation of the protein at –20°C, the incubations were centrifuged at 870g for 10 min. The supernatant was evaporated to dryness under nitrogen. It was reconstituted in 0.1 M ammonium acetate buffer, pH 8.33 (200 μl), centrifuged (18,400g for 5 min), and the final supernatant was analyzed by LC-MS or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Irreversible binding of [3H]MP to the protein pellets was measured by exhaustive solvent extraction as described for hepatocytes but using 3 ml of solvent rather than 5 ml. Incubations of RLM were also carried out in the presence of SKF-525A (50 μM), ABT (1 mM), thiols [1 mM β-mercaptoethanol (βME), GSH, GSH-EE, or NAC], N-acetyllysine (1 mM), and semicarbazide (5 mM) or sodium cyanide (1 mM) with appropriate controls. Microsomes coincubated with ABT were preincubated with the inhibitor and NADPH-regenerating system for 15 min before addition of [3H]MP (20 μM, 0.25 μCi) and then supplementary NADPH-regenerating system equal to the first portion.
Incubations with CYP2A2, CYP3A2, CYP2B1, CYP2C6, CYP2C11, CYP2C12, CYP2C13, and CYP2E1 Supersomes (10 pmol P450) were carried out in a final volume of 1 ml of Tris-HCl buffer (0.2 M), pH 7.4, containing [3H]MP (20 μM, 0.25 μCi) and the NADPH-regenerating system that was used for microsomal incubations. Incubations were processed for LC-MS in the same way as microsomal incubations.
Animal Experiments. Adult male Wistar rats (200–300 g) were dosed orally with 150 mg/kg MP in 0.9% saline (25 mg/ml) once daily for three consecutive days. For P450 inhibition (Mugford et al., 1992), animals received an i.p. injection of ABT in 0.9% saline (50 mg/kg; 50 mg/ml) 1 h before each MP dosing. Control animals received vehicle only. Animals were killed 24 h after the final dose of MP or saline. Blood was drawn by cardiac puncture, and the serum was prepared for assays of alanine amino transferase (ALT) and glutamate dehydrogenase (GLDH). Serum ALT was measured using the ThermoTrace Infinity ALT Liquid stable reagent (Alpha Laboratories, Eastleigh, Hampshire, UK) according to the manufacturer's instructions. Serum GLDH was measured using a protocol based on the method of Ellis and Goldberg (1972). The assay buffer, pH 7.4, contained 0.18 mM NADH, 0.95 mM ADP, 0.1% Triton X-100, 75 mM triethanolamine, and 75 mM ammonium sulfate. In a 96-well plate, 60 μl of serum was added to 180 μl of the assay buffer, mixed, and incubated at 37°C for 15 min. Reaction was initiated by adding 60 μl of α-ketoglutaric acid (50 mM) and mixing. The rate of fall of absorbance at 340 nm over 5 min was measured immediately. For determination of hepatic tGSH levels, approximately 50 mg of tissue was homogenized in a mixture of 5-sulfosalicyclic acid solution (200 μl; 6.5% w/v) and GSH stock buffer (800 μl; 143 mM NaH2PO4, 6.3 mM EDTA, pH 7.4), and the protein was allowed to precipitate on ice for 10 min before centrifugation at 18,400g for 5 min. The supernatants were removed to determine tGSH by the microtiter plate assay used for hepatocyte samples (Vandeputte et al., 1994). The sedimented pellets were solubilized in 1 M NaOH at 60°C before protein content was determined (Bradford, 1976).
For i.v. drug administration, adult male Wistar rats (200–300 g) were anesthetized with urethane (1.4 g/ml in isotonic saline; 1.0 ml/kg i.p.) and cannulated via the trachea, femoral vein, and common bile duct. [3H]MP (17.5 mg/kg, 10 μCi) dissolved in 0.9% saline (17.5 mg/ml) was injected into the femoral vein over 20 min. This dose was lower than the 150 mg/kg given orally to avoid the acute neurotoxicity of MP. Bile was collected as hourly fractions for 5 h. Aliquots were either assayed for radioactivity by liquid scintillation counting (10 μl) or analyzed by parallel LC-MS and radiochromatography (50 μl).
LC-MS, LC-MS/MS, and Radiometric Detection. A Quattro II mass spectrometer (Waters, Manchester, UK) fitted with the standard coaxial electrospray source was used for LC-MS in the positiveion mode. The LC system consisted of two Jasco PU980 pumps (Jasco UK, Great Dunmow, Essex, UK) and a Jasco HG-980-30 mixing module. Analytes were resolved on a Columbus 5-μm C-18 column (250 × 4.60 mm; Phenomenex, Macclesfield, Cheshire, UK) with a gradient of acetonitrile (0% for 5 min, 10–40% over 20 min) in ammonium acetate (0.1 M; pH 8.33) at a flow rate of 0.9 ml/min. The analytes were monitored at 254 nm. Eluate split-flow to the LC-MS interface was approximately 50 μl/min. Nitrogen was used as the nebulizing and drying gas. The interface temperature was 80°C, and the capillary voltage was 3.9 kV. Spectra were acquired between m/z 100 and 1050 over a scan duration of 5 s. In-source fragmentation of analyte ions was achieved at a cone voltage of 50 V. Data were processed with MassLynx 3.5 software (Waters, Manchester, UK). The remainder of the column eluate was mixed with Ultima Flo AP scintillant (1.0 ml/min) in a flow scintillation analyzer [Radiomatic Flo-Oneβ A250; PerkinElmer LAS (UK), Seer Green, Beaconsfield, Buckinghamshire, UK], and the radiolabeled components were quantified using A250-1.6 software.
LC-MS/MS was carried out with a PerkinElmer Series 200 autosampler and HPLC pump coupled to an Applied Biosystems/MDS Sciex 4000 Q TRAP (Applied Biosystems, Foster City, CA) equipped with a TurboIonSpray source probe. The column and elution conditions were those used for LC-MS. The instrument was controlled, and data were analyzed via Analyst 1.4.1. Electrospray ionization was performed with a source temperature of 450°C and an ion spray voltage of 5.5 kV in the positive-ion mode. Nitrogen was used as curtain gas (10 psi) and collision gas (35 psi). The declustering potential was 30 V; the entrance potential was 10 V; and the collision cell exit potential was 15 V.
Characterization of Glutathione Adduct Metabolite M8 by Deuterium Exchange. For postincubation hydrogen/deuterium (H/D) exchange, MP (100 μM) was incubated at 37°C with RLM (protein concentration, 1 mg/ml) in a final volume of 1 ml of Tris-HCl buffer (0.2 M), pH 7.4, containing NADPH-regenerating system and GSH (1 mM). After 60 min, an equal volume of ice-cold acetonitrile was added to the incubation, and the mixture was stored at –20°C until further use. After centrifugation at 870g for 10 min, the supernatant was evaporated to dryness under nitrogen, and the residue was reconstituted immediately at room temperature. Residues of single incubations were reconstituted in either methanol or MeOD (200 μl), and residues of combined pairs of incubations were reconstituted in either water or D2O (500 μl). Each solution was clarified by centrifugation at 10,000g for 5 min. For in situ H/D exchange, the buffer and all of the solutions of reagents were prepared in D2O. The composition of the microsomal mixture was otherwise unchanged, and the conditions of incubation were as before. The supernatant obtained by centrifugation after addition of ice-cold acetonitrile to the incubation was used for mass spectral analysis. Reconstituted residues and supernatant were immediately infused (20 μl/min) directly into the electrospray ionization source of an LTQ Orbitrap mass spectrometer (ThermoFisher Scientific, Hemel Hempstead, Hertfordshire, UK). The capillary and spray voltages were 4 V and 3.8 kV, respectively, and the capillary temperature was 275°C. Full-scanning (m/z 100–900) and selected MS/MS acquisitions in positiveion mode were performed at 100,000 resolution through Xcaliber 2.0 SR2 (Thermo Fisher Scientific, Hemel Hempstead, Hertfordshire, UK). The instrument was calibrated for accurate mass measurements on the day of analysis.
Statistical Analysis. All results are expressed as the mean ± S.E.M. Values to be compared were analyzed for non-normality using a Shapiro-Wilk test. Student's t tests were used when normality was indicated. A Mann-Whitney U test was used for nonparametric data. One-way ANOVA tests were used for multiple comparisons. All calculations were performed using Arcus Quickstat statistical software (StatsDirect Altrincham, Cheshire, UK). Results were considered to be significant when p < 0.05.
Results
Toxicity of MP and Side-Chain Ring Analogs in Hepatocytes. Incubation of MP (20–500 μM) with rat hepatocyte suspensions for 6 h resulted in marked concentration-dependent cytotoxicity represented by decreases in both tetrazolium dye (MTS) reduction and trypan blue exclusion (Fig. 2). The drug was not significantly cytotoxic to mouse hepatocytes at concentrations up to 500 μM. The side-chain ring analogs (Fig. 1), namely pyrilamine (p-methoxy substituent) and tripelennamine (phenyl substituent), were much less toxic than MP to rat hepatocytes (Fig. 3).
Biochemical Characteristics and Metabolic Dependence of Cytotoxicity. The tGSH content of rat hepatocytes decreased with increasing concentrations of MP (20–500 μM) over 6 h (Fig. 4), and the decline attained statistical significance at 100 μM MP. When the simultaneous time courses of tGSH depletion and cytotoxicity at 200 μM MP were plotted, the depletion of tGSH reached approximately 65% before a significant decrease in cell viability became apparent between the 2nd and 4th hours of incubation (Fig. 5).
Prior incubation of the rat hepatocytes with either of two general P450 inhibitors (1 mM ABT or 50 μM SKF-525A) reduced the cytotoxicity of 200 μM MP (Table 1), but only ABT, which rapidly and efficiently destroys P450 in rat hepatic microsomes (Mugford et al., 1992), attenuated the depletion of tGSH significantly. Coincubation with a 5-fold molar excess of a thiol (1 mM GSH, GSH-EE, or NAC), in contrast to the coincubation with dithiothreitol, which is known to have a cytoprotective action (Ratra et al., 1998b), did not affect the cytotoxicity of MP (data not shown).
Metabolic Correlates of Cytotoxicity in Hepatocytes. [3H]MP underwent irreversible binding to rat hepatocytes that was both concentration- and time-dependent over 6 h (Figs. 4 and 5), with mean values (±S.E.M.) for 200 μM drug increasing from 0.04 nmol equivalent (equiv)/mg protein at 0 h to 6.3 ± 2.5 nmol equiv/mg protein at 6 h. The latter value was significantly (p < 0.01) greater than the irreversible binding to mouse hepatocytes at 6 h: 2.5 ± 0.9 nmol equiv/mg protein. In rat hepatocytes, the irreversible binding of radiolabeled material increased significantly before the appearance of cytotoxicity after the 2nd hour of incubation (Fig. 5). The irreversible binding over 6 h was attenuated by both ABT and SKF-252A (Table 1).
Turnover of [3H]MP in the rat hepatocyte suspensions after 6 h was 76.0 ± 2.4% (mean ± S.E.M., n = 3). The major radiolabeled metabolites resolved by HPLC (M1–M8; Fig. 6) were quantified radiometrically and characterized by MS and MS/MS.
Unchanged [3H]MP (retention time, 33.0 min) was identified, through comparisons with authentic standard, by coelution and its electrospray mass spectrum: m/z 262 ([M + 1]+), 217 ([M + 1-HN(CH3)2]+), 121 ([C5H4N.HNCHCH2+1]+; pyridyl ring fragment), 119 ([C5H4N.NCHCH2]+; pyridyl ring fragment), 97 ([C4H3S.CH2]+; thiophenyl ring fragment), and 72 ([CH2CH2N(CH3)2])+.
M1 (28.0 min; 27.0 ± 4.0% of eluted radioactivity) yielded an abundant ion at m/z 248 that was assigned to [M + 1]+ of desmethyl MP (Fig. 1) and fragmented extensively to m/z 217 ([M + 1-H2NCH3]+), which corresponded to the generic fragment of MP derivatives modified only in the N, N-dimethylamino side chain (Cerniglia et al., 1988).
M3 (26.0 min; 12.7 ± 3.3%; [M + 1]+, m/z 278), a monooxygenated derivative, was assigned to the known N-oxide metabolite of MP (Kelly et al., 1992) due to its spectrum retaining m/z 217 ([M + 1-HNO(CH3)2]+), 119, and 97, indicating that oxygenation had occurred on the dialkyl side chain rather than the pyridyl or thiophenyl ring (Cerniglia et al., 1988), respectively.
M2 (27.0 min; 7.2 ± 2.9%) yielded m/z 278 for [M + 1]+ of another oxygenated metabolite and a Q1 full-scan spectrum comprised of m/z 233 (oxygenated analog of m/z 217), 137 (oxygenated analog of m/z 121), 97, and 72, suggesting oxygenation of the pyridyl ring. However, this position of functionalization was not confirmed by the product ions of m/z 278 obtained through MS/MS, which included m/z 233 and m/z 97 but m/z 113 (oxygenated analog of thiophenyl fragment m/z 97) instead of m/z 137. The shoulder of M2 contained, in lesser abundance, bisdesmethyl MP: m/z 234 ([M + 1]+), 217 ([M + 1-NH3]+), 121, and 97.
M4 (24.5 min; 2.1 ± 0.5%) and M5 (23.0 min; 2.0 ± 0.4%) corresponded to peaks in the mass chromatogram for m/z 294 and were assigned tentatively to dioxygenated derivatives of MP. In both cases, fragmentation to m/z 233, 137, and 97 suggested oxygenation of the dialkyl side chain and oxygenation of the pyridyl ring.
M6 (20.5 min; 6.1 ± 0.8%; [M + 1]+, m/z 454) was assigned to an O-glucuronide of monooxygenated MP from the indicative neutral loss of dehydroglucuronic acid (m/z 278, [M + 1–176]+). This metabolite has been identified previously (Kelly et al., 1992), and its fragmentation suggested hydroxylation of the pyridyl ring: m/z 409 ([M + 1-HN(CH3)2]+), 233, 137, 97, and 72.
M7 (19.8 min; [M + 1]+, m/z 278) yielded a spectrum that contained m/z 233, and M7 seemed to be one or more monooxygenated metabolites functionalized in the pyridyl-thiophenyl ring system, but it could not be characterized further.
M8 (14.5 min; 16.0 ± 1.7%; [M + 1]+, m/z 585) was identified as a GSH conjugate of monooxygenated MP. The mass chromatogram for m/z 585 contained one major peak. The product ion spectrum of the metabolite obtained by LC-MS/MS included ions indicative of the generic neutral loss of pyroglutamate (m/z 456, [M + 1–129]+) and GSH (m/z 278, [M + 1–307]+) from a GSH adduct (Fig. 7). The fragmentation also indicated thioether conjugation at an oxygenated thiophene moiety, principally from the formation of ions at m/z 540 ([M + 1-HN(CH3)2]+) and m/z 411 ([456-HN(CH3)2]+) attributable to an unmodified N-dialkyl side chain and ions at m/z 121 ([C5H4N.HNCHCH2+1]+) and m/z 166 ([C5H4N.HNCH2CH2.N(CH3)2+1]+) attributable to an unmodified pyridyl ring. Apparent dehydration products were found at m/z 567 ([M + 1-H2O]+), 522 ([540-H2O]+), and 260 ([278-H2O]+). The spectrum gave no evidence of a neutral loss of a unit of 48 atomic mass units (amu), which would have been tentatively attributable to sulfur monoxide derived from an S-oxide structure (Dansette et al., 2005). It is known that parent-ion dehydration can be a feature of the electrospray mass spectrum of both a dihydrohydroxythiophenyl-S-glutathionyl adduct of an epoxythiophene and the glutathione adduct of a thiophene S-oxide (Dansette et al., 2005). The chemical structure and origin of M8 were investigated further by H/D exchange analysis as described below.
In rat hepatocyte suspensions that had been pretreated with 1 mM ABT for 30 min, turnover of [3H]MP was significantly reduced from 76.0 ± 2.4 to 43.0 ± 6.3% (n = 3). It is noteworthy that formation of glutathione adduct M8 was suppressed completely, and only M1 (desmethyl MP) and M3 (MP N-oxide) were detected (Fig. 6); a selective blocking of biotransformations that conforms generically with ABT's selective inhibition of hepatic P450 (Emoto et al., 2005). Incubations with SKF-525A showed 55.2 ± 5.5% turnover. In the latter case, M3 was the major metabolite, but M8 still accounted for approximately 5% of recovered radioactivity (data not shown).
Mouse hepatocyte incubations showed less turnover of [3H]MP (54.1 ± 6.9%, n = 3) compared with rat. The metabolite profile (data not shown) was qualitatively similar to the rat profile, but the amount of GSH conjugate M8 formed was significantly less than in rat hepatocytes: 4.3 ± 0.5% of eluted radioactivity compared with 16.0 ± 1.7%.
Metabolic Activation by Hepatic Microsomes. RLM, in the presence of NADPH, achieved a 89.3 ± 3.5% (n = 3) turnover of [3H]MP and produced several radiolabeled metabolites (Fig. 8), two of which were identified as M1 and M3 (2.3 ± 0.6 and 13.9 ± 1.2% of eluted radioactivity, respectively) by LC-MS. Neither M10 (19.4 min, 52.6 ± 4.1%), M11 (22.0 min), M12 (15.0 min), nor M13 (4.3 min) could be identified. In the presence of ABT, the turnover of [3H]MP in the RLM incubations was decreased from 89.3 ± 3.5 to 44.1 ± 2.1%. Formation of M10 reduced from 52.6 ± 4.1% of recovered radioactivity to 4.4 ± 2.2%.
Metabolism of [3H]MP by HLM (29.5 ± 4.5%) and MLM (39.3 ± 8.9%) was significantly less extensive than the turnover by RLM. MLM and HLM produced only M1 and M3 (Fig. 8).
Incubations of [3H]MP with CYP2C11 Supersomes for 60–120 min yielded only M1. Incubations with CYP2C6 yielded M1 and M2, whereas CYP2C12 produced only M2. None of the other five recombinant rat P450 that were tested (CYP2A2, CYP3A2, CYP2B1, CYP2C13, and CYP2E1) produced detectable turnover of [3H]MP.
[3H]MP (20 μM) incubated with RLM, HLM, and MLM for 60 min in the presence of NADPH underwent irreversible binding to the microsomal protein that was equivalent to 0.80 ± 0.17, 0.23 ± 0.05, and 0.17 ± 0.06 nmol equiv/mg protein (n = 3), respectively (Fig. 8). Preincubation of RLM with ABT significantly reduced the irreversible binding from 0.70 ± 0.12 to 0.19 ± 0.13 nmol equiv/mg protein. SKF-525A (50 μM) did not have a significant effect on the irreversible binding of [3H]MP to RLM (data not shown); notwithstanding, it caused a 90% reduction in bioactivation of [thienyl methylene-14C]MP by RLM when metabolic activation was assessed through irreversible binding of radiolabel to coincubated DNA (Lampe and Kammerer, 1987). This unexpected finding contrasted with the significant inhibition by the compound of irreversible binding to rat hepatocytes. The divergent effects of ABT and SKF-525A on microsomal irreversible binding, and on cytotoxicity, were analogous to the different P450 isoform selectivities of the inhibitors (Emoto et al., 2005).
Upon the addition of GSH to the RLM incubations, the major radiolabeled metabolite was M8, accounting for 54.9 ± 4.0% of recovered radioactivity. (Fig. 9A). The formation of M8 was principally associated with diminution of the stable but unidentified metabolite M10, the production of which was inhibited by ABT. These observations implied that M10 and M8 were derived from a common reactive intermediate. The evidence for oxidative metabolic activation of MP at the thiophene ring, derived from the characterization of M8, immediately suggested two possibilities for the identity of M10: 1) that it was by analogy with a major metabolite of tienilic acid (Belghazi et al., 2001) 5-hydroxythiophene MP formed by rearrangement of either MP S-oxide or the 4,5-epoxide, or 2) that it was by analogy with a metabolite of 2-phenylthiophene (Dansette et al., 2005) the cycloadditive dimer of MP S-oxide. However, neither of these possibilities was confirmed by LC-MS analyses of hepatic microsomal incubations of MP.
A single conjugate of a monooxygenated reactive intermediate of [3H]MP and the coincubated thiol was also formed in substantial amounts in incubations of RLM including GSH-EE (52.6 ± 4.8% of recovered radioactivity), NAC (65.2 ± 5.1%), or βME (75.2 ± 2.5%), and the mass spectra of the products supported conjugation on the thiophene ring in a manner analogous to the spectrum of the GSH adduct (data not shown). The spectrum of the βMP conjugate included m/z 113 (oxygenated analog of m/z 97), a fragment suggestive of an oxygenated thiophene ring. Very little M8 was formed in incubations of either MLM or HLM with GSH: 2.0 ± 0.8 and 4.9 ± 1.1% of turnover, respectively. All of the thiol trapping agents significantly reduced the irreversible binding of radiolabeled material to RLM by approximately 80–85% (Fig. 9B).
Incubations of [3H]MP with RLM and either N-acetyllysine (a hard nucleophile), semicarbazide for trapping aldehydes, or NaCN for trapping iminium ions showed no variation in the metabolite profile and no difference in the irreversible binding compared with [3H]MP alone (data not shown).
Characterization of Glutathione Adduct Metabolite M8 by Deuterium Exchange. The mass spectral identification of metabolite M8 formed by RLM incubated with MP and GSH was confirmed using the LTQ Orbitrap. In addition to MS2 fragments at m/z 540, 456, 411, and 278 obtained previously by LC-MS and by LC-MS/MS on a Q TRAP instrument, m/z 522 ([540-H2O]+), 411 ([540–129]+), 393 ([411-H2O]+), 267 ([233+S + 2H]+; from scission of cysteine Cβ-S bond), and 233 ([540-GSH]+) were obtained as fragments of m/z 540 in an MS3 product-ion scan.
It was first established that GSH dissolved in MeOD rapidly exchanged all seven of its labile amine, amide, carboxyl, and sulfhydryl protons with a consequential increase in the mass of the protonated molecule from 308.0917 ([M+H]+; Δ +0.1 ppm) to 316.1419 ([MD+D]+; Δ+0.1 ppm; MD represents the molecular weight of the fully deuterated analyte). The mass of the protonated microsomal metabolite increased from 585.2173 (C24H37N6O7S2; Δ +0.8 ppm) to 592.2618 [C24H30D7N6O7S2; ([MD+D]+; Δ+5.2 ppm)] when the compound was deuterated under the same conditions (Fig. 10, B and C). This was interpreted as representing exchange of the six amine, amide, and carboxyl protons on the glutathionyl residue (Fig. 11). Six H/D exchanges are attributable to the glutathionyl residues of other adducts (Erve et al., 2004). The ion at m/z 593.2648 was identified as [MD+D]+ for the mono-13C isotopomer of hexadeutero-M8 by comparison of the measured mass with the calculated mass: 593.2599 (Fig. 10C). The ions at 590.2491 and 591.2553 were due to incomplete deuteration of M8 in MeOD. The M8 recovered from microsomal incubations also underwent six H/D exchanges when dissolved in D2O. The diagnostic MS2 product ions of M8, namely [M + 1-HN(CH3)2]+, [M + 1-pyroglutamate]+, and [M + 1-GSH]+, were subject to mass shifts of six, five, and two amu, respectively, that were consistent with the six proton exchanges being confined to the glutathionyl residue (Fig. 11, A and B).
When the microsomal incubation of MP and GSH was performed in D2O to attempt deuterolabeling of metabolic intermediates as well as M8, both heptadeutero- and hexadeutero-labeled M8 ([MD+D] at m/z 593 and 592, respectively) were obtained. Heptadeutero-M8 yielded analogous [M + 1-HN(CH3)2]+, [M + 1-pyroglutamate]+, and [M + 1-GSH]+ fragments (Fig. 11A) at m/z 547, 462, and 281, respectively, representing mass shifts of seven, six, and three amu. This can be ascribed to monodeuteration of the oxygenated reactive metabolite of MP at the thiophene ring during addition of heptadeutero-GSH generated from GSH in situ.
The results of these deuterium exchange experiments can be tested against the three proposed mechanisms of oxidative bioactivation of monocyclic 2-thiophenes (Kalgutkar et al., 2005): S-oxidation (Belghazi et al., 2001; Dansette et al., 2005), 4,5-epoxidation without opening of the thiophene ring (O'Donnell et al., 2003; Dansette et al., 2005), and 4,5-epoxidation with concerted opening of the epoxide and thiophene rings to produce a γ-thioketo-α,β-unsaturated aldehyde (O'Donnell et al., 2003) (Fig. 12). A dihydrohydroxythiophenyl-S-glutathionyl adduct formed by addition of GSH to a thiophene epoxide of MP would be expected to exchange seven protons, including the hydroxyl proton on the thiophene ring, and yield a monoisotopic [MD+D]+ ion at m/z 593.2662 (calculated). However, isomeric thioether adducts formed by addition of GSH to MP S-oxide and the α,β-unsaturated aldehyde intermediate (Fig. 12) would both have structures consistent with the observed six H/D exchanges in MeOD: H/D exchange on the MP-derived moiety would not be expected in either case. Nevertheless, the following observations suggest that a thioketo aldehyde is an improbable precursor of M8: 1) in microsomal incubations of suprofen, a 2-aroylthiophene, the putative 4,5-epoxide precursor of the γ-thioketo-α,β-unsaturated aldehyde intermediate, and not the aldehyde, was trapped with GSH as a thioether adduct; the aldehyde was stabilized separately, as a pyridazine derivative, with semicarbazide (O'Donnell et al., 2003); 2) semicarbazide neither inhibited the irreversible binding of [3H]MP to liver microsomes nor yielded an additional metabolite—and specifically no pyridazine derivative; and 3) furan and pyrrole epoxides can also conjugate with GSH without opening of the primary heterocycle (Kalgutkar et al., 2005; Williams et al., 2007), and, in general, it seems that α,β-unsaturated carbonyls derived from heteroarene epoxides by ring opening are not trapped by GSH in vitro. The glutathione adduct of a γ-thioketo-α,β-unsaturated aldehyde might also be formed directly, by concerted reaction between GSH and a thiophene epoxide of MP, but no published precedent for such a reaction has been found. Therefore, the available analytical data are most readily understood in terms of M8 being a glutathione adduct of MP S-oxide.
Hepatotoxicity and Metabolic Activation of MP in Vivo. Rats that had received oral doses of MP (150 mg/kg/day) for 3 days showed evidence 24 h later of necrotic liver injury in the form of a 2- and 18-fold increase in serum ALT and GLDH activity, respectively (Table 2). This toxicity was accompanied by a significant 1.9-fold elevation in hepatic tGSH content. Predosing with ABT (50 mg/kg i.p.) 1 h before each administration of MP (150 mg/kg) blocked the increases of ALT, GLDH, and hepatic tGSH.
Anesthetized rats administered [3H]MP (17.5 mg/kg) i.v. eliminated 5.06 ± 3.02 and 23.2 ± 6.04% (n = 3) of the dose in bile over 1 and 5 h, respectively. Radiolabeled metabolites M6 and M8 were identified by LC-MS as described for the corresponding metabolites produced by rat hepatocytes. M8, the GSH conjugate of monooxygenated MP, constituted 19.9 ± 3.7% (n = 3) of the radioactivity in the 0–1 h bile collection (Fig. 13).
Discussion
Thiophene rings are present in a number of compounds associated with toxicity and are known to undergo bioactivation (Kalgutkar et al., 2005). Nevertheless, experimental verification that the heterocycle can act as a bioactivation-dependent toxicophore has proved elusive. The findings of the present investigations into the metabolic activation and cytotoxicity of MP represent the first confirmation of this relationship.
MP produces an acute hepatotoxicity in male rats at relatively low doses (Graichen et al., 1985; Ratra et al., 1998a, 2000; Huang et al., 2004; Craig et al., 2006). The comparative mildness of this injury more typically resembles drug-induced clinical hepatotoxicities than the severe damage produced in experimental animals by many other reference hepatotoxicants. These characteristics have favored MP as a benchmark compound for toxicogenomic (Huang et al., 2004; Beekman et al., 2006), toxicoproteomic, and metabonomic analysis (Craig et al., 2006). Paradoxically, the biomolecular perturbations associated with the hepatotoxicity of the drug have been analyzed without knowledge of either the toxicophore or its mechanistic involvement. The bioactivation of MP to cytotoxic reactive intermediates was implied by earlier observations, but it has previously defied chemical definition and biological correlations.
The binding of [3H]MP to mitochondria in periportal hepatocytes (Reznik-Schüller and Lijinsky, 1981), charge modification of proteins (Craig et al., 2006), opening of the mitochondrial transition permeability pore, and loss of intracellular Ca2+ homeostasis (Ratra et al., 1998b) have identified an organellar target for MP. Glutathione depletion in hepatocytes and cytoprotection by dithiothreitol (Ratra et al., 1998b) suggest a toxicogenesis involving oxidative stress. MP stimulates lipid peroxidation in rat liver (Hernandez and Lijinsky, 1989), but neither we nor others (Hernandez and Lijinsky, 1989; Ratra et al., 1998a) observed depletion of hepatic glutathione in vivo. Nevertheless, the numerous genomic and proteomic effects of the drug include oxidative stress responses (Huang et al., 2004; Craig et al., 2006). Whether these effects are causal or consequential is unclear.
Necrotic death of hepatocytes exposed to [3H]MP was preceded by irreversible binding of radiolabel and the depletion of tGSH reported previously (Ratra et al., 1998b). This depletion might be a precondition for the cytotoxicity of MP, but the progression of hepatotoxicity in vivo, despite an increase in hepatic GSH, suggests otherwise. The increase in hepatic GSH, also reported previously (Ratra et al., 2000), is attributable to induction by MP of a cell defense pathway that produces significant up-regulation of glutamate-cysteine ligase catalytic subunit (C. M. Hirst, A. E. Mercer, E. E. Graham, S. L. Regan, D. J. Antoine, C. A. Benson, D. P. Williams, J. Foster, J. G. Kenna, and B. K. Park, manuscript in preparation). Tienilic acid, a 2-aroylthiophene associated with hepatotoxicity, depletes GSH in rat hepatocytes without causing cytotoxicity (López-García et al., 2005).
The only previously published indication for cytotoxic MP metabolites is an attenuation of toxicity in male rat hepatocytes by the nonspecific P450 inhibitor metyrapone (Ratra et al., 1998b). This concentration-dependent cytoprotection correlated with metyrapone's inhibition of CYP2C11, but not CYP3A2, in liver microsomes of male rats. Furthermore, CYP2C11-deficient hepatocytes were resistant to MP-medicated cytotoxicity. Wrighton et al. (1991) hypothesized that the selective, MP-induced destruction of hepatic CYP2C11 in rats might be attributable to a reactive product of thiophene ring oxidation. Whereas these observations implicated a particular isoform, the transformation catalyzed by CYP2C11 was not identified, and inhibition by metyrapone of CYP2C11 in hepatocytes was not determined. We have confirmed the metabolism dependence of cytotoxicity in hepatocytes with nonspecific P450 inhibitors ABT (Mugford et al., 1992) and SKF-525A, and we have shown that the only transformation of [3H]MP catalyzed by CYP2C11 is N-demethylation; a reaction that was catalyzed to a lesser extent by CYP2C6, although not by any of the other six rat hepatic isoforms that were tested. An iminium ion intermediate of N-demethylation has been trapped with cyanide in incubations of rabbit liver microsomes (Ziegler et al., 1981), and, hitherto, it was the only reactive metabolite of MP to be characterized, although no attempt has been made to connect N-demethylation with cytotoxicity. Notwithstanding the fact that we achieved considerable [3H]MP N-demethylation with rat liver microsomes, no evidence of an iminium ion was forthcoming: coincubated cyanide did not yield the stable N-(cyanomethyl)-normethapyrilene adduct obtained by Ziegler et al. (1981), and it did not have an apparent effect on the profile of stable drug metabolites or inhibit irreversible binding of radiolabel to microsomes. In rat hepatocytes, the extensive attenuation by ABT of cytotoxicity, tGSH depletion, and turnover and irreversible binding of [3H]MP was not associated with a comparable inhibition of N-demethylation. It seems that neither CYP2C11 nor N-demethylation can be causally connected with hepatocytotoxicity.
Other metabolites, such as amines and aldehydes and further oxidation products produced by microsomes (Ziegler et al., 1981), liver homogenate (Kammerer and Schmitz, 1986), or rats (Kammerer et al., 1988), are putatively formed from four possible iminium ions of MP (Singer et al., 1987). However, with the exception of derivatives resulting from dealkylation of the N-thienylmethyl group, none of them has been linked with toxicity. The end product of side-chain oxidation, 2-thiophene carboxylic acid, was tentatively identified as a minor MP metabolite in mouse hepatocytes and the principal metabolite in rat hepatocytes (Kelly et al., 1992). Because rat hepatocytes are more sensitive to MP, the acid has been regarded as a marker of reactive and cytotoxic intermediates. Whereas this species difference in sensitivity has now been characterized, we were unable to find 2-thiophene carboxylic acid in hepatocyte incubations (data not shown). The metabolic precursor of 2-thiophene carboxylic acid, 2-thiophene-carboxaldehyde, reacted with thiols in aqueous solution to yield thioacetal and thiazolidine conjugates (data not shown), but it is not an identified MP metabolite (Kammerer and Schmitz, 1986), and neither it nor 2-thiophenemethanol, at 50 μM, was toxic to rat hepatocytes (data not shown). An aldehyde trapping agent, semicarbazide (O'Donnell et al., 2003), coincubated with rat liver microsomes, did not produce identifiable metabolite conjugates or influence irreversible binding of [3H]MP.
Evidence for hepatic bioactivation of MP in rats has previously comprised the irreversible binding of [3H(G)]MP to liver protein in vivo (Lijinsky and Muschik, 1982), of [thienyl methylene-14C]MP to microsomes and coincubated DNA (Lampe and Kammerer, 1987, 1990), and of [5-3H-pyridyl]MP to microsomes (Singer et al., 1987). When taken with our finding that [3H]MP labeled at C-2 of the 1,2-diaminoethane moiety also undergoes irreversible binding to hepatocytes and microsomes, it would seem that the bound structure(s) incorporates the alkyldiamine backbone as well as the thiophene and pyridine rings. NADPH-dependent binding to DNA in microsomal incubations was reduced by metyrapone, SKF-525A, GSH, and NAC but only equaled approximately 25% of the (uncharacterized) binding to the microsomes (Lampe and Kammerer, 1987). We have now established by similar methods of inhibition that the radiolabeled metabolites of [3H]MP binding to hepatic microsomes are also at least predominantly P450-generated soft electrophiles.
Reduction of cytotoxicity in rat hepatocytes by ABT was coincident with inhibition of the two major indicators of the metabolic activation of [3H]MP: irreversible binding of radiolabel to the cells and formation of glutathione adduct M8. The metabolite was characterized by mass spectrometry as a novel thioether adduct of monooxygenated MP substituted on the thiophene ring. This preliminary identification of a bioactivation-dependent toxicophore was reinforced by the lower sensitivity of mouse hepatocytes, which, as also noted by Kelly et al. (1992), effect a much lower turnover of MP and, as reported here, bioactivate MP to a lesser extent. In addition, the arene analogs of MP, pyrilamine and tripelennamine, were much less hepatocytotoxic, notwithstanding the fact that all three compounds are metabolized predominantly via demethylation, N-oxidation, and pyridine-ring hydroxylation (Kelly and Slikker, 1987; Kammerer and Schmitz, 1988).
H/D exchange analyses of M8 indicated that the precursor of the adduct was probably MP S-oxide rather than a thiophene epoxide; although both types of intermediate are known as reactive metabolites of other monosubstituted thiophenes. Tienilic acid (Belghazi et al., 2001) and its 3-thiophene isomer (Valadon et al., 1996) are reported to be metabolized to S-oxides by hepatic microsomes. Tienilic acid forms numerous protein adducts in rat hepatocytes, although no GSH adduct has been found (López-García et al., 2005). 2-Phenylthiophene is uniquely metabolized by rat liver microsomes to both S-oxide and thiophene epoxide intermediates (Dansette et al., 2005). Suprofen, another 2-aroylthiophene, apparently yields a thiophene epoxide that partly rearranges to a γ-thioketo-α, β-unsaturated aldehyde, but only the epoxide forms a glutathione adduct (O'Donnell et al., 2003). Thiophene itself forms an S-oxide in vivo (Dansette et al., 1992).
The hepatotoxicity of these thiophenes is more variable or uncertain than their bioactivation. Tienilic acid seems to be less toxic than MP to rat hepatocytes (Higaki et al., 1989; López-García et al., 2005). The C-3 isomer, unlike tienilic acid, is hepatotoxic in rats (Bonierbale et al., 1999), but its hepatocytotoxicity is unknown. Thenyldiamine, the C-3 isomer of MP, like MP, was not hepatotoxic in rats at 100–200 mg/kg (Hernandez and Lijinsky, 1989), but it induced greater lipid peroxidation. Neither the bioactivation nor cytotoxicity of thenyldiamine is documented. Suprofen was associated with renal damage in patients, but it is not toxic to rat hepatocytes (Masubuchi et al., 1998). The toxicity of 2-phenylthiophene has not been reported.
While the generic status of the thiophene ring as a bioactivation-dependent toxicophore awaits confirmation, it is clear from the present findings that the status of this heterocycle as a structural alert for toxicity is justified and invites further metabolic and mechanistic investigations.
Acknowledgments
We are indebted to Philip J. Roberts (Department of Pharmacology and Therapeutics, University of Liverpool) for assistance with the cannulated rat experiments; Nick Bushby and David A. Killick (Isotope Chemistry Unit, AstraZeneca) for preparation of [3H]MP; Christopher Smith and Sunil Sarda (AstraZeneca) for LC-MS/MS analyses; and Charles J. O'Donnell and Richard T. Gallagher (AstraZeneca).
Footnotes
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This work was supported by studentships funded by AstraZeneca (to E.E.G. and R.J.W.) and AstraZeneca/Biotechnology and Biological Sciences Research Council (to C.M.H.).
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The data presented here have appeared in abstract form as follows: Dalton-Brown E, Hirst C, Maggs J, Williams D, Wild MJ, Wilson I, Harding J, Kenna G, and Park K (2006) The metabolic basis of methapyrilene-induced hepatotoxicity. Drug Metab Rev 38 (Suppl 1):175–176; and Hirst C, Dalton-Brown E, Williams D, Maggs J, Powell H, Harding J, Wilson I, Wild MJ, Kenna G, and Park K (2006) Chemical, biological, and toxicological mechanisms of hepatotoxins. Drug Metab Rev 38 (Suppl 1):169–170.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.135483.
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ABBREVIATIONS: MP, methapyrilene; P450, cytochrome P450; tGSH, total glutathione (GSH plus glutathione disulfide); MeOD, monodeuteromethanol; HPLC, high-performance liquid chromatography; [3H]MP, methapyrilene tritiated at C-2 of the diaminoethane moiety; GSH, glutathione; GSH-EE, glutathione O-ethyl ester; NAC, N-acetylcysteine; ABT, 1-aminobenzotriazole; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; LC-MS, liquid chromatography-mass spectrometry; RLM, rat liver microsomes; MLM, mouse liver microsomes; HLM, human liver microsomes; LC-MS/MS, liquid chromatography-tandem mass spectrometry; βME, β-mercaptoethanol; ALT, alanine amino transferase; GLDH, glutamate dehydrogenase; H/D, hydrogen/deuterium exchange; equiv, equivalent; amu, atomic mass unit; MD, molecular weight of fully deuterated analyte; SKF-525A, 2-diethylaminoethyl 2,2-diphenyl pentanoate.
- Received December 17, 2007.
- Accepted April 29, 2008.
- The American Society for Pharmacology and Experimental Therapeutics