0022-3565/07/3223-1208–1220$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 322, No. 3 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 125302/3242892 JPET 322:1208–1220, 2007 Printed in U.S.A.

The and Toxicity of in the Wistar Rat and CD-1 Mouse: a Chemical and Biochemical Definition of the Toxicophore

Dominic P. Williams, Daniel J. Antoine, Philip J. Butler, Russell Jones, Laura Randle, Anthony Payne, Martin Howard, Iain Gardner, Julian Blagg, and B. Kevin Park The Drug Safety Research Group, Department of Pharmacology, University of Liverpool, Liverpool, United Kingdom (D.P.W., D.J.A., L.R., B.K.P.); Pharmacokinetics Dynamics and Metabolism (R.J., A.P., M.H., I.G.) and Sexual Health Chemistry (J.B.), Pfizer, Sandwich, United Kingdom; and Cyprotex, Nacclesfield, United Kingdom (P.J.B.) Downloaded from Received May 4, 2007; accepted June 6, 2007

ABSTRACT Furosemide, a loop diuretic, causes hepatic necrosis in mice. lic acid metabolite (22.1 Ϯ 3.3%), N-dealkylated metabolite Previous evidence suggested hepatotoxicity arises from meta- (21.1 Ϯ 2.9%), and furosemide glucuronide (12.8 Ϯ 1.8%). jpet.aspetjournals.org bolic bioactivation to a chemically reactive metabolite that Furosemide-glutathione conjugate was not observed in bile binds to hepatic proteins. To define the nature of the toxic from mice dosed with [14C]furosemide. The novel ␥-ketocar- metabolite, we examined the relationship between furosemide boxylic acid, identified by nuclear magnetic resonance spec- metabolism in CD-1 mice and Wistar rats. Furosemide (1.21 troscopy, indicates bioactivation of the furan ring. Formation of mmol/kg) was shown to cause toxicity in mice, but not rats, at ␥-ketocarboxylic acid was P450-dependent. In mouse liver mi- 24 h, without resulting in glutathione depletion. In vivo covalent crosomes, a ␥-ketoenal furosemide metabolite was trapped, binding to hepatic protein was 6-fold higher in the mouse forming an N-acetylcysteine/N-acetyl lysine furosemide ad- (1.57 Ϯ 0.98 nmol equivalent bound/mg protein) than rat duct. Furosemide (1 mM, 6 h) became irreversibly bound to at ASPET Journals on January 15, 2020 (0.26 Ϯ 0.13 nmol equivalent bound/mg protein). In vivo cova- primary mouse and rat hepatocytes, 0.73 Ϯ 0.1 and 2.44 Ϯ 0.3 lent binding to mouse hepatic protein was reduced 14-fold by nmol equivalent bound/mg protein, respectively, which was a predose of the cytochrome P450 (P450) inhibitor, 1-amino- significantly reduced in the presence of ABT, 0.11 Ϯ 0.03 and benzotriazole (ABT; 0.11 Ϯ 0.04 nmol equivalent bound/mg 0.21 Ϯ 0.1 nmol equivalent bound/mg protein, respectively. protein), which also reduced hepatotoxicity. Administration of Furan rings are part of new chemical entities, and mechanisms [14C]furosemide to bile duct-cannulated rats demonstrated underlying species differences in toxicity are important to un- turnover to glutathione conjugate (8.8 Ϯ 2.8%), ␥-ketocarboxy- derstand to decrease the drug attrition rate.

Furosemide (4-chloro-N-furfuryl-5-sulfamoyl-anthranilic al., 2000), and there may be a potential risk of hepatotoxicity acid; FS) is a highly potent loop diuretic frequently used in when administered in large doses to patients with acute and the treatment of edematous states associated with cardiac, chronic renal failure (Mitchell et al., 1974). renal, and hepatic failure and for the treatment of hyperten- It has been postulated that the hepatocellular damage sion (Ponto and Schoenwald, 1990). FS has been shown to caused by FS is a result of cytochrome P450-mediated forma- produce massive hepatic necrosis in mice by a mechanism tion of a toxic metabolite (Mitchell et al., 1974, 1976) rather independent of its diuretic action (Mitchell et al., 1974; Liu et than the parent drug. FS can be converted by cytochrome al., 1995; Wong et al., 2000). At therapeutic doses, FS is not P450 to a chemically reactive metabolite, which binds co- toxic to humans, although it has been associated with jaun- valently with hepatic macromolecules both in vivo and in dice (Dargie and Dollery, 1975) and hypersensitivity (Noce et vitro (Mitchell et al., 1976). Incubation with inhibitors of P450 decreased the incidence and severity of FS- Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. induced hepatic necrosis (Mitchell et al., 1974). This hepato- doi:10.1124/jpet.107.125302. toxicity is reproducible in mice with at least eight other

ABBREVIATIONS: FS, furosemide; P450, cytochrome P450; NAC, N-acetyl cysteine; NAL, N-acetyl lysine; GSH, reduced glutathione; ABT, 1-aminobenzotriazole; PEG, polyethylene glycol; DMSO, dimethyl sulfoxide; ALT, ; HPLC, high-pressure/performance liquid chromatography; GSSG, oxidized glutathione; LC-MS, liquid chromatography-mass spectrometry; MS/MS, tandem mass spectrometry; NMR, nuclear magnetic resonance spectroscopy; MTS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; amu, atomic mass unit; SG, glutathione adduct. 1208 Furosemide Metabolism and Hepatotoxicity 1209 furan-containing compounds including 2-acetylfuran and fu- ries Ltd. (Hemel Hempstead, Hertfordshire, UK). All solvents were ran itself (Mitchell et al., 1974). Experiments using radiola- of HPLC grade and were the products of Fischer Scientific plc beled FS demonstrate that the entire drug molecule becomes (Loughborough, UK). covalently bound to protein. It was suggested that this was a Experimental Animals. The protocols described were under- consequence of metabolic activation of the furan ring, possi- taken in accordance with criteria outlined in a license granted under the Animals (Scientific Procedures) Act 1986 and approved by the bly through epoxidation (Mitchell et al., 1976). It has more University of Liverpool Animal Ethics Committee. recently been demonstrated with 4-ipomeanol that a double Investigation of the Hepatotoxicity of FS in the Wistar Rat N-acetyl cysteine (NAC) and N-acetyl lysine (NAL) conjugate and CD-1 Mouse. Male CD-1 mice (25–35 g) and male Wistar rats can be formed from in vitro incubations. This structure sup- (200–250 g) were administered a single i.p. injection of FS (1.21 ports the formation of an 1,4-enedial metabolite of the furan mmol/kg; n ϭ 4 per group) in PEG. Animals treated with vehicle ring (Baer et al., 2005). This has also been shown to be the alone were used as controls. Either 5 or 24 h after administration, case for teucrin A, a furan-containing diterpenoid germander animals were killed by cervical dislocation, and blood was collected extract associated with hepatotoxicity in man (Druckova and by cardiac puncture. Blood samples were stored at 4°C and allowed Marnett, 2006). Enhancement of FS-protein covalent bind- to clot overnight. Livers were collected, rinsed, and stored in 10% ing, in the presence of an epoxide hydrolase inhibitor, has neutral buffered formalin for histological analysis and/or snap-fro- been reported (Wirth et al., 1976). However, chemical defini- zen. Furthermore, a group of male CD-1 mice (25–35 g) were admin- istered a single injection of ABT (745 ␮mol/kg in PEG i.p.) 1 h before tion of the ultimate toxic metabolite of FS has not been the dose of FS. reported. Determination of the in Vivo Levels of Covalent Binding of Downloaded from Previous studies have demonstrated that hepatic glutathi- FS to Hepatic Tissue in the Wistar Rat and CD-1 Mouse. To one (GSH) levels were not altered by the administration of a assess in vivo levels of hepatic covalent binding, male CD-1 mice toxic dose of FS to mice (Mitchell et al., 1974). FS has also (25–35 g) were administered FS [1.21 mmol/kg (400 mg/kg); n ϭ 4 been shown to have no effect on the cytosolic or mitochondrial per group] and [14C]FS (10 ␮Ci) i.p. An injection of ABT (745 ␮mol/kg GSH content in CD-1 mice (Wong et al., 2000). Furthermore, in PEG i.p.) was given to an additional group of mice 1 h before the administration of [14C]FS and unlabeled FS. After 1, 5, or 24 h, no GSH depletion was observed in rat hepatocytes treated jpet.aspetjournals.org with FS (Takagi et al., 1991). However, 70% depletion in animals were killed by cervical dislocation, and blood was collected cellular GSH content has been reported in isolated CD-1 as described previously. Livers were removed, rinsed, and stored at Ϫ80°C before use. To determine the levels of covalent binding, a mouse hepatocytes (Grewal et al., 1996). The postulated portion (ϳ50 mg) of liver was taken from mice and Wistar rats that mechanisms of toxicity induced by the reactive metabolite(s) had been administered [14C]FS (10 ␮Ci) and 1.21 mmol/kg FS. This of FS include binding to critical cellular macromolecules and was homogenized and subjected to an exhaustive solvent extraction subsequent disruption of calcium homeostasis (Mitchell et procedure as described previously (Pirmohamed et al., 1995b). The

al., 1974; Massey et al., 1987). amount of radiolabeled FS bound irreversibly to hepatic protein in at ASPET Journals on January 15, 2020 The objective of the current study is to fully clarify the vivo was determined. Aliquots of the solubilized protein were taken metabolism of FS in vivo in Wistar rats and CD-1 mice and to for quantification of radioactivity by liquid scintillation counting. identify the nature of chemically reactive species formed and Determination of Serum Alanine Transaminase Levels. Se- attempt to define the role these species play in initiation of rum was separated by pulse centrifugation (4000 rpm) from blood hepatotoxicity. We have investigated the role of cytochrome that had been allowed to clot overnight at 4°C. Serum ALT levels were determined using ThermoTrace Infinity ALT Liquid stable P450 in the formation of reactive metabolite(s) and made an reagent according to the manufacturer’s instructions. Hepatotoxicity assessment of the role of furan bioactivation in species dif- was indicated by ALT levels greater than 200 U/l (Goldring et al., ferences in susceptibility to furosemide hepatotoxicity. In 2004). vivo biliary studies have been employed to identify cyto- Histological Examination of Murine Hepatic Sections. A chrome P450-dependent metabolite formation and species transverse section of the left hepatic lobe was collected from forma- differences in metabolism between the Wistar rat and CD-1 lin-fixed 24-h livers. Paraffin-embedded tissue sections were pre- mouse to define the relationship in susceptibility to FS hep- pared at a thickness of 4 ␮m and were stained with hematoxylin and atotoxicity. Rodent hepatocyte investigations have been used eosin for evaluation. All histological examinations were performed to demonstrate a relationship among metabolism, covalent by a single pathologist (blinded to drug treatment) evaluating one binding, GSH depletion, and cytotoxicity in vitro. Finally, liver section per animal, using a DMLB microscope [Leica Microsys- tem (UK) Ltd., Milton Keynes, UK]. Hepatic sections were scored species differences in covalent binding are demonstrated in according to predefined criteria. In brief, grade 0 represented normal the Wistar rat, CD-1 mouse, and human to define the relative histology, and grade 4 represented massive hepatocellular necrosis hazard of protein modification FS poses to hepatic protein of involving entire lobules. each species. Determination of Glutathione Levels in Vivo in Wistar Rats and CD-1 Mice. Total hepatic glutathione (GSH ϩ GSSG) levels Materials and Methods were determined spectrophotometrically using a microtiter plate assay (Vandeputte et al., 1994) based upon the 5,5Ј-dithiobis(2-ni- Materials. [14C]FS (specific activity, 629 MBq/mmol) and unla- trobenzoic acid)-glutathione disulfate recycling procedure first re- beled FS were provided by Pfizer (Kent, UK) and determined to be ported by Owens and Belcher (1965). Ͼ98% pure. The 14C label was incorporated into the carboxyl carbon Investigation of the Biliary Metabolites of FS in the Wistar atom of FS and thus expected to be incorporated in all major metab- Rat and CD-1 Mouse. Adult male Wistar rats (200–400 g) and olites. GSH, NADPH (tetrasodium salt), 1-aminobenzotriazole male CD-1 mice (30–35 g) were terminally anesthetized with ure- (ABT), polyethylene glycol (PEG), Hanks’ balanced salt solution, pH thane (1.4 g/ml in isotonic saline; 1.0 ml/kg i.p.) and pentobarbital 7.4, and dimethyl sulfoxide (DMSO) were purchased from Sigma sodium (60 mg/kg in isotonic saline i.p.), respectively, and cannu- (Poole, UK). Infinity alanine transaminase (ALT) (GPA) liquid kits lated via the trachea, jugular vein, and the common bile duct. were purchased from Alpha Laboratories (Eastleigh, UK). Bio-Rad [14C]FS (10 ␮Ci) and 151 ␮mol/kg unlabeled FS dissolved in DMSO Protein Assay Dye Reagent was purchased from Bio-Rad Laborato- were administered i.v. over 10 min. One group of Wistar rats and one 1210 Williams et al. group of mice (n ϭ 4 per group) were treated with ABT (745 ␮mol/kg The LC-MS fraction collection system consisted of an 1100 binary i.v.) in DMSO 1 h before administration of FS. Drug-free bile was pump (Agilent Tech. Inc.), 1100 diode array detector (Agilent Tech. collected for 30 min before administration of FS. Bile was then Inc.), Accurate 5:1 splitter box (LC Packings, Sunnyvale, CA), LCQ collected hourly for 3 h into preweighed Eppendorfs. Protein within Classic mass spectrometer (Thermo Electron Corp., Waltham, MA), the bile samples was precipitated with addition of ice-cold acetoni- and a 202 fraction collector (Gilson Inc., Middleton, WI). The HPLC trile. The samples were centrifuged at 2200 rpm for 10 min. The system used a HIRB-250SP (250 ϫ 7.75 mm) column (Hichrom, resultant supernatant was collected, transferred to fresh tubes, and Berks, UK) with two mobile phases; water containing 0.1% formic evaporated to dryness under a stream of nitrogen at 37°C. The acid (v/v) and acetonitrile containing 0.1% formic acid (v/v), at a flow residue was stored at Ϫ20°C until analysis, when it was reconsti- rate of 2.0 ml/min. Two gradient systems were used to purify the tuted in methanol (100 ␮l) and water (100 ␮l) for analysis by radio- ␥-ketocarboxylic acid metabolite. Multiple injections (3 ϫ 300 ␮l) of metric LC-MS. the semipurified bile extract were made, a gradient elution was HPLC and LC-MS Analysis of Wistar Rat and CD-1 Mouse performed (5% of the organic mobile phase for 1 min, increasing to Bile. The configuration of the LC-MS system and parallel radioac- 40% over 12 min), and fractions were collected. The resulting frac- tivity detector (Radiomatic A250; Canberra Industries, Meriden, CT) tions were pooled, diluted 2-fold with 0.1% (v/v) formic acid in water, has been described previously (Madden et al., 1996). A Quattro II and reinjected (4 ϫ 500 ␮l) onto the second gradient system (5% of tandem quadruple instrument (Micromass Ltd., Manchester, UK) the organic mobile phase for 1 min, increasing to 40% over 30 min). fitted with the standard in-line LC-MS interface and electrospray In each case, the column eluant was split with the high flow directed source was employed in both positive and negative ion mode for full to the fraction collector and the low flow to the MS. The mass scanning acquisitions between m/z 100 and 1050 at 1 scan/5 s. The spectrometer was operated in Ϫve ion mode with an electrospray LC system consisted of two Jasco PU980 pumps [Jasco (UK), Great ionization source. The capillary temperature was 250°C; the source Downloaded from Dunmow, Essex, UK] and a Jasco HG-980-30 mixing module. Biliary voltage, 4.5 kV; the capillary voltage, Ϫ47 V; and the sheath gas flow and microsomal metabolites were resolved on an Ultracarb 5-␮m C-8 and auxiliary gas flow were both 50 (arbitrary units). Full scan data column (250 ϫ 4.60 mm) with a gradient of acetonitrile (10–60% were acquired with the fraction collector being triggered to collect over 30 min) in ammonium formate, pH 3.5 (10 mM) at a flow rate of when the ␥-ketocarboxylic acid metabolite of FS (m/z 363) was de- 0.9 ml/min. Eluate split flow to the LC-MS interface was ϳ40 ␮l/min. tected. The collected fractions were combined, the solvent was re- The remainder of the column eluent was mixed with Ultima-Flo for moved at 30°C under a stream of nitrogen (Turbovap; Caliper Life jpet.aspetjournals.org detection of radioactivity. Nitrogen was used as both the nebulizing Sciences), and the residue was stored at Ϫ20°C until analysis by and drying gas. The interface temperature was 70°C; the capillary NMR. voltage, 3.9 kV; the HV and RF lens voltage, 0.5 kV and 0.1 V, Deuterium Exchange for Determination of ␥-Ketocarboxy- respectively; and the photomultiplier voltage, 650 V. The cone volt- lic Acid Formation. Metabolite III was reconstituted in 50/50 (v/v) age was 30 V. MS/MS analysis was carried out at a collision energy deuterated methanol/deuterium oxide (100% D; Euristop, Gif-sur- of 25 eV using Argon as collision gas. Data were processed with Yvette, France), allowed to exchange for 24 h at room temperature, MassLynx 3.5 software (Micromass Ltd.). and then analyzed by mass spectrometry. ␥ Accurate Mass Analysis of Wistar Rat Bile. Accurate mass Nuclear Magnetic Resonance Spectroscopy of FS- -Keto- at ASPET Journals on January 15, 2020 determinations of parent compound and metabolites were carried carboxylic Acid Metabolite Isolated from Wistar Rat Bile. The out on the Waters quadrupole time of flight Premier (Waters Ltd., sample was dissolved in 0.5 ml of d4-methanol (100% D; Euristop) Elstree, Herts, UK) operating in “V” mode using leuenkephalin (m/z and transferred to a 3-ml NMR tube. The sample was analyzed using 556.2771) as lock spray internal mass calibrant. The instrument was a Bruker 600 MHz Ultrashield Magnet (Bruker, Newark, DE) with a operated under both positive and negative ion mode for full scanning cryoprobe accessory (BrukerA) at 30°C utilizing proton, proton-pro- acquisitions between m/z 100 and 800 at 1 scan/s. The LC system ton (correlation spectroscopy), and proton-carbon (heteronuclear sin- consisted of an 1100 binary pump (Agilent Tech. Inc., Palo Alto, CA) gle quantum correlation and heteronuclear multiple-bond correla- and a CTC PAL autosampler (Presearch Ltd., Basingstoke, Hants, tion spectroscopy) correlations. UK). Biliary metabolites were resolved on a Sunfire 3.5-␮m C18 Hepatocyte Isolation from Male Wistar Rats and CD-1 Mice. (100 ϫ 2.1 mm i.d.) column (Waters Ltd.) with a linear gradient of Rat and mouse hepatocytes were isolated using a modified two-step acetonitrile (5–98% over 8 min) in water, both containing 0.1% (v/v) in situ collagenase profusion based on Seglen (1976). Male Wistar formic acid, at a flow rate of 0.2 ml/min. The column eluate passed rats (150–170 g) and male CD-1 mice (20–30 g) were anesthetized directly into the MS source. Nitrogen was used as both the nebuliz- with sodium pentobarbital (60 mg/ml, 1 ␮l/g i.p.). The hepatic portal ing and desolvation gas. The source and desolvation temperatures vein was cannulated, and the liver was perfused for 7 min at a were 120 and 300°C, respectively; the capillary voltage was 3.2 kV; constant flow rate of 12 ml/min for mice and 40 ml/min for rats, with and the sample cone voltage was 25 V. MS/MS analysis was carried a wash buffer containing Hanks’ balanced salt solution (from 10ϫ out at a collision energy of 25 eV using Argon as collision gas. Data stock: magnesium chloride, magnesium sulfate, sodium bicarbonate) were processed with MassLynx 4.0 sp4 (Waters Ltd.). (Invitrogen, Paisley, UK). Wash buffer was also supplemented with ␥ Mass-Directed Fraction Collection of FS -Ketocarboxylic 5.8 mM HEPES and 4.5 mM NaHCO3. The wash step was followed Acid Metabolite from Wistar Rat Bile. Rat bile was pre-extracted by a collagenase perfusion using wash buffer supplemented with 5 using solid phase extraction. A Bakerbond C18 (1000 mg) solid-phase mM CaCl2, 0.5 mg/ml collagenase A (Roche Applied Science, India- extraction column (J.T. Baker, Deventer, The Netherlands) was pre- napolis, IN), and 0.072 mg/ml trypsin inhibitor until the liver con- conditioned with 20 ml of acetonitrile followed by 20 ml of 0.1% (v/v) stitute had been digested. All wash and digestion solutions were kept formic acid in water. Bile (500-␮l aliquot) was loaded onto the col- at 37°C throughout the perfusion. After in situ digestion, the liver umn, the column was washed with 10 ml of 0.1% formic acid in was excised, and the liver capsule was removed to release parenchy- water, and the eluant was collected. The ␥-ketocarboxylic acid me- mal and nonparenchymal cells. The cells were suspended in 50 ml of tabolite of furosemide was eluted with a step-wise gradient of in- wash buffer supplemented with DNase I (0.1 mg/ml) and filtered creasing percent of acetonitrile in water containing 0.1% (v/v) formic through a 125-␮m mesh (Lockertex, Cheshire, UK). suspensions acid. The fractions (10–20% acetonitrile) containing the ␥-ketocar- were immediately centrifuged at 30g for 2 min, the resulting super- boxylic acid metabolite were pooled and evaporated to dryness under natant containing nonparenchymal cells was removed, and the cells a stream of nitrogen at 30°C (Turbovap; Caliper Life Sciences, Hop- were resuspended in wash buffer containing DNase I. This centri- kinton, MA). The extract was reconstituted in 90:10 (v/v) water/ fuge step was repeated twice at 50g with wash buffer alone; follow- acetonitrile containing 0.1% (v/v) formic acid and further purified by ing, the cells were resuspended in an incubation buffer (wash buffer ⅐ LC-MS fraction collection. supplemented with 1 mM MgSO4 7H2O). Hepatocyte viability was Furosemide Metabolism and Hepatotoxicity 1211 assessed by trypan blue exclusion and only used when viability was determine the levels of [14C]FS bound to the hepatocyte protein. Ͼ85%. Experimental hepatocyte incubations contained a total vol- Irreversible binding was determined by an exhaustive solvent ex- ume of 6 ml at a cell concentration of 2 ϫ 106 cells/ml. traction procedure using 70% methanol as described previously (Pir- Assessment of FS-Induced Cytotoxicity in Freshly Isolated mohamed et al., 1995a). Aliquots of the solubilized protein (dissolved Rodent Hepatocytes. Hepatocyte incubations were carried out in in 1 ml of 1 M NaOH at 60°C) were taken for quantification of an orbital shaker set at 190 rpm for 0 to 6 h with furosemide [0–2 radioactivity by liquid scintillation counting and normalized to pro- mM in MeOH (1% v/v) and 0.1 ␮Ci of [14C]FS in ethanol]. Some tein concentration. incubations contained were preincubated with the nonspecific P450 In Vitro Bioactivation of FS in CD-1 Mouse, Wistar Rat, and inhibitor ABT (5 mM, 1 h). Following incubations, cytotoxicity was Human Liver Microsomes. FS (100 ␮M) was incubated with determined by trypan blue exclusion (100-␮l cell suspension and 20 mouse liver microsomes (2 mg/ml), NADPH (1 mM), NAL (1 mM), ␮l of 0.4% trypan blue solution at 0, 0.5, 1, 2, 4, and 6 h). Cytotoxicity and NAC (1 mM) at 37°C for 1 h. After 1 h, the reaction was was also determined by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5- terminated by the addition of 3 ml of ice-cold acetonitrile. The sam- diphenyl tetrazolium bromide (MTS) solution using the CellTiter 96 ples were centrifuged at 2200 rpm for 10 min. The resultant super-

AQueous One Solution Cell Proliferation Assay kit (Promega, Madi- natant was collected, transferred to fresh tubes, and evaporated to son, WI), according to the manufacturer’s instructions. Cell suspen- dryness under a stream of nitrogen at 37°C. The residue was stored sion (100 ␮l) was combined with MTS solution (20 ␮l), incubated at at Ϫ20°C until being reconstituted in methanol (100 ␮l) and water 37°C for 1 h, and centrifuged at 50g for 2 min. The absorbance of the (100 ␮l) for analysis by LC-MS as described earlier. For covalent resulting supernatant was measured at 490 nm using a microtiter- binding studies, [14C]FS (0.1 ␮Ci, 0.6 ␮M) was incubated with CD-1 plate reader (Dynex Technologies, Chantilly, VA). mouse, Wistar rat, and human liver microsomes (2 mg) in the pres- Determination of GSH Content of Primary Rodent Hepato- ence and absence of NADPH (1 mM) and the presence and absence of Downloaded from cytes. From the same incubations used for cytotoxicity assessment, GSH (1 mM). the effect of FS on total cellular levels of GSH was determined. Cells Determination of Protein Concentration. Soluble protein con- (1 ϫ 106) from the hepatocyte incubation were centrifuged at 50g for centration was measured using Bio-Rad protein assay reagent ac- 2 min. The resulting pellet was resuspended in 10 mM HCl. One-fifth cording to the manufacturer’s instructions. of lysed hepatocytes was removed for protein content determination. Statistical Analysis. All results are expressed as mean Ϯ S.E.M. The remaining hepatocyte lysate was combined with 6.5% (w/v) Values to be compared were analyzed for non-normality using a jpet.aspetjournals.org 5-sulfosalicylic acid (four parts hepatocyte lysate and one part 5-sul- Shapiro-Wilk test. Student’s t tests were used when normality was fosalicylic acid) and stored on ice for 10 min before centrifugation at indicated. A Mann-Whitney U test was used for nonparametric data. 18,400g for 5 min. The total GSH and GSSG content was measured For multiple comparisons, one-way analysis of variance tests were at 412 nm from the resulting supernatant (Vandeputte et al., 1994). used. All calculations were performed using Arcus Quickstat statis- GSH content was compared with known standards ranging from 0 to tical software (Altrincham, UK), results were considered to be sig- 40 nmol/ml. nificant when p Ͻ 0.05. Investigation of FS Metabolism and Irreversible Binding in

Primary Rodent Hepatocytes. The remaining portion of the he- at ASPET Journals on January 15, 2020 patocyte incubation (after cytotoxicity and GSH assessment) was Results placed in an equal volume of ice-cold acetonitrile and centrifuged at Investigation of the Hepatotoxicity of FS in the Wistar Rat 880g for 10 min. The resulting supernatant was evaporated under a and CD-1 Mouse steady stream of N2 in a 50°C water bath. The residue was reconsti- tuted in 100 ␮l of methanol and 100 ␮l of water for radiochromato- FS (1.21 mmol/kg i.p.) was found to be significantly toxic to graphic analysis. The protein pellet from the 880g spin was used to mice at both 5 and 24 h (Fig. 1A), compared with control-

Fig. 1. A, effect of a 1.21 mmol/kg i.p. dose of furosemide on serum ALT activity in male CD-1 mice. B, effect of the cytochrome P450 inhibitor 1-aminobenzotriazole on FS toxic- ity in mice. Serum was collected 24 h after dose, and serum ALT activity was used as a measure of toxicity. Controls received vehi- cle (PEG) only. The ABT-only group received 745 ␮mol/kg ABT i.p. The FS-only group re- ceived 1.21 mmol/kg FS i.p. The FS ϩ ABT group received a 1-h predose of ABT (745 ␮mol/kg i.p.) before administration of 1.21 mmol/kg FS i.p. C, in vivo covalent binding (f) of FS (1.21 mmol/kg; 10 ␮Ci) in the pres- ence and absence of the cytochrome P450 inhibitor, 1-aminobenzotriazole (745 ␮mol/ kg; 1-h predose), to murine hepatic protein. Mean serum ALT activity (units per liter) from the serum of the same groups of mice ,p Ͻ 0.01 ,ءء .ࡗ). Values are mean Ϯ S.E.M) Mann-Whitney U test. 1212 Williams et al. dosed mice. Administration of a 1-h predose of ABT (745 male Wistar rats, mean hepatic GSH levels were not signif- ␮mol/kg i.p.) before a toxic dose of FS provided complete icantly altered 1 h following a 1.21 mmol/kg i.p. dose of FS. protection from toxicity at 24 h. There was no increase in serum ALT levels (24 h) in mice dosed with ABT and then Biliary Metabolism of FS in the Wistar Rat and CD-1 toxic FS compared with control-dosed mice (Fig. 1B). Admin- Mouse istration of FS (1.21 mmol/kg i.p.) to male Wistar rats (data [14C]FS (10 ␮Ci; 151 ␮mol/kg) administered i.v. to anes- not shown) failed to elicit any increase in serum ALT levels at thetized male Wistar rats was rapidly excreted into bile. either 5 or 24 h after dosing.

Irreversible Binding of FS to Hepatic Protein in Vivo Over a period of 24 h, the level of FS bound covalently to hepatic protein increased steadily following administration of [14C]FS (10 ␮Ci) and 1.21 mmol/kg FS to mice (0.35 Ϯ 0.16, 0.80 Ϯ 0.28, and 1.57 Ϯ 0.98 nmol equivalent bound/mg protein at 1, 5, and 24 h, respectively). The level of covalent binding was paralleled by the mean ALT activity at each time point (68 Ϯ 28.5, 213 Ϯ 48.8, and 1285.3 Ϯ 766.9 U/l at 1, 5, and 24 h, respectively). A predose of ABT (745 ␮mol/kg i.p.) Downloaded from was shown to prevent, significantly, the binding of FS to hepatic protein at 5 and 24 h (0.09 Ϯ 0.04 and 0.11 Ϯ 0.04 nmol equivalent bound/mg protein). ABT also significantly reduced the level of ALT release at 5 and 24 h (160 Ϯ 93.6 and 20.8 Ϯ 35.5 U/l). Interestingly, a predose of ABT also

dramatically reduced the amount of radiolabeled material jpet.aspetjournals.org recovered from hepatic tissue (Fig. 1C). In Wistar rats, the level of FS bound covalently to hepatic protein was 0.26 Ϯ 0.13 nmol equivalent bound/mg protein following a 24-h dose of [14C]FS (10 ␮Ci) and 1.21 mmol/kg FS. Administration of ABT (745 ␮mol/kg i.p.) 1 h before administration of FS resulted in covalent binding levels of Ϯ

0.22 0.06 nmol equivalent bound/mg protein. Serum ALT at ASPET Journals on January 15, 2020 levels in these rats were measured as 16.4 Ϯ 13.5 and 4.6 Ϯ 8.1 U/l, respectively. This data are summarized in Table 1.

Histological Examination of Murine Hepatic Sections The histopathology data corroborated serological evidence and confirmed that FS-treated mice developed mild to mod- erate multifocal hepatic necrosis, predominantly within the centrilobular to midzonal regions (Fig. 2). These livers were graded using predefined criteria and had a mean histopathol- ogy score (n ϭ 4) of 2.2. Control livers were graded 0.

Effect of FS on Hepatic GSH Levels in Vivo in Wistar Rats and Mice FS did not have any significant effect on hepatic GSH Fig. 2. Representative hematoxylin and eosin-stained hepatic sections levels in either rats or mice. Mean hepatic GSH levels were from mice that had received vehicle (PEG) only (A) and FS (1.21 mmol/kg; 24 h) (B). Arrows indicate: bold right arrow, intact hepatocytes; medium not significantly altered compared with control 1, 5, and 24 h right arrow, congested sinusoids; and light right arrow, necrotic area. CV, following a 1.21 mmol/kg i.p. dose of FS to male CD-1 mice. In central vein.

TABLE 1 Overview of in vivo metabolism and bioactivation of FS CD-1 mice and Wistar rats received ͓14C͔FS (151 ␮mol/kg; 10 ␮Ci) with or without a 1-h predose of ABT (745 ␮mol/kg). Values are expressed as mean percentage turnover Ϯ S.E.M. of excreted dose in 0- to 1-h collection of bile. Covalent binding values are in vivo levels of FS bound to hepatic tissue expressed as nanomoles of equivalent bound per milligram of protein following 24-h dose of FS (1.21 mmol/kg; 10 ␮Ci), in the presence and absence of ABT (745 ␮mol/kg; 1-h predose)

FS-Glutathione N-Dealkylated % ␥-Ketocarboxylic FS Covalent Binding of FS Dose of FS FS Conjugate FS Acid Glucuronide to Hepatic Tissue

␮mol % nmol equivalent bound/mg protein Wistar rat FS 37.8 34.4 Ϯ 3.8 8.7 Ϯ 2.8 21.1 Ϯ 2.9 22.1 Ϯ 3.3 12.8 Ϯ 1.8 0.26 Ϯ 0.13a FS ϩ ABT 37.8 52.6 Ϯ 5.2 20.8 Ϯ 0.6 7.4 Ϯ 1.1 19.1 Ϯ 1.1 0.22 Ϯ 0.06a CD-1 mouse FS 5.3 89.6 Ϯ 24.7 3.1 Ϯ 1.5 4.5 Ϯ 1.7 2.7 Ϯ 1.1 1.57 Ϯ 0.98a FS ϩ ABT 5.3 89.6 Ϯ 25.1 2.2 Ϯ 0.2 7.9 Ϯ 0.9 0.11 Ϯ 0.04a a Dose of FS used to assess covalent binding was 1.21 mmol/kg. Furosemide Metabolism and Hepatotoxicity 1213

Over 3 h, 21.2 Ϯ 2.6% (mean Ϯ S.E.M., n ϭ 4) of the dose (37.76 ␮mol) was excreted into bile. Wistar rat bile (0–1-h collection) contained four prominent radiolabeled metabo- lites (Fig. 3A; summarized in Table 1), also detectable by MS (Fig. 3, B–F), in addition to the parent compound (34.4 Ϯ 3.8% of excreted dose). FS (V) corresponded to the single peak in the m/z 329-ion chromatogram (Fig. 3F). MS/MS daughter ion spectra (positive ion) of FS alone showed a major frag- ment at m/z 81, attributable to methyl furan (data not shown). Negative ion MS/MS daughter ion spectra of FS alone showed fragments at m/z 285 (loss of 44 amu; carbox- ylic acid) and at m/z 205 (further loss of 80 amu; methyl furan; data not shown). The most polar metabolite (I) corre- sponded to the single peak in the m/z 636-ion chromatogram (Fig. 3B) and was identified as a novel FS GSH conjugate (FS-SG; 8.7 Ϯ 2.8% of excreted dose). MS/MS daughter ion spectra (positive ion; Fig. 4A) yielded indications of GSH Downloaded from jpet.aspetjournals.org at ASPET Journals on January 15, 2020

Fig. 4. MS/MS daughter ion spectra: positive ion spectrum of FS-GSH conjugate (m/z 636) (A), negative ion spectrum of N-dealkylated metab- olite of FS (m/z 249) (B), and negative ion spectrum of FS-glucuronide (m/z 505) (C).

conjugation. A fragment at m/z 308 was attributable to GSH. A loss of 129 amu resulting in the fragments at m/z 507 and 179 is characteristic of cleavage at the ␥-glutamyl-cysteinyl peptide linkage within a GSH residue (Maggs et al., 1995). The second most polar metabolite (II) corresponded to the single peak in the m/z 249-ion chromatogram (Fig. 3C). This was identified as an N-dealkylated metabolite of FS (21.1 Ϯ 2.9% of excreted dose). MS/MS daughter ion spectra (nega- tive ion; Fig. 4B) showed fragments at m/z 205 (loss of 44

amu; CO2) and 78 (NSO2), confirming the presence of these groups with the metabolite and indicating metabolic loss of the alkyl group. A second novel metabolite (III) was also identified (22.1 Ϯ 3.3% of excreted dose; Fig. 3D) and corre- sponded to the single peak in the m/z 363-ion chromatogram (Fig. 5). Accurate mass analysis of the ion indicated m/z 363.0041 (Fig. 5), consistent with an elemental composition

of C12H12N2O7SCl, suggesting the addition of H2 and O2 to furosemide. Accurate mass MS/MS daughter ion spectra (negative ion, Fig. 5) identified fragments at m/z 319.0155 (loss of 43.9871 amu; COO) and m/z 275.0257 (loss of 87.9767; 2ϫ COO), respectively. The detected loss of 2ϫ COO suggests that two carboxylic acid groups are present in the metabolite. A fragment ion at m/z 204.9828 indicated a molecular for- Ϫ mula of C6H6N2O2SCl ( 1.1-mDa error) consistent with the Fig. 3. Radiochromatogram (A) and LC-MS ion-current chromatograms ϩ (B–F): B, m/z 636; C, m/z 249; D, m/z 363; E, m/z 505; and F, m/z 329 for loss of 114.0342 (loss of methyl furan H2O2) from the [14C]FS (V) and its metabolites (I–IV) in male Wistar rat bile (0–1 h). fragment at m/z 319. The loss of 114 amu is evidence of FS 1214 Williams et al.

Fig. 5. Accurate mass MS/MS daugh- ter ion spectra of ␥-ketocarboxylic acid metabolite of FS from Wistar rat bile. Downloaded from jpet.aspetjournals.org

hydrolysis to either a dihydrodiol or a ␥-ketocarboxylic acid. interesting to note that no FS-SG conjugate was observed to Further confirmation of the metabolic alteration to the furan be formed in the CD-1 mouse. ring was demonstrated by absence of an m/z 81 fragment, attributable to methyl furan, seen in the positive MS/MS Deuterium Exchange for Determination of daughter ion spectra (data not shown). However, MS/MS ␥-Ketocarboxylic Acid Formation at ASPET Journals on January 15, 2020 analysis could not differentiate between a dihydrodiol or a If metabolism of FS forms a ␥-ketocarboxylic acid, this ␥-ketocarboxylic acid metabolite of FS (see NMR analysis). would contain four exchangeable protons, whereas a dihydro- Glucuronidation of FS was also evident in analysis of Wistar diol metabolite would contain five exchangeable protons un- rat bile. FS glucuronide (IV; 12.8 Ϯ 1.8% of excreted dose) der negative ion MS. Negative ion MS of metabolite III gave was the least polar of the four identified metabolites and pre-exchange peaks at 363.003 and 364.9962 (chlorine iso- ϩ ϩ corresponded to the single peak in the m/z 505-ion chromato- tope). Postdeuterium exchange ( D2O CD3OD), peaks gram (Fig. 3E). MS/MS daughter ion spectra (negative ion; were observed at 367.0222 and 369.0211, giving a strong Fig. 4C) demonstrated a loss of 176 amu to provide a frag- indication that a ␥-ketocarboxylic acid metabolite of FS was ment at m/z 329. A fragment at m/z 175 was attributable to formed. glucuronic acid with a loss of 18 amu (water), also producing a fragment at m/z 113 following further loss of 18 (water) and Nuclear Magnetic Resonance Analysis of the 44 (COO) amu. The glucuronic acid was shown not to have ␥-Ketocarboxylic Acid Metabolite of FS from Wistar Rat Bile conjugated on the furan ring by the presence of a fragment at The 1H NMR spectrum of the metabolite (Fig. 6, A and B) m/z 81, attributable to methyl furan, in positive MS/MS shows the absence of the furan ring and the presence of two ␮ daughter ion spectra. A predose of ABT (745 mol/kg), 1 h mutually coupling CH2 groups. This suggests that the me- before FS administration, demonstrated complete inhibition tabolism has affected the furan ring. The [13C]NMR shifts of ␥ of P450-mediated metabolism of FS to the -ketocarboxylic these two CH2 groups, obtained from an heteronuclear single acid metabolite and GSH conjugate (data not shown). quantum correlation experiment, are observed at 35.0 and Anesthetized male CD-1 mice demonstrated lower levels of 29.1 ppm, respectively. Analysis of the heteronuclear multi- biliary excretion in comparison with Wistar rats when ad- ple-bond correlation spectroscopy data shows correlations 14 ␮ ␮ ministered i.v. with [ C]FS (10 Ci; 151 mol/kg). Over 3 h, from both of these CH2 groups to carbonyl carbons at 175.5 11.01 Ϯ 2.60% (mean Ϯ S.E.M.; n ϭ 4) of the dose (5.3 ␮mol) and 204.7 ppm. A further correlation to the carbonyl at 204.7

was excreted into bile. However, of this dose, 89.6 Ϯ 24.7% ppm is observed from a CH2 singlet at 4.386 ppm. These was the parent compound. Radiometric HPLC demonstrated observations are consistent with metabolism of the furan

FS turnover to three minor radiolabeled metabolites with ring to a C(O)CH2CH2COOH moiety (data not shown). similar retention times to ␥-ketocarboxylic acid (4.5 Ϯ 1.7%), FS glucuronide (2.7 Ϯ 1.1), and the N-dealkylated metabolite Metabolism and Cytotoxicity of FS toward Primary Wistar of FS (3.1 Ϯ 1.5%) identified in Wistar rat bile. Following a Rat and CD-1 Mouse Hepatocytes predose of ABT (745 ␮mol/kg), 1 h before FS administration, Murine Hepatocytes. FS was metabolized to N-dealkyl- formation of ␥-ketocarboxylic acid was entirely inhibited. It is ated FS, ␥-ketocarboxylic acid, and FS glucuronide by murine Furosemide Metabolism and Hepatotoxicity 1215

Fig. 6. A, 1H NMR spectrum of isolated ␥-ketocar-

boxylic acid metabolite of FS in d4-MeOH. Inset, magnification of the mutually coupled protons of the 1 1 methyl groups from C19 and C20.B, H- H NMR spectrum (correlation spectroscopy) of isolated ␥-ke-

tocarboxylic acid metabolite of FS in d4-MeOH. The mutually coupled protons from the methyl groups

(20-H2 and 19-H2) of the metabolized furan ring. Downloaded from jpet.aspetjournals.org

TABLE 2 Overview of in vitro metabolism and bioactivation of FS in CD-1 mouse hepatocytes Primary CD-1 mouse hepatocytes were exposed to FS (1 mM FS for both metabolism and toxicity studies; this included 0.1 ␮Ci/ml ͓14C͔FS for metabolism and covalent binding studies) for 6 h. Metabolism and toxicity dependence upon CYP450 was assessed through the use of ABT (5 mM, 1 h predose). Values are expressed as mean Ϯ S.E.M. of four independent hepatocyte isolations. For the metabolic studies, values are expressed as mean percentage of total metabolites following radiochromatographic integration of peaks: *, p Ͻ 0.05, FS compared with control; and †, p Ͻ 0.05 FS and ABT compared with FS alone (Mann-Whitney U test).

Metabolism FS FS-Glutathione N-Dealkylated FS ␥-Ketocarboxylic Acid FS Glucuronide

Conjugate at ASPET Journals on January 15, 2020

% FS 33.3 Ϯ 6.0 11.0 Ϯ 1.7 19.3 Ϯ 3.9 33.3 Ϯ 5.8 FS ϩ ABT 68.2 Ϯ 3.8 38.2 Ϯ 8.0 Cytotoxicity Toxicity Total GSH Content Covalent Binding of FS to Hepatic Tissue Trypan Blue Dye Exclusion MTS Reduction

% control nmol equivalent bound/mg protein FS 63.1 Ϯ 4.3* 65.9 Ϯ 4.8* 77.6 Ϯ 19.6 0.73 Ϯ 0.1* FS ϩ ABT 93.6 Ϯ 3.8† 102.2 Ϯ 5.2† 89.4 Ϯ 10.1 0.11 Ϯ 0.03† hepatocytes. In hepatocyte incubations in the presence of FS (Table 3; Fig. 8, A and C). Significant dose-and time- ABT, only the FS glucuronide metabolite could be detected dependent decreases in cell viability were observed both by (see Table 2). Similar to the in vivo situation, there was no trypan blue dye exclusion and the MTS assay. Cytotoxicity significant dose- or time-dependent GSH depletion observed was correlated with dose- and time-dependent increases in in CD-1 mouse hepatocytes incubated with FS (Table 2; Fig. irreversible binding of FS to hepatocyte protein (Fig. 8, B and 7, A and C). Significant dose-and time-dependent decreases D). In the presence of ABT, there was a significant decrease in cell viability were observed both by trypan blue dye exclu- in irreversible binding, and cell viability returned to control sion and the MTS assay. Cytotoxicity was correlated with values (Table 3). dose- and time-dependent increases in irreversible binding of FS to hepatocyte protein. Covalent binding was observed to In Vitro Bioactivation of FS in CD-1 Mouse Liver plateau after approximately 1 h and at FS concentrations Microsomes and Covalent Binding to Mouse, Rat, and above 1 mM (Fig. 7, B and D). In the presence of ABT, there Human Liver Microsomes was a significant decrease in irreversible binding, and cell Covalent binding was observed in CD-1 mouse, Wistar rat, viability returned to control values (Table 2). and human microsomal preparations in the presence of Wistar Rat Hepatocytes. FS was metabolized to a FS-SG NADPH. Inclusion of GSH in these incubations returned the conjugate, N-dealkylated FS, ␥-ketocarboxylic acid, and FS level of irreversible binding to background level, as observed glucuronide by rat hepatocytes. Hepatocyte incubations in in the absence of NADPH (Fig. 9A). the presence of ABT, only the FS glucuronide metabolite Coincubation of NAL and NAC with FS in a NADPH- could be detected (see Table 3). In contrast to the in vivo dependent CD-1 mouse liver microsomal system led to the situation, there was significant dose- or time-dependent GSH identification of a FS NAL/NAC double adduct. MS/MS depletion observed in Wistar rat hepatocytes incubated with daughter ion spectra analysis of m/z 660 (negative ion; Fig. 1216 Williams et al. Downloaded from jpet.aspetjournals.org

Fig. 7. Relationship between dose- and time-dependent FS cytotoxicity with total GSH content and irreversible binding in the CD-1 mouse hepatocyte. A, demonstration of the dose-dependent cytotoxic effect of FS (0–2 mM; 6 h) to freshly isolated CD-1 mouse hepatocytes (f) compared with total 14 cellular GSH levels (F). B, dose-dependent relationship between cell viability and irreversible binding of FS (6 h; 0–2 mM; 0.1 ␮Ci [ C]FS) (F) to CD-1 at ASPET Journals on January 15, 2020 mouse hepatocyte protein with resulting cytotoxicity (f). C, time-dependent relationship between cell viability and cellular GSH content after FS (1 mM) administration. D, time-dependent relationship between cell viability and irreversible binding of FS (1 mM; 0.1 ␮Ci [14C]FS) (F) to CD-1 mouse hepatocyte protein with resulting cytotoxicity (f). Cytotoxicity was determined by trypan blue exclusion and MTS reduction. Cytotoxicity and GSH content are expressed as percent control. Irreversible binding is expressed as nanomoles of equivalent bound per milligram of protein. Data are given p Ͻ 0.01 when comparing incubations for FS cytotoxicity or ,ءء ;p Ͻ 0.05 ,ء .as mean (ϮS.E.M.) of five to eight independent hepatocyte isolations irreversible binding with control incubations (Mann-Whitney U test).

TABLE 3 Overview of in vitro metabolism and bioactivation of FS in Wistar rat hepatocytes Primary Wistar rat hepatocytes were exposed to FS (1 mM FS for both metabolism and toxicity studies; this included 0.1 ␮Ci/ml ͓14C͔FS for metabolism and covalent binding studies) for 6 h. Metabolism and toxicity dependence upon CYP450 was assessed through the use of ABT (5 mM, 1 h predose). Values are expressed as mean Ϯ S.E.M. of four independent hepatocyte isolations.

Metabolism FS FS-Glutathione Conjugate N-Dealkylated FS ␥-Ketocarboxylic Acid FS Glucuronide

% FS 4.2 Ϯ 2.1 5.3 Ϯ 2.6 17.6 Ϯ 1.4 16.6 Ϯ 2.3 62.3 Ϯ 4.0 FS ϩ ABT 46.0 Ϯ 6 60.3 Ϯ 4.7 Cytotoxicity Toxicity Total GSH Content Covalent Binding of FS to Hepatic Tissue Trypan Blue Dye Exclusion MTS Reduction

% control nmol equivalent bound/mg protein FS 50.2 Ϯ 7.4** 53.9 Ϯ 4.2** 48.6 Ϯ 6.9** 2.4 Ϯ 0.3*** FS ϩ ABT 87.8 Ϯ 5.1†† 95.2 Ϯ 3.6†† 86.8 Ϯ 2.8†† 0.2 Ϯ 0.1††† For the metabolic studies, values are expressed as mean percentage of total metabolites following radiochromatographic integration of peaks: *, p Ͻ 0.05; **, p Ͻ 0.01, FS compared with control; and †, p Ͻ 0.05; ††, p Ͻ 0.01; †††, p Ͻ 0.005, FS and ABT compared with FS alone (Mann-Whitney U test).

9B) showed that the loss of 412 amu was a prominent peak pyrrole ring structure as well as the loss of the carboxylic in the spectra (m/z 249), indicating loss of the NAL and acid group. NAC groups, along with the pyrrole ring, effectively leav- ing N-dealkylated FS. The peak at m/z 487 is most proba- Discussion bly the result of loss of the NAC group (loss of 173 amu), retaining the pyrrole structure and NAL group. The loss of We have demonstrated that bioactivation of the furan ring 129 amu provided a fragment at m/z 532 that was attrib- represents the mechanism of metabolic activation and hepa- utable to cleavage adjacent to the cysteine sulfur atom. totoxicity of FS. Characterization of three key novel metab- The fragment at m/z 205 indicated loss of the adduct- olites from in vivo and in vitro studies, FS ␥-ketocarboxylic Furosemide Metabolism and Hepatotoxicity 1217 Downloaded from jpet.aspetjournals.org

Fig. 8. Relationship between dose- and time-dependent FS cytotoxicity with total GSH content and irreversible binding in the Wistar rat hepatocyte. A, demonstration of the dose-dependent cytotoxic effect of FS (0–2 mM; 6 h) to freshly isolated Wistar rat hepatocytes (f) compared with total cellular GSH levels (F). B, dose-dependent relationship between cell viability and irreversible binding of FS (6 h; 0–2 mM; 0.1 ␮Ci [14C]FS) (F) to Wistar rat hepatocyte protein with resulting cytotoxicity (f). C, time-dependent relationship between cell viability and cellular GSH content after FS (1 mM) at ASPET Journals on January 15, 2020 administration. D, time-dependent relationship between cell viability and irreversible binding of FS (1 mM; 0.1 ␮Ci [14C]FS) (F) to Wistar rat hepatocyte protein with resulting cytotoxicity (f). Cytotoxicity was determined by trypan blue exclusion and MTS reduction. Cytotoxicity and GSH content are expressed as percent control. Irreversible binding is expressed as nanomoles of equivalent bound per milligram of protein. Data are given p Ͻ 0.01 when comparing incubations for FS cytotoxicity or ,ءء ;p Ͻ 0.05 ,ء .as mean (ϮS.E.M.) of five to eight independent hepatocyte isolations irreversible binding to control incubations (Mann-Whitney U test). acid, FS-SG conjugate, and a mixed N-acetyl lysine and N- study. The glucuronide of FS has been shown to be unreac- acetyl cysteine conjugate, is primary evidence (Fig. 10). Bio- tive in terms of covalent binding (Bolze et al., 2002), and activation was dependent upon cytochrome P450 enzymes, compared with other acyl glucuronides, we have observed led to hepatocellular necrosis, in the absence of glutathione this using the synthetic FS acyl glucuronide, which remained depletion. Biliary, hepatocyte, and microsomal studies have stable for up to 96 h at physiological pH. provided evidence of two reactive, potentially toxic, metabo- Previous work suggested that an epoxide of FS is respon- lites, FS epoxide and FS enedial (Fig. 10). The relative con- sible for hepatotoxicity observed in the mouse. FS covalently tribution of each metabolite to covalent binding and toxicity binds to microsomal protein, and the level of binding paral- remains unknown. lels severity of necrosis. Binding is decreased by cysteine, The major metabolic route of FS in man is glucuronidation. GSH, and by the presence of P450 inhibitors, sug- The glucuronide metabolite of FS has been found in human gesting bioactivation of the FS molecule (Mitchell et al., urine and plasma (Beermann et al., 1975, 1977; Benet, 1979; 1974). Covalent binding was found to be increased in the Perez et al., 1979; Smith et al., 1980; Kerremans et al., 1982; presence of an epoxide hydrolase inhibitor (Wirth et al., Smith and Benet, 1983; Hammarlund-Udenaes and Benet, 1976), and no covalent binding was detected with tetrahydro- 1989). We demonstrate the formation of FS-glucuronide in furosemide, an analog in which the furan ring is reduced and both Wistar rat and CD-1 mouse bile. We confirmed the cannot form an epoxide (Wirth et al., 1976). structure of FS-glucuronide through synthesis of the authen- Demonstration of in vivo metabolism of FS to ␥-ketocar- tic standard, which coeluted with the glucuronide found in boxylic acid and GSH adduct are novel findings with respect bile (data not shown). Our findings are supported by reports to understanding the mechanism of FS toxicity. Both metab- that have identified FS-glucuronide by MS at m/z [M-H]Ϫ 505 olites directly indicate the formation of an epoxide and a and 507 (Mizuma et al., 1998). FS-1-O-acyl glucuronide has ␥-ketoenal reactive intermediate. FS can be metabolized to been reported to be formed by rat liver microsomes incubated ␥-ketocarboxylic acid, which is excreted in bile, which further with FS (Rachmel et al., 1985). This report observed an indicates that FS epoxide is the reactive intermediate. The [M-1]Ϫ ion at mass 505, the aglycone fragment at m/z 329, instability of FS epoxide is evident in the formation of ␥-ke- and the sugar fragment ion of mass 175, as they were in this tocarboxylic acid and the levels of covalent binding observed. 1218 Williams et al.

covalent binding to protein. Murine hepatocytes do not form FS-SG and show no signs of GSH depletion; however, cytotoxicity is observed in murine hepatocytes. We con- clude that covalent binding is the cause of cytotoxicity in the murine hepatocyte. In rat hepatocytes, GSH depletion and covalent binding may contribute to cytotoxicity, hence greater cytotoxicity seen in the rat hepatocytes, compared with mouse hepatocytes. In vitro, there is more covalent binding intermediate formed in the rat, compared with mouse. At lower doses (0.5–1 mM) and earlier time points (1–2 h), there are differences in the cytotoxicity between the rat and the mouse. This is attributable to the de- creased levels of GSH in the rat hepatocytes, due to GSH conjugation of FS, allowing more conjugation to mouse hepatocyte protein, making them more susceptible to in vitro cytotoxicity. At higher doses and later time points, cytotoxicity is similar between the species, with the rat

hepatocytes having greater covalently bound material as- Downloaded from sociated. To define the ultimate hepatotoxin, we employed the simultaneous assessment of both metabolism and toxicity in vivo with a sensitive and nonsensitive species. FS was shown to be toxic to mice at 5 h, with ALTs significantly

increased (Wong et al., 2000), whereas severe toxicity ap- jpet.aspetjournals.org pears at 24 h (Mitchell et al., 1974). However, the same dose of FS in rats had no effect on serum ALT levels. The CYP450 inhibitor, ABT, prevented ␥-ketocarboxylic acid formation, indicating formation of ␥-ketocarboxylic acid and FS-epoxide is CYP450 mediated. In vivo, there was Fig. 9. A, graph showing NADPH-dependent irreversible binding of FS to 6-fold higher covalent binding of FS to mouse liver com- CD-1 mouse, Wistar rat, and human liver microsomes (2 mg). Microsomal

pared with the rat. ABT reduced, by 14-fold, the level of at ASPET Journals on January 15, 2020 incubations were carried out in the absence of NADPH (gray bar), in the presence of NADPH (1 mM; black bar), and in the presence of NADPH (1 covalently bound FS in vivo to mouse hepatic protein. ABT mM) and GSH (1 mM; white bar) for 1 h. B, MS/MS daughter ion spectra: protected against FS toxicity, demonstrating that forma- negative ion spectrum of NAL/NAC FS-adduct (m/z 660). tion of the toxic metabolite is P450-dependent. Table 1 provides an overview of turnover of FS in mouse and rat, Electrophilicity of FS epoxide results in attacks on nucleo- alongside levels of covalent binding in vivo. philic macromolecules, hence the covalent binding, which An important observation is the correlation between irre- may allow for GSH conjugation. FS has no effect on hepatic versible binding to hepatic protein and serum ALT activity, GSH levels in vivo. GSH depletion was observed in rat pri- in light of the lack of GSH depletion. In the mouse, FS mary hepatocytes. In agreement with the literature, hepatic epoxide can bypass the hepatic GSH pools and bind to pro- GSH levels and cytosolic and mitochondrial GSH content tein. In contrast to that seen previously with FS, predeple- were not affected by a 1.21 mmol/kg dose of FS in mice tion of hepatic GSH in mice did not lead to exacerbation of (Mitchell et al., 1974; Wong et al., 2000). Previously, FS was hepatotoxicity (Mitchell et al., 1973). The observation of a shown to have no effect on GSH content or cell viability in rat GSH conjugate in rats, yet not in mice, may help explain hepatocytes (Takagi et al., 1991). Depletion (70%) in cellular species susceptibility. In addition, the overall turnover to GSH has been reported in mouse hepatocytes (Grewal et al., stable metabolites is much greater in the rat (66% turnover) 1996), in agreement with other in vitro mouse hepatocyte than the mouse (10% turnover). This would lead to a contin- studies (Massey et al., 1987). Depletion was observed at 3 h, uously higher hepatic load of FS in the mouse liver compared with lactate dehydrogenase leakage, cytosolic [Ca2ϩ] appear- with the rat liver, allowing covalent binding, initiating cell ance, and protein sulfhydryl depletion being observed at 4 h. death. This discrepancy could be due to more rapid elimination in We incubated FS with NAL and NAC in a mouse liver vivo. However, in these previous studies, with only 1-h lag microsomal system to examine whether FS undergoes N- between GSH depletion and cytotoxicity, the GSH depletion substituted cysteinyl pyrrole formation, similar to ipomeanol seen in mice in vitro is a consequence of the cytotoxicity (Baer et al., 2005). The detection of an NAL/NAC-FS adduct rather than the cause. Importantly, in the previous studies, in vitro provides further evidence of FS bioactivation and an neither FS metabolism nor bioactivation were assessed. alternative metabolic pathway to that seen in vivo. In vitro, In rat hepatocytes, which are able to form a GSH conju- FS forms a highly unstable ␥-ketoenal that can be trapped by gate, GSH depletion became significant at 100 ␮M(6h) equimolar amounts of NAL and NAC, further evidence of and at 1 mM (1 h), and significant cytotoxicity became epoxide formation. evident at 750 ␮M (6 h) and 1 mM (4 h). This suggests that Screening systems are required to identify the potential of formation of FS-SG leading to rat hepatocyte GSH deple- novel compounds to cause toxicity in humans at the earliest tion was the cause of the in vitro cytotoxicity, along with stage of drug development. Although FS is not a toxic threat Furosemide Metabolism and Hepatotoxicity 1219 Downloaded from jpet.aspetjournals.org at ASPET Journals on January 15, 2020

Fig. 10. Major pathways of metabolism of furosemide in vivo. Broken arrow, postulated pathway for ␥-ketocarboxylic acid via a reactive epoxide intermediate. Roman numerals, peaks from MS identification in Fig. 3. to humans, we provide evidence that furan moieties can be organ, similar to that observed with the hepatotoxicity of bioactivated to epoxide intermediates and that an unsubsti- tamoxifen in rodents versus humans (Boocock et al., 2000). tuted furan, of the type found in FS, should be regarded as a safety issue. The lack of hepatotoxicity observed in humans with FS will be a function of the extent of epoxidation of the Acknowledgments furan ring and the dose of FS required for efficacy (1–2 mg/kg We acknowledge the Medical Research Council and the generosity human) relative to the dose required for toxicity [400 mg/kg of Safety Sciences Europe, Pfizer (Sandwich, UK) for the generation of the histopathology slides, in particular, Virgile Richard and Peter in mice; compare clozapine with olanzapine (Uetrecht, O’Brien. 2001)]. Certain substructures are considered structural alerts and a potential hazard, and the risk of hepatotoxicity References will be a function of dose, fractional clearance of reactive Baer BR, Rettie AE, and Henne KR (2005) Bioactivation of 4-ipomeanol by CYP4B1: metabolite, efficiency of metabolic biotransformation, and adduct characterization and evidence for an enedial intermediate. Chem Res downstream events, such as induction of cell defense. It is Toxicol 18:855–864. Beermann B, Dalen E, and Lindstrom B (1977) Elimination of furosemide in healthy imperative that downstream functional biological markers subjects and in those with renal failure. Clin Pharmacol Ther 22:70–78. are assessed during preclinical screening, on top of assess- Beermann B, Dalen E, Lindstrom B, and Rosen A (1975) On the fate of furosemide in man. Eur J Clin Pharmacol 9:51–61. ment of GSH conjugate formation and/or covalent binding to Benet LZ (1979) Pharmacokinetics/pharmacodynamics of furosemide in man: a re- protein. In the case of FS, in vitro hepatocyte models and the view. J Pharmacokinet Biopharm 7:1–27. Bolze S, Bromet N, Gay-Feutry C, Massiere F, Boulieu R, and Hulot T (2002) CD-1 mouse are incorrect predictors of the safety profile of FS Development of an in vitro screening model for the biosynthesis of acyl glucuronide in humans. The lack of FS-induced hepatotoxicity in man can metabolites and the assessment of their reactivity toward human serum albumin. Drug Metab Dispos 30:404–413. be explained through a rigorous assessment of both qualita- Boocock DJ, Maggs JL, Brown K, White INH, and Park BK (2000) Major inter- tive and quantitative drug metabolism within the target species differences in the rates of O-sulphonation and O-glucuronylation of alpha- 1220 Williams et al.

hydroxytamoxifen in vitro: a metabolic disparity protecting human liver from the exanthematic pustulosis (AGEP) in a patient treated with furosemide. Am J Med formation of tamoxifen-DNA adducts. Carcinogenesis 21:1851–1858. Sci 320:331–333. Dargie HJ and Dollery CT (1975) Adverse reactions to diuretic drugs, in Meyler’s Owens CW and Belcher RV (1965) A colorimetric micro-method for the determina- Side Effects of Drugs (Dukes MNG ed) p 483, Exerpta Medica, Amsterdam, The tion of glutathione. Biochem J 94:705–711. Netherlands. Perez J, Sitar DS, and Ogilvie RI (1979) Biotransformation of furosemide in patients Druckova A and Marnett LJ (2006) Characterization of the amino acid adducts of the with acute pulmonary edema. Drug Metab Dispos 7:383–387. enedial derivative of teucrin A. Chem Res Toxicol 19:1330–1340. Pirmohamed M, Williams D, Madden S, Templeton E, and Park BK (1995a) Metab- Goldring CE, Kitteringham NR, Elsby R, Randle LE, Clement YN, Williams DP, olism and bioactivation of clozapine by human liver in vitro. J Pharmacol Exp Ther McMahon M, Hayes JD, Itoh K, Yamamoto M, et al. (2004) Activation of hepatic 272:984–990. Nrf2 in vivo by acetaminophen in CD-1 mice. Hepatology 39:1267–1276. Pirmohamed M, Williams DP, Madden S, Templeton E, and Park BK (1995b) Grewal KK, Rafeiro E, and Racz WJ (1996) Bromobenzene and furosemide hepato- Metabolism and bioactivation of clozapine by human liver in vitro. J Pharmacol toxicity: alterations in glutathione, protein thiols, and calcium. Can J Physiol Exp Ther 272:984–990. Pharmacol 74:257–264. Ponto LL and Schoenwald RD (1990) Furosemide (frusemide): I. A pharmacokinetic/ Hammarlund-Udenaes M and Benet LZ (1989) Furosemide pharmacokinetics and pharmacodynamic review. Clin Pharmacokinet 18:381–408. pharmacodynamics in health and disease–an update. J Pharmacokinet Biopharm Rachmel A, Hazelton GA, Yergey AL, and Liberato DJ (1985) Furosemide 1-O-acyl 17:1–46. glucuronide: in vitro biosynthesis and pH-dependent isomerization to beta- Kerremans AL, Tan Y, Van Ginneken CA, and Gribnau FW (1982) Specimen han- glucuronidase-resistant forms. Drug Metab Dispos 13:705–710. dling and high-performance liquid chromatographic determination of furosemide. Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83. J Chromatogr 229:129–139. Smith DE and Benet LZ (1983) Biotransformation of furosemide in kidney trans- Liu J, Liu Y, Parkinson A, and Klaassen CD (1995) Effect of oleanolic acid on hepatic plant patients. Eur J Clin Pharmacol 24:787–790. toxicant-activating and detoxifying systems in mice. J Pharmacol Exp Ther 275: Smith DE, Lin ET, and Benet LZ (1980) Absorption and disposition of furosemide in 768–774. healthy volunteers, measured with a metabolite-specific assay. Drug Metab Dispos Madden S, Maggs JL, and Park BK (1996) Bioactivation of carbamazepine in the rat 8:337–342. in vivo: evidence for the formation of reactive arene oxide(s). Drug Metab Dispos Takagi S, Takayama S, and Onodera T (1991) Hepatotoxicity of DR-3438, tienilic 24:469–479. acid, indacrinone and furosemide studied in vitro. Toxicol Lett 55:287–293.

Maggs JL, Williams D, Pirmohamed M, and Park BK (1995) The metabolic formation Uetrecht J (2001) Prediction of a new drug’s potential to cause idiosyncratic reac- Downloaded from of reactive intermediates from clozapine, a drug associated with agranulocytosis in tions. Curr Opin Drug Discov Dev 4:55–59. man. J Pharmacol Exp Ther 275:1463–1475. Vandeputte C, Guizon I, Genestie-Denis I, Vannier B, and Lorenzon G (1994) A Massey TE, Walker RM, McElligott TF, and Racz WJ (1987) Furosemide toxicity in microtiter plate assay for total glutathione and glutathione disulfide contents in isolated mouse hepatocyte suspensions. Toxicology 43:149–160. cultured/isolated cells: performance study of a new miniaturized protocol. Cell Biol Mitchell JR, Jollow DJ, Potter WZ, Davis DC, Gillette JR, and Brodie BB (1973) Toxicol 10:415–421. Acetaminophen-induced hepatic necrosis: I. Role of drug metabolism. J Pharmacol Wirth PJ, Bettis CJ, and Nelson WL (1976) Microsomal metabolism of furosemide Exp Ther 187:185–194. evidence for the nature of the reactive intermediate involved in covalent binding. Mitchell JR, Potter WZ, Hinson JA, and Jollow DJ (1974) Hepatic necrosis caused by Mol Pharmacol 12:759–768.

furosemide. Nature 251:508–511. Wong SG, Card JW, and Racz WJ (2000) The role of mitochondrial injury in bromo- jpet.aspetjournals.org Mitchell JR, Snodgrass WR, and Gillette JR (1976) The role of biotransformation in benzene and furosemide induced hepatotoxicity. Toxicol Lett 116:171–181. chemical-induced liver injury. Environ Health Perspect 15:27–38. Mizuma T, Benet LZ, and Lin ET (1998) High-performance liquid chromatographic Address correspondence to: Dr. Dominic Williams, Drug Safety Research determination and identification of acyl migration and photodegradation products Group, Department of Pharmacology and Therapeutics, University of Liver- of furosemide 1-O-acyl glucuronide. J Chromatogr B Biomed Sci Appl 718:153– pool, Sherrington Buildings, Ashton Street, Liverpool, Merseyside L69 3GE, 162. UK. E-mail: [email protected] Noce R, Paredes BE, Pichler WJ, and Krahenbuhl S (2000) Acute generalized at ASPET Journals on January 15, 2020