Cytochrome P450 11A1 Bioactivation of a Kinase Inhibitor in Rats

Article pubs.acs.org/crt

Cytochrome P450 11A1 Bioactivation of a Kinase Inhibitor in Rats: Use of Radioprofiling, Modulation of Metabolism, and Adrenocortical Cell Lines to Evaluate Adrenal Toxicity Donglu Zhang,† Oliver Flint,† Lifei Wang, Ashok Gupta,§ Richard A. Westhouse,† Weiping Zhao,† Nirmala Raghavan,† Janet Caceres-Cortes,† Punit Marathe,† Guoxiang Shen,† Yueping Zhang,† Alban Allentoff,‡ Jonathan Josephs,† Jinping Gan, Robert Borzilleri,‡ and W. Griffith Humphreys*,† †

Department of Pharmaceutical Candidate Optimization, ‡Chemistry, and §Discovery Medicine and Clinical Pharmacology, Bristol-Myers Squibb Research & Development, Princeton, New Jersey 08543, United States S Supporting Information *

ABSTRACT: A drug candidate, BMS-A ((N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl) 2oxo-1,2-dihydropyridine- 3-carboxamide)), was associated with dose- and time-dependent vacuolar degeneration and necrosis of the adrenal cortex following oral administration to rats. Pretreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, ameliorated the toxicity. In vivo and in vitro systems, including adrenal cortex-derived cell lines, were used to study the mechanism responsible for the observed toxicity. Following an oral dose of the C-14 labeled compound, two hydroxylated metabolites of the parent (M2 and M3) were identified as prominent species found only in adrenal glands and testes, two steroidogenic organs. In addition, a high level of radioactivity was covalently bound to adrenal tissue proteins, 40% of which was localized in the mitochondrial fraction. ABT pretreatment reduced localization of radioactivity in the adrenal gland. Low levels of radioactivity bound to proteins were also observed in testes. Both M3 and covalent binding to proteins were found in incubations with mitochondrial fraction isolated from adrenal tissue in the presence of NADPH. In vitro formation of M3 and covalent binding to proteins were not affected by addition of GSH or a CYP11B1/2 inhibitor, metyrapone (MTY), but were inhibited by ketoconazole (KTZ) and a CYP11A1 inhibitor, R-(+)-aminoglutethimide (R-AGT). BMS-A induced apoptosis in a mouse adrenocortical cell line (Y-1) but not in a human cell line (H295R). Metabolite M3 and covalent binding to proteins were also produced in Y-1 and to a lesser extent in H295R cells. The cell toxicity, formation of M3, and covalent binding to proteins were all diminished by R-AGT but not by MTY. These results are consistent with a CYP11A1-mediated bioactivation to generate a reactive species, covalent binding to proteins, and subsequently rat adrenal toxicity. The thorough understanding of the metabolism-dependent adrenal toxicity was useful to evaluate cross-species adrenal toxicity potential of this compound and related analogues.

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reproduction, and development.3 The adrenal gland is histologically and functionally divided into three zones: the outer zona glomerulosa, the intermediate zona fasciculata, and the inner zona reticularis.4 Each zone is selectively responsible for the synthesis of mineralocorticoid, and glucocorticoid steroid hormones and some androgens.5 Steroid production is regulated by, among others, the adrenocorticotrophic hormone (ACTH) via cAMP-mediated protein kinase A pathways6,7 that

INTRODUCTION BMS-A is an analogue from a series of potent MET kinase inhibitors intended for use in the treatment of cancer.1,2 The compound was found to induce atrophy of the adrenal cortex (zona fasciculata and zona reticularis) after administration to rats in early efficacy and toxicology evaluations. The objective of this study was to determine whether the adrenal toxicity was mediated by metabolic bioactivation using in vitro incubations with adrenal fractions and cell cultures to aid in cross-species risk assessment for BMS-A and other related MET kinase inhibitors. The adrenal gland is the most important steroidogenic tissue among the three endocrine organs (along with the testes and ovaries) that synthesize all steroids, and is vital for health, © 2012 American Chemical Society

Special Issue: Use of Radioisotope-Labeled Compounds in Drug Metabolism and Safety Studies Received: December 2, 2011 Published: February 1, 2012 556

dx.doi.org/10.1021/tx200524d | Chem. Res. Toxicol. 2012, 25, 556−571

Chemical Research in Toxicology

Article

(Grand Island, NY). Apo-ONE Homogeneous caspase-3/7 Assay Kit was purchased from Promega (Madison, WI, #G7791). Water was prepared with a PICOpure ultrapure water system from Hydro Service and Supplies (Research Triangle Park, NC). Sucrose-Tris buffer contained 0.25 M sucrose and 15 mM Tris-HCl (pH 7.4), and glycerol-Tris buffer contained 20% glycerol and 25 mM Tris-HCl (pH 7.4). All other chemicals used were of reagent grade or better. Protein concentrations were determined by the Lowry method.20 Animals. Male Sprague−Dawley rats (200−300 g) were purchased from Charles River Laboratories (Wilmington, MA) and were housed and maintained under standard conditions recommended by Animal Care and Use Committee (ACUC) in a 12 h light/12 h dark, constant temperature environment with free access to rat chow and drinking water. Rat Studies for Histopathology Evaluation. Animals were orally administered a 10 or 30 mg/kg dose of BMS-A as a suspension in 70% PEG400 in water. Rats were evaluated after a single dose or after 7 daily doses (n = 3 for each group). For the pretreatment group, rats were administered 50 mg/kg of ABT solution in water by intraperitoneal injection 2 h before drug dose. At study termination (24 h after last dose), adrenal glands were collected, fixed in 10% neutral buffered formalin, processed to hematoxylin and eosin stained slides, and evaluated by light microscopy for drug-related changes. BDC Rat Studies. Animals were acclimatized for 1 week prior to surgery and were subjected to bile duct/jugular vein cannulation under light anesthesia. Animals were allowed to recover for at least 24 h prior to compound administration. Bile was collected 24 h prior to experiment and was infused into the duodenum via a duodenal cannula during the experiment. Rats were fasted overnight and fed 4 h post dose. Experiments were performed on fully conscious, freely moving animals. Two groups of rats were administered a single dose of [14C]BMS-A (15 mg/kg, 199.5 μCi/kg) by oral gavage. Group 1 rats (n = 3) received only [14C]BMS-A (3 mg/mL as a suspension in 70% PEG400 in water) and group 2 rats (n = 3) were pretreated with ABT (50 mg/ kg, IP) 2 h before receiving [14C]BMS-A. Urine, bile, and feces were collected for 0−24 h. Plasma samples were collected from the rats at 1, 2, 4, 8, and 24 h after dosing. At study termination (24 h), adrenal glands, livers, brains, testes, and GI and contents were collected for metabolite characterization. Radioactivity Determination of in Vitro and in Vivo Samples. Individual bile and urine radioactivity was measured in duplicate by adding 50 μL aliquots of each sample to 15 mL of Ecolite liquid scintillation cocktail. Duplicate aliquots of the individually homogenized GI tract and fecal samples (approximately 0.2 g each) and other tissues (approximately 0.075 g each) were weighed accurately then digested through the addition of 1 mL of Soluene-350. The samples were incubated overnight with gentle shaking in a 60 °C water bath. Upon cooling, 0.2 mL of 30% hydrogen peroxide and 15 mL of Ecolite were added to each sample. Radioactivity in all samples was determined by counting on a Packard Tri-Carb model 2250CA liquid scintillation counter (Packard Biosciences, Downers Grove, IL). Radioactivity in bile, urine, and feces, and GI content samples was calculated by scaling the average radioactivity in aliquots to the total samples collected over each time interval in each matrix. Radioactivity levels from in vitro samples were determined after mixing aliquots of incubations, extraction supernatants, or digested or dissolved protein solutions with 15 mL of Ecolite before counting. Sample Preparation and Analysis. Tissue Homogenates. Homogenates of brain, testes, liver, feces, and GI contents were prepared in 5 volumes of 50:50 (v/v) ACN and water. Pooled homogenates of liver, brain, testes, and GI content were prepared by combining equal volumes from individual homogenate samples of each animal in the same group. Pooled bile, urine, and fecal samples were prepared by combining a constant volume percentage from each sample excreted during 0−24 h for each treatment group. Pooled plasma samples were prepared by combining equal volumes of the three samples collected at each time point (1, 2, 4, 8, and 24 h) for each group. Adrenal glands were homogenized in 0.25 M sucrose-

selectively increase mineralocorticoid synthesis from cholesterol. For this synthesis, cholesterol must be transported from the outer to the inner mitochondrial membrane by the steroidogenic acute regulatory proteins (StAR).8 ACTH activation of steroid hormone synthesis can be mimicked by the addition of cAMP or forskolin, a diterpene derived from C. forskohkii, that is a specific adenylate cyclase inhibitor that activates the protein kinase A pathway.9 Angiotensin II activates the protein kinase C pathway resulting in increased production of aldosterone.10 Activation of protein kinase A or C pathways will result in induced expression of genes encoding steroidogenic enzymes and transporter proteins and consequently increase secretion of cortisol and aldosterone, respectively.11 Steroidogenic enzymes are responsible for the synthesis of the active steroid hormones corticosterone, cortisol, and aldosterone in adrenals, and estradiol and testerosterone in gonads.12 These enzymes are hydroxysteroid dehydrogenases and membrane-bound P450 enzymes that include the mitochondrial P450 enzymes (CYP11A1 and 11B1/2) and microsomal P450 enzymes (CYP21, CYP17, and CYP19). In contrast to microsomal P450 enzymes that use flavincontaining cytochrome P450 oxidoreductase to transfer electrons, mitochondrial P450 enzymes, CYP11A1 and CYP11B1/2, use a flavin−protein adrenodoxin reductase (ferredoxin reductase) and a nonheme iron−sulfur protein adrendoxin (ferredoxin) to transfer electrons from NADPH.13 CYP11A1, also known as P450scc, is a cholesterol side chain cleavage enzyme responsible for the first step to convert cholesterol to all steroids, and is expressed in adrenal glands, gonads, placenta, and brain.12 The general steroidogenic pathways are listed in Supporting Information, Figure 1S. Most biosynthetic steroidogenic steps in the adrenal cortex are generally similar among rats, mice, and humans, supporting the usefulness of rodents as a toxicology model. However, the predominant glucocorticoid in the rodent is corticosterone compared to cortisol in humans and other higher mammals. H295R cells express all key steroidogenic enzymes and produce all three classes of steroid hormones.14,15 In this study, we report an analysis of the toxicity and metabolism of BMS-A in rodents and in vitro adrenal cell lines, including a comparison of the toxicity of BMS-A in human (H295R) and mouse (Y-1) adrenocortical cell lines. The human H295R cell line has been widely used to study adrenocortical functional regulation of steroidogenesis and screening for enzyme inhibitor.16−18 Mouse Y-1 cells express the functional enzymes implicated in both the glucocorticoid and mineralocorticoid pathways.19

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MATERIALS AND METHODS

Materials. BMS-A (N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3fluorophenyl)-1-(4-fluorophenyl) 2-oxo-1,2-dihydropyridine-3-carboxamide) and [14C]BMS-A (13.3 μCi/mg, radiochemical purity 98.0%) as well as metabolites M1 and M25 were synthesized by Medicinal Chemistry, Bristol-Myers Squibb. Ecolite liquid scintillation cocktail was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). Soluene-350 was purchased from Packard Biosciences (Downers Grove, IL). Deepwell LumaPlate-96-well plates were purchased from PerkinElmer Life Sciences (Boston, MA). ABT, R-(+) and S(−)-AGT, MTY, KTZ, NADPH, GSH, hydrogen peroxide (30%), NMR solvents, and Ham’s F-12 medium were purchased form SigmaAldrich Co. (St. Louis, MO), and formic acid was purchased from J.T. Baker (Phillipsburg, NJ). NuSerum and ITS+ Premix were from BD Biosciences (San Diego, CA) and penicillin−streptomycin and Dulbecco’s modified Eagle’s medium were from Life Technologies 557



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Tris buffer (1 mL) and centrifuged as described in the adrenal gland fractionation section found below. Each fraction was suspended in 1 mL of 20% glycerol-Tris buffer, and the protein concentration was determined. Plasma Extraction. Pooled rat plasma samples (0.3 mL) were extracted two times with 1 mL of 50:50 (v/v) methanol/ACN. After the addition of organic solvents from each extraction step, samples were vortexed to resuspend the protein precipitate and then sonicated for 5 min. The extracted samples were centrifuged at 4,000g for 10 min at 5 °C, and the supernatants from each centrifugation step were combined. The radioactivity in the combined supernatants was determined. The protein pellet was digested in 1 N NaOH overnight and counted for radioactivity. The combined supernatants were evaporated to dryness at room temperature under a stream of nitrogen gas. The dried residues were reconstituted in 100 μL of ACN/water (65:35, v/v). The samples were then centrifuged at 4,000g for 10 min at 5 °C prior to analysis. Adrenal Gland, Liver, Brain, and Testes Homogenate Extraction. To each homogenate sample (0.2 mL for adrenal gland, 0.2 mL for livers, and 1 mL for brains and testes) were added 2 volumes of methanol and 30 volumes of ACN containing 1% acetic acid (v/v). The samples were centrifuged at 4000g for 60 min. The extraction was repeated twice. Radioactivity was determined in the supernatants from each extraction step and the combined supernatants. The radioactivity in the second extracted supernatant was generally at background level. The protein pellet was dissolved in 0.5 mL of 4 M urea, and the protein concentration was determined. Aliquots of the supernatants of each extraction and protein solution were counted for radioactivity. The combined supernatant was concentrated and reconstituted in 100 μL of ACN/water (65:35, v/v) prior to analysis. Bile. Aliquots of pooled rat bile samples (200 μL) were diluted with 0.8 mL of water prior to analysis. Urine. Aliquots of pooled rat urine (1 mL) were centrifuged at 4,000g for 10 min at 5 °C prior to analysis. Feces. Pooled fecal homogenate (1.0 mL) was extracted by the addition of 4 mL of methanol/ACN (1:1, v/v) and mixed on a vortex mixer for 10 min then centrifuged at 4000g at 5 °C for 15 min. The extraction was repeated one more time, and supernatants were combined. The recovery of radioactivity from the extraction was quantitative. The extract was evaporated under nitrogen to dryness. The residue was reconstituted in 1.0 mL of the buffer, vortexed, and centrifuged for 5 min at 4000g prior to analysis. Fractionation of Adrenal Glands for in Vitro Incubations and Incubation Conditions. Adrenal glands from 15 male 6-week old rats were collected (approximately 2 g tissue/rat) and homogenized in 12 mL of sucrose-Tris buffer using a tissue homogenizer. After centrifugation at 600g for 15 min at 4 °C, the cell debris/lysosome pellet was resuspended in 2.7 mL of glycerol-Tris buffer. The homogenate supernatant was centrifuged at 9000g for 15 min. The mitochondrial pellet was washed with 10 mL of sucrose-Tris buffer and suspended in 3.2 mL of the glycerol-Tris buffer to give a preparation containing 17.2 mg protein/mL. An S9 fraction was prepared by centrifugation at 100,000g for 60 min. The microsome pellet was washed with 5 mL of the sucrose buffer and suspended in 2.5 mL of the glycerol-Tris buffer to give a preparation containing 11 mg protein/mL. The cytosol fraction (9.5 mL) contained 13.3 mg protein/mL. [14C]BMS-A (15 μM) was incubated in triplicate with rat adrenal fractions (1 mg protein/ml) of homogenate, nuclei/cell debris/ lysosomes, mitochondria, cytosol, and microsomes in 0.1 M sodium phosphate buffer (pH 7.4) in the presence of NADPH (1.5 mM) for 1 h at 37 °C. Inhibitors of P450 metabolism, MTY (50 μM) and ABT (1 mM), were included in some incubations. The final volume of ACN in the incubation mixtures of 0.25 mL was 0.5% (v/v). The metabolismdependent inhibitor ABT was preincubated in the presence of NADPH for 15 min before the substrate was added. Incubation mixtures were extensively extracted with 2 volumes of methanol and 30 volumes of ACN containing 1% acetic acid (v/v). ACNprecipitated protein was washedand dissolved in 4 M urea, and radioactivity and protein concentrations were determined. The

combined supernatant was concentrated, reconstituted, and analyzed by HPLC and LC/MS. Further incubations of [14C]BMS-A (15 μM) with rat mitochondrial fractions (1 mg/mL protein) were performed in sodium phosphate buffer (0.1 M, pH 7.4) for 1 h at 37 °C in the presence of NADPH (1.5 mM), GSH (5 mM), KTZ (2 μM), S- or R-AGT (30 μM), or M1 (30 μM). The final volume of ACN in the incubation mixtures was 0.5% (v/v). Samples were prepared for analysis as in the preceding section. In addition, [14C]BMS-A was incubated in triplicate with pooled rat liver microsomes, and a combination of rat liver microsome and adrenal mitochondrial fractions. The incubation mixtures (0.25 mL) contained sodium phosphate buffer (0.1 M, pH 7.4), liver microsomes (1 mg protein/mL), or mitochondria (1 mg/mL), [14C]BMS-A (15 μM), and NADPH (1.5 mM). The final ACN content in incubations was 0.25% (v/v). Reactions were initiated with the addition of NADPH and incubations continued for 30 min at 37 °C with shaking (90 rpm). After incubation, ice-cold ACN (0.5 mL) was added to stop the reaction and samples centrifuged at 4000g for 5 min prior to analysis. Incubations with Y-1 and H295 Cell Cultures. H295R (ATCC CRL-2128) and Y-1 (ATCC CCL-79) cell lines were grown at 37 °C, respectively, in Dulbecco’s modified Eagle’s medium and Ham’s F12 medium containing 2.5% NuSerum, 1% ITS+ premix, and 1% penicillin−streptomycin under 5% CO2 in air following a published procedure.21 Culture passages were done by trypsin digestion and dilution of 1 to 4 every 2 weeks (just prior to reaching confluency) with medium changes of 2 to 3 times per week. Cultures of mouse Y-1 and human H295R adrenocortical cell lines were exposed to increasing concentrations of BMS-A (1−200 μM) in the presence or absence of general CYP inhibitors (ABT, KTZ), a specific inhibitor of CYP11A1 (R-AGT) or CYP11B1/2 (MTY), or P450 inducers (ACTH or forskolin). Caspase 3/7 activity, a measure of the induction of apoptosis, was more sensitive than other measures such as total cell ATP. Caspase-3/ 7 activity was assayed using the Apo-ONE Homogeneous caspase-3/7 Assay Kit following the manufacturer’s instructions.22 Metabolite Isolation from Y-1 Cells. Incubations of [14C]BMS-A (10 μM) was performed in Y1 cells for 24 h in 5 × 75 cm culture flasks. The combined supernatant was concentrated and metabolites separated using HPLC conditions as described below. Fractions containing M3 were collected, evaporated under a stream of nitrogen, and prepared for NMR analysis. Approximately 50 μg of M3 was isolated from a total of 10 injections, based on radioactivity determination. Proton NMR data and TOCSY with wet solvent suppression were collected on a Bruker 600 MHz instrument in 3 mm tubes (Bruker Corp., Billerica, MA). Cell pellets were prepared and analyzed using the conditions described in the adrenal fractions section. HPLC and Radioprofiling. HPLC analysis was performed on an Agilent 1100 series system equipped with two pumps, an autoinjector, and a UV detector (Agilent Technologies, Santa Clara, CA). Samples for radioactivity profiling (bile, urine, plasma, tissue homogenate extracts, in vitro incubations, and cell culture supernatants) were injected onto an ACE-3 C18 column (4.6 mm × 150 mm, 3 μm). The column was maintained at room temperature and monitored at a wavelength of 330 nm. Separation of metabolites was achieved using a linear-gradient consisting of mobile phase A and B:A (0.1% formic acid in water and B, 0.1% formic acid in ACN). The mobile phase flow rate was 1 mL/min. The gradient program used to elute the samples from the column started from 5% B and was increased to 20% in 5 min, 45% in 55 min, 50% in 5 min, 90% in 2 min, and then decreased to 5% B in 3 min before equilibration. The retention times of reference standards were confirmed by their UV absorbance and LC/MS/MS analysis. For quantification of radioactivity, the HPLC eluate was collected in 0.25min intervals on Deepwell LumaPlate-96-well plates with a Gilson Model FC 204 fraction collector (Gilson, Middleton, WI). Fractions of column eluates were evaporated to dryness on a Savant Speed-Vac (Savant Instruments Inc., Holbrook, NY) and were counted for 558



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Figure 1. (a) Adrenal gland cortex from a Sprague−Dawley rat treated with vehicle and (b) single-oral dose of BMS-A (10 mg/kg). (c) Adrenal gland cortex from a Spraque-Dawley rat pretreated with ABT (50 mg/kg, IP) 2 h prior to the administration of BMS-A (10 mg/kg). Data Analysis. Disintegrations per minute for the LSC were determined from measured CPM values with a manufacturer provided quench curve. For each radiochromatographic profile, the average CPM values of several early wells were designated as background (approximately 3 CPM) and subtracted from each subsequent fraction. Profiles were prepared by plotting the resulting net CPM values against time-after-injection. A constant quench was assumed for all fractions. Radioactive peaks in the chromatographic profiles were reported as a percentage of the total radioactivity recovered during the HPLC run. The mean plasma concentration versus time data for radioactivity and unchanged parent were analyzed with a noncompartmental method.23 The peak plasma concentration (Cmax) and the time to reach peak concentration (Tmax) were recorded directly from experimental observations. Total radioactivity was converted to parent ng-equivalents based on the radioactive specific activity of 13.3 μCi/

radioactivity for 10 min with a Packard Top Count microplate scintillation analyzer (Packard Biosciences, Downers Grove, IL). Identification of Metabolites by LC/MS/MS. LC/MS and LC/ MS/MS analyses were performed on pooled bile, urine, cell culture supernatants, and extracts of feces, plasma, and tissue homogenates. Analyses were performed on a Finnigan LTQ ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The samples were analyzed in the electrospray ionization (ESI) mode (positive ion mode). The HPLC separation was performed using conditions as described above. The HPLC flow was split by an Accurate splitter (LC Packings, San Francisco, CA), and only one-fourth of HPLC effluent was directed to the mass spectrometer through a valve set to divert the flow to waste from 0 to 2 min. The capillary temperature used for analysis was set at 250 °C. The nitrogen gas flow rate, spray current, and voltages were adjusted to give maximum sensitivity for the parent compound. 559



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Figure 2. Concentration- and time-dependent toxicity in adrenal cell lines (Y-1 and H295R) as measured by caspase activity (panels A and B). The results from a 24-h incubation of the cell lines with increasing concentrations of BMS-A are shown in panel A. The results of a time course experiment examining the activity upon exposure to 100 μM BMS-A are shown in panel B. Enzyme inhibitor-dependent toxicity of BMS-A in adrenal cell lines of Y-1 and H295R measured by caspase activity (panels C and D). The results found with R-AGT (2 h pretreatment, followed by 18 h coincubation with 0 or 50 μM BMS-A) are shown in panel C, and the results found with MTY (2 h pretreatment, followed by 18 h coincubation) are shown in panel D. mg. Parent concentrations were based on the conversion of CPM values contained in the HPLC fraction corresponding to the parent retention time. The area under the plasma concentration versus time curve from 0 to 24 h (AUC0−24), was calculated by a combination of conventional trapezoidal and log-trapezoidal methods by Kinetica (version 4.4). Protein covalent binding as pmol radioactivity-equivalent/mg proteins was calculated based on radioactivity counts, amount of the protein in the sample, and radiospecific activity of the compound. Incubations were generally run in triplicate measurements, and results are expressed as the mean ± standard deviation. Statistical analysis was performed using one-way analysis for variance (ANOVA) followed by Dunnett’s test to compare the findings in different groups, and values of P < 0.05 are considered significant.

dose withdrawal in rats at low multiples of the anticipated efficacious exposures. After 7-daily doses at 10 mg/kg, toxicity observations included moderately decreased cortical thickness, loss of zona glomerulosa, and laminar fibrosis and contraction of the zona reticularis. At 3 mg/kg, there were no histological findings after a single dose, but moderate vacuolar degeneration in all layers of the cortex with the most significant in the zonae glomerulosa and reticularis was observed after 7-daily doses. At 30 mg/kg, cortical necrosis and hemorrhage were observed in the zona reticularis even after a single dose. The histology results from rat adrenal tissue following multiple and single high level oral doses of BMS-A are shown in Supporting Information Figures 3S and 4S. In addition, urine corticosterone concentrations decreased with increased single doses of 3, 10, and 30 mg/kg, and the decreases were >95% from normal levels after seven daily doses at 10 and 30 mg/kg. This finding was not reversible after a 7-day recovery period. The adrenal toxicity was recapitulated in vitro in Y-1 and H295R adrenal cell lines with a higher sensitivity in Y-1 cells. Drug-induced alterations in the caspase 3/7 activities in Y-1 and H295R cells are shown in Figure 2A, B. The compound induced caspase activity in a dose- and time-dependent manner

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RESULTS Toxicity Observations in Rat Adrenal Gland and Adrenocortical Cell Lines. Vacuolar degeneration with single cell necrosis in the adrenal gland cortex was observed after a single oral dose of BMS-A at 10 mg/kg (Figure 1b) and was absent in control rats (Figure 1a). Pretreatment with ABT prevented adrenal toxicity (Figure 1c). Adrenal toxicity was time- and dose-dependent and irreversible with compound 560



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Table 1. (A) Radioactivity Concentrations in Plasma and Tissues of Ratsa and (B) Radioactivity Distribution and Protein Covalent Binding in Adrenal Tissue Fractions of Ratsb (A) concentrations in μg/g or μg-eq/g (parent or metabolites, total radioactivity) (% of extractable radioactivityc) rat groupd time total radioactivity M1 parent M2 M3

1 2 1 2 1 2 1 2 1 2

plasma 1h 9.6 5.6 7.17 (74.7) 1.18 (21.2) 1.58 (16.5) 4.18 (74.6) tracee trace trace trace

2h 13.3 6.6 11.1 2.32 1.53 4.17

(83.6) (35.2) (11.5) (63.2)

4h 17.8 8.9 15.0 3.96 1.14 4.54

(84.2) (44.5) (6.4) (51.0)

8h 21.9 11.1 20.4 6.58 1.27 4.15

(93.1) (59.3) (5.8) (37.4)

24 h 25.9 19.8 24.6 (94.9) 14.9 (75.2) 0.13 (0.5) 4.06 (20.5)

adrenal gland

liver

brain

testes

24 h 470.5 144.7 4.55 (17.6) 3.36 (15) 3.88 (6) 7.16 (31.9) 8.28 (32.5) 4.84 (21.6) 0.26 (1) 1.26 (5.6)

24 h 14.6 18.5 8.75 (67) 6.11 (33) 0.22 (1.7) 7.96 (43) NDf ND ND ND

24 h 1.2 3.4 0.92 (90.3) 0.51 (15.7) 0.06 (6.3) 2.59 (80.2) ND ND ND ND

24 h 10.7 13.6 1.97 (34.5) 1.60 (14.6) 0.03 (0.6) 4.98 (45.8) 0.76 (13.3) 0.37 (3.4) 0.02 (0.3) 0.21 (1.9)

(B) % radioactivity distribution in isolated subcellular fractions

protein covalent binding (pmol/mg)

fractions

control

ABT

control

ABT

debris/nuclei mitochondria cytosol microsome

31 39 27 3.0

20 39 36 4.9

7990 10700 4910 74% of radioactivity covalently bound to proteins, MTY did not seem to have any effect. Irreversible binding was proportional to the formation of metabolite M3, which represented approximately 25% of the total metabolism of this compound in mitochondrial incubations. Incubations of [14C]BMS-A in mitochondrial fractions provided results that demonstrated that radioactivity covalently bound to proteins was inhibited by >90% by KTZ and R-AGT (Table 2B). KTZ and R-AGT also inhibited the formation of M3. M1 inhibited the amount of radioactivity covalently bound to proteins from the parent compound by approximately 65%. S-AGT and GSH were not effective inhibitors. In addition, no

in mouse (Y-1) but not in human (H-295) cells at concentrations seen in the rat adrenal gland. The cell toxicity levels were detected in Y-1 cell cultures at drug concentrations ≥3 μM (Figure 2A). The toxicity was observed in Y-1 cells after only 2 h of exposure to concentrations equivalent to those measured in vivo in the adrenal gland (Figure 2B). The most sensitive measure of toxicity in vitro was caspase activation, indicating induction of apoptosis. Dose Recovery and Tissue Distribution in Rats. Approximately 29% and 8% of the dose was recovered in BMS-A-treated and BMS-A/ABT-treated rat bile, respectively. Urine recovery was low from both groups. Since the study was conducted only for 0−24 h in order to collect tissues including adrenal glands and testes for metabolite profiling, recovery of radioactivity was not complete. The individual recoveries of radioactivity in bile, urine, feces, and GI tracts of rats administered single oral doses of [14C]BMS-A are shown in the Supporting Information, Table 1S. As shown in Table 1A and B, radioactivity appeared to accumulate in adrenal tissue, and nonextractable radioactivity was 443 and 122 μg/g (3.5-fold decrease) in adrenal glands from BMS-A-treated and BMS-A/ABT-treated rats, respectively. Nonextractable radioactivity was approximately 30-fold higher in adrenal tissue relative to total radioactivity in liver. The level of nonextractable radioactivity in testes was 24.5 and 16.5 pmol/mg from BMS-A-treated and BMS-A/ABT-treated rats, respectively. Upon fractionation of the adrenal glands from rats administered [14C]BMS-A, approximately 40% of radioactivity was found in the mitochondrial fraction in both BMS-A-treated and BMS-A/ABT-treated groups. Approximately 20−30% of 561



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and mass-spectral fragmentation patterns of metabolites to those of synthetic standards provided additional verification of the identity of metabolites M1 and M25, respectively. The major MS fragmentation for the molecule was cleavage of the amide bond; thus, the two fragments of the molecule resulting from amide cleavage were denoted “A” and “B”, as shown in Table 4, to facilitate description of metabolite structural assignments. The positions of conjugation with glucuronic acid were not identified. P. Parent peak P showed a molecular ion [M+H]+ at m/z 459 and major fragment ions at m/z 234, 216, 188, and 160 in LC/MS/MS analysis (Table 4). The major fragmentation was the cleavage of the amide bond to form a fragment ion at m/z 216 that included the A substructure. M1. Metabolite M1 was identified as a lactam metabolite of BMS-A. M1 had a molecular ion [M+H]+ at m/z 475 and showed fragment ions at m/z 234, 216, 188, and 160. This metabolite had the same retention and fragmentation pattern as a synthetic standard. M2. Metabolite M2 had a molecular ion [M+H]+ at m/z 491 and showed fragment ions at m/z 473 (491 − 18), 250 (234 + 16), 232 (216 + 16), and 176 (160 + 16). Metabolite M2 was identified as a hydroxylated lactam (proposed structure shown in Table 4). M3. Metabolite M3 had a molecular ion at m/z 475 and major fragment ions at m/z 232 (216 + 16), 204 (232 − 28), 176 (204 − 28), 148 (176 − 28), and 122, several of which were CO-loss fragment ions. Since M3 was a major metabolite in incubations with Y-1 cell lines, the compound was incubated in Y-1 cells, and the monohydroxy metabolite M3 was isolated from cell culture incubations. Mass-spectral analysis indicated that this isolate had the same mass spectral properties as M3 from rat samples. The structure of M3 was characterized using proton and TOCSY experiments. Loss of resonance H25 is observed in the proton spectrum of the monohydroxylated metabolite as compared to that of the parent. Proton 24 has shifted upfield by 0.6 ppm, and proton 26 has shifted upfield by 0.3 ppm in the metabolite spectrum as compared to that of the parent. The absence of proton resonance 25 in the metabolite spectrum and the upfield chemical shift change of H24 and H26 pointed to hydroxylation at position 25 of the pyridinone ring. These data are consistent with hydroxylation at position 25 of the pyridinone ring. 1H−1H correlations are observed

Table 2. (A) Evaluation of Protein Covalent Binding in Incubations of [14C]BMS-A with Rat Adrenal Fractions and (B) Effects of Enzyme Cofactors and Enzyme Inhibitors on Protein Covalent Binding of [14C]BMS-A in Incubations with Rat Adrenal Mitochondrial Fractionsa (A) protein covalent binding (DPM/mg protein/min) fraction homogenate debris/lysosomes mitochondria microsomes cytosol

(−) NADPH

(+) NADPH

± ± ± ± ±

259 ± 13 271 ± 12 575 ± 28 91.2 ± 2.8 16.9 ± 3.4 (B)

15.5 24.3 42.5 44.8 11.0

4.6 3.9 3.1 5.1 0.7

(+) MTY

(+) ABT

246 ± 33 ND 630 ± 42 ND ND

194 ± 9 140 ± 1 154 ± 31 29.2 ± 5.0 6.0 ± 0.3

covalent binding (pmol/mg protein/h) to rat adrenal mitochondrial proteins condition

pmol/mg/h

% inhibition

no NADPH (+) NADPH (+) GSH (+) KTZ (+) R-AGT (+) S-AGT (+) M1

19.8 ± 4.3 543 ± 58 529 ± 27 25.8 ± 1.6 60.0 ± 1.3 362 ± 31 200 ± 42

NA NA 2.6 98.8 92.3 34.6 65.6

a

ND, not determined. NA = not applicable. NADPH added to all incubations unless otherwise noted.

GSH adducts were identified from the incubations by radioactivity or LC/MS analysis. Results found in Table 3 demonstrate that radioactivity appeared to be covalently bound to cell proteins, which was inhibited by KTZ and to a lesser extent by MTY. Similarly, M3 formation and parent disappearance were also inhibited by KTZ and R-AGT. The metabolism profiles of [14C]BMS-A in Y-1 cells in the absence and presence of inhibitors are presented in Supporting Information, Figure 6S. Metabolite Identification. The metabolite structures were assigned based on LC/MS and LC/MS/MS analysis. A summary of the major ions observed in the mass-spectra, proposed structures, and occurrence of the metabolites are presented in Table 4. Comparison of HPLC retention times

Table 3. Protein Covalent Binding and Metabolite Formation of [14C]BMS-A in Y-1 Cell Cultures in the Absence and Presence of P450 Inhibitors Y-1 Protein covalent binding (pmol/mg) and % inhibition of protein covalent binding conditions

cells

% inhibition

0 min control 24 h incubation (+) KTZ (+) R-AGT (+) MTY

5.7 ± 1.0 390 ± 72 204 ± 32 140 ± 18 268 ± 54

NA NA 48.3 65.0 31.8

24 h incubation (+) KTZ (+) R-AGT (+) MTY

supernatant

% inhibition

% in supernatant

3.6 ± 0.4 NA 45.7 ± 7.1 NA 23.7 ± 3.6 52.2 10.7 ± 2.3 82.5 42.6 ± 5.1 7.0 inhibition of metabolite formation and parent disappearance

NA 12.8 9.4 8.2 13.6

M3

M3a

parent disappearance

NA 59.5 84.9 28.7

NA 59.2 80.3 19.7

NA 60.4 88.4 19.4

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Table 4. LC/MS/MS Characterization (MH+, Major Fragments), Proposed Structures, and Occurrence of BMS-A and Its Metabolitesa

a

M3a was identified in Y-1 and H295R cell incubations (Figure 6), which had a molecular ion at m/z 491 and major fragment ions at m/z 232 and 176.

major fragment ions at m/z 216, 188, and 179, consistent with a trioxygenation metabolite with all modifications on the B substructure. Information on assignment of additional metabolites can be found in Supporting Information, Metabolite Identification. Retention times for all metabolites can be found in Supporting Information Tables 2SA and 2SB. In summary, the metabolic pathways of BMS-A in BDC rats and in vitro incubations include monooxygenation (M1, M3, M5, M11, M14, M16, and M26), dioxygenation (M2, M18, M19, M20, and M21), trioxygenation (M17 and M12), glucuronidation of parent compound (M8 and M9), glucur-

among H25, H24, and H26 in the TOCSY spectrum of the parent compound. The 1H−1H correlations between H24 and H26 are present in the TOCSY spectrum of the monohydroxy metabolite, but correlations among H25 with H24 and H26 are not observed in the spectrum of the metabolite (data not shown). These results support assignment of position 25 as the site of hydroxylation (Figure 3). M3a and M27. These two metabolites were found in culture incubations with Y-1 and H295R cells. M3a had a molecular ion at m/z 491 and major fragment ions at m/z 232, and 176, consistent with a dioxygenation metabolite with one oxygen on the A substructure. M27 had a molecular ion at m/z 507 and 563



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treated rats, M2, M1, and the parent accounted for 21.6%, 15%, and 31.9% of the radioactivity in these samples. In addition, M3 represented 5.6%. M2 and M3 were new metabolites not found in the liver homogenate. In the brain homogenate, the predominant metabolite identified in BMS-A-treated rats was M1 (90.3% of radioactivity). The parent compound represented 6.3% of radioactivity. In BMS-A/ABT-treated rats, M1 was 16%, and the parent accounted for 80% of the total radioactivity. No significant levels of M2 and M3 were found in the brain samples. In the testes homogenate, the predominant metabolite identified in BMS-A-treated rats was M1 (35% of radioactivity). M2 accounted for approximately 14% of the radioactivity. The parent and M3 were minor components. In BMS-A/ABTtreated, the parent and M1 accounted for 46% and 15% of the radioactivity. M2 and M3 were minor components. In adrenal glands and testes, ABT appeared to slow down the conversion of the parent to M1 and to prevent the complete further metabolism of M3. Bile, Urine, and Feces. The predominant components in bile were the parent, M4, M5, and M6 (Tables 2SA and 2SB, Supporting Information). Metabolites accounted for over 70% of the total radioactivity in bile samples. The major metabolite identified in urine was M5. The predominant components identified in feces were the parent and M1. Incubations with Liver Microsomes. Formation of M1 was identified as the major species formed in incubations with rat liver microsomes. Incubations in Mitochondrial Fractions. Incubations of [14C]BMS-A in mitochondrial fraction produced M3 and the formation of M3 was reduced by inclusion of KTZ or R-AGT in incubations (data not shown). An incubation with combined rat liver microsomes and rat adrenal mitochondrial fraction produced M2 in addition to M1 and M3, suggesting that M3 required the action of both liver and adrenal enzymes for formation (Figure 5). Incubations in Cell Cultures. Incubations of [14C]BMS-A in Y-1 and H295R cells produced M3 and minor metabolites M3a and M27 (Figure 6). There was more M3 formation in Y-1 cultures than H295R cultures under the similar culture conditions. Formation of M3 was inhibited by R-AGT and KTZ and to a much less extent by MTY (Figure 5S, Supporting Information). The level of M3 formation and its reduced formation by KTZ and R-AGT correlated with the levels of radioactivity covalent bound to proteins in Y-1 cells (Table 3). Cell Culture Incubations in the Presence of Inhibitors. The drug-induced toxicity in Y-1 cells was diminished by ABT, KTZ, and R-AGT (Figure 2C), and augmented by ACTH that stimulates mitochondrial P450 enzymes (CYP11A1 and 11B1/ 2) (data not shown). The toxicity and metabolism were induced significantly (>100%) by ACTH in Y-1 cells and abrogated dose-dependently by R-AGT but not by MTY (Figure 2D). In contrast, H295R cells were unaffected by the drug, and caspase activity was induced modestly by forskolin but not by ACTH (data not shown). Steroid synthetic activity was slightly greater in H295R than in Y-1 cells. Additional experiments were conducted with siRNAs against P45011A1 and showed a significant amelioration of caspase activation in cell culture and that BMS-A attenuates steroid synthesis in both mouse and human adrenocortical cells with ≥50% reduction in H295R cell cultures at drug concentrations >12 μM (data not shown).

Figure 3. 1D 1H NMR of BMS-A and M3.

onidation of the monooxygenated metabolites (M4, M6, M7, M13, and M15), glucuronidation of the dioxygenated metabolites (M22, M23, and M24), amide hydrolysis of the parent (M25), and O-dearylation of the parent (M10). Dioxygenated metabolite M3a and trioxygenated metabolite M27 were identified in cell culture incubations. Metabolite Profiles. The radiochromatographic profiles of homogenates of plasma, adrenal gland, liver, brains, and testes tissue are shown in Figures 4−5. The radioactivity profiles of bile, urine, and feces are shown in Supporting Information Figures 3SA and 3SB. The distribution profiles of radioactive components in 0−24 h pooled urine, bile, and fecal samples are shown in Supporting Information Tables 2SA and 2SB. Plasma. The radioactivity distribution of the parent and metabolites is shown in Table 1 and Figures 4a, 4SA, and 4SB (Supporting Information). The radiochromatographic profiles are significantly different for the BMS-A-treated and BMS-A/ ABT-treated rats. M1 was the largest radioactive peak at all time points from the BMS-A-treated group and ranged from 75 to 95% of total radioactivity contained in the sample. The parent compound ranged from 0.5 to 16.5%. The parent was the major peak and accounted for about 75−21% of radioactivity in samples from BMS-A/ABT-treated rats; M1 accounted for 21− 75%. The AUC values for the parent and M1 were 41.0 and 960 μM·h and 197.0 and 406 μM·h in BMS-A-treated and BMS-A/ ABT-treated groups, respectively. M1 was identified as a major circulating component (>20× parent AUC). BMS-A/ABTtreatment increased the parent Cmax and AUC by 2.3- and 4.8fold and decreased M1 Cmax and AUC by approximately 40% (Table 1). Tissues Homogenate Profiles. The predominant metabolite identified in BMS-A-treated rat liver homogenate was M1, and the parent compound was less than 2% of the total radioactivity. In BMS-A/ABT-treated samples, M1 and the parent accounted for 33 and 43% of total radioactivity, respectively. In the adrenal gland homogenate from BMS-A-treated rats, the predominant metabolites identified were M2 (32.5% of sample) and M1 (17.6% of sample). The parent and M3 only represented minor components (6 and 1%). In BMS-A/ABT564



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Figure 4. Metabolite profiles from rats following oral administration of [14C]BMS-A (15 mg/kg, 199.5 μCi/kg) (a) Plasma profile without (panel A, 1 hr; panel C, 24 hr) and with pretreatment of ABT (panel B, 1hr; panel D, 24 hr). (b) Liver (panels A, B) and adrenal (panels C, D) tissue profiles at 24 hr without (panels A, C) and with pretreatment of ABT (panels B, D). (c) Brain (panels A, B) and testes (panels C, D) tissue profiles at 24 hr without (panels A, B) and with pretreatment of ABT (panels B, D).

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DISCUSSION

the adrenal toxicity, suggesting that the toxicity might be dependent on metabolism of the compound. To investigate a mechanism that is responsible for the adrenal toxicity, we conducted several sequential experiments. First, detailed metabolic pathways were studied in bile-duct cannulated rats

BMS-A caused adrenal toxicity in rats following oral administration. These toxic effects were seen (albeit with reduced severity) in monkeys and mice, suggesting a common mode of action for the effect. ABT pretreatment ameliorated 565



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Figure 5. Metabolite profiles in incubations of [14C]BMS-A with rat liver microsomes (A), adrenal mitochondrial fraction (B), and a combination of both (C).

Figure 6. Metabolite profiles of [14C]BMS-A in Y-1 and H295R cell cultures. The compound was incubated at 10 μM in the cell cultures for 24 h, and the media were analyzed by HPLC and LC/MS.

following an oral dose of [14C]BMS-A. Following oral

were qualitatively similar among plasma samples at all

14

administration of [ C]BMS-A, the biotransformation profiles

collection time points in BMS-A-treated and BMS-A/ABT566



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Figure 7. Proposed bioactivation pathway of [14C]BMS-A in rat adrenal gland.

treated rats, except that relative quantities of M1 decreased by ABT pretreatment, consistent with a reduction in formation of M1 by the P450 inhibitor. In bile and urine, the biotransformation profiles were also qualitatively similar between the groups. The major metabolism pathways of BMS-A in rats were oxidation and glucuronidation. Approximately 3−7% of the radioactivity in bile and less than 1% of the radioactivity in the urine was attributed to parent compound, indicating that the compound was cleared primarily through metabolism. As metabolite profiles were similar with and without ABT and no metabolites were identified that provided any direction for further investigation of a mechanism for the observed adrenal toxicity, adrenal tissue was profiled directly. Profiling work did reveal unique metabolites not found in liver or plasma. In addition, radioactivity was accumulated in the adrenal gland and covalently bound selectively to mitochondrial proteins. The organ-specific metabolites and radioactivity covalent bound to proteins were both reduced by ABT pretreatment. Subsequently, adrenal specific metabolism of the compound was studied in rat adrenal gland fractions. In vitro incubations in adrenal mitochondrial fraction showed that

metabolite M3, a metabolite that was specific to adrenal gland homogenate, was also formed in mitochondrial fraction in the presence of NADPH. Another metabolite, M2, that was also specific to the rat adrenal gland, was produced in an incubation containing both rat liver microsomes and rat adrenal gland mitochondrial fraction, suggesting that M1 was formed in the liver and delivered to the adrenal gland and further metabolized to M2. Radioactivity covalently bound to proteins was also found in the in vitro incubations. Both M3 formation and radioactivity covalent bound to proteins were inhibited by KTZ and R-AGT, a CYP11A1 inhibitor,24 but not by MTY, an inhibitor for CYP11B1/2.25 These in vivo and in vitro metabolism studies with [14C]BMS-A suggest CYP11A1 mediate biotransformation and bioactivation of the compound in adrenal mitochondria that generate a reactive species leading to compound covalent bound to proteins and subsequent rat adrenal toxicity. Interestingly, adrenal-specific metabolites M2 and M3 were also found in testes homogenates of rats following oral administration of [14C]BMS-A. In addition, a low level of radioactivity covalent bound to proteins was observed in testes. This finding is also consistent with bioactivation of the 567



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Table 5. Comparison of Selected Adrenal Toxicants That Affect Steroidogenic Enzymes

where it was converted to M2 in the adrenal also via an epoxide intermediate. The epoxide intermediates could rearrange to yield M2 and M3 or bind to macromolecules. The proposed site of epoxidation is based on the assignment of the structure of M3, which stemmed from NMR characterization of the metabolite. The understanding of the site of bioactivation was important for efforts to limit this property in subsequent analogues. While the liver metabolite M1 was easily transferred to the adrenal gland, only very trace levels of adrenal-formed metabolites were found in the circulation. A similar mechanism

compound by CYP11A1. CYP11A1 is also expressed in brain tissue.26 However, lack of detectable M2 or M3 in brain tissues is consistent with a much lower level of expression of CYP11A1 in brain than in testes or adrenal glands.10 The proposed bioactivation pathways of BMS-A in the rat adrenal gland is shown in Figure 7. This scheme is similar to that previously proposed for a structurally related substituted pyrazinone.27 The parent could be directly converted to M3 in the adrenal gland via an epoxide intermediate or be converted to M1 in the liver, then M1 was transferred to the adrenal gland 568



Article

dependent with mitochondrial disorganization and disappearance of central cristae observed at 12 and 24 h following a single 3 mg/kg dose; at 6−12 mg/kg, mitochondria vacuolization was followed by mitochondria disappearance at 6 h; at 25 mg/kg, mitochondria lesion led to degeneration/ necrosis of zona fasciculata.40,45 For the Pfizer compound, the 7-day exploratory toxicology studies conducted in rats and dogs resulted in an off-target dose-dependent adrenal finding, characterized by increased cortical vacuolation and adrenal weights. In vitro studies in adrenal microsomes showed the compound inhibited CYP21-hydroxylase that might result in accumulation of an intermediate steroidogenesis metabolite, leading to cytoplasmic vacuoles and increase adrenal weight.38 When considering a clinical development plan for this compound, the absence of sensitive, predictive biomarkers for detection of adrenal toxicity raised concerns. In addition, functional effects often lag the onset and severity of histological effect. Because of high lipid contents, high blood perfusion, high concentrations of P450 enzymes, and containing all steroidogenic enzymes, adrenal glands may be vulnerable to xenobiotics-induced toxicity.26,46 Suppression of adrenocortical function and steroidogenesis may have fatal consequences as demonstrated by etomidate, an anesthetic agent that caused severe adrenal insufficiency followed by patient deaths.47 The role of adrenal enzymes in off-target toxicity effects is poorly understood and under investigated, and specifically, the contibution of alterations of steroidogenic pathways by xenobiotic compounds is not well characterized. Investigation of metabolism by steriodogenic enzymes and cell toxicity in adrenocortical cell lines may offer a screen to help assess adrenal toxicity potential. The results of thorough metabolite identification and covalent binding to protein studies made possible with the use of a radiolabeled version of BMS-A, as well as enzyme inhibitor studies in vitro, in vivo, and in cell culture, support the hypothesis that organ-specific CYP11A1-mediated bioactivation is a key mediator of cellular toxicity of BMS-A. The studies also helped to provide a mechanism-based risk assessment for adrenal toxicity to support the development of a drug candidate and a method to evaluate the potential for adrenal toxicity of BMS-A analogues.

could be followed in the testes leading to radioactivity covalently bound to proteins. Although many epoxide intermediates are trapped by GSH, there were no adducts detected in these experiments. This may be due to lack of thiolreactivity or instability of the GSH adduct. To further investigate the adrenal toxicity of BMS-A and to develop cell models for evaluating the adrenal toxicity potential of subsequent compounds, mouse and human adrenocortical cell lines were tested for their sensitivity to BMS-A exposure. BMS-A induced caspase activity in Y-1 cells but not in H295R cells. While it is possible that the compound might induce some level of caspase activity through the inhibition of MET kinase signaling, the sensitivity of the caspase signal to both specific and general modulators of P450 activity lead to the conclusion that the majority of the signal is produced via the compoundspecific bioactivation pathway. The cell toxicity was abrogated by R-AGT but not by MTY. Y-1 and H295R cells also produced the unique metabolites found in rat adrenal gland homogenate and radioactivity covalent bound to proteins. Therefore, damaged proteins probably contributed to the degradation of biochemical pathways involved in hormone secretion that led to cell apoptosis, and inhibition of CYP11A1mediated bioactivation seemed to correlate with cell toxicity. There was much lower formation of M3 and radioactivity covalent bound to proteins in H295R cells than in Y-1 cells, and there appeared to be a correlation between cell toxicity and the levels of metabolism of this compound. Y-1 and H295R cells were found to be a useful tool to evaluate CYP11A1dependent adrenal toxicity potential, and the result from this study aided in the overall risk assessment of the potential for adrenal toxicity for BMS-A in humans. Protein covalently bound radioactivity was found in Y-1 cells and was reduced by R-AGT, but to a much lower extent by MTY in both culture supernatants and cells. The human H295R cells were much more resistant to BMS-A induced toxicity than the mouse Y-1 cells, and the dose-dependent abrogation of toxicity was demonstrated by R-AGT but not by MTY. Activated caspase-3 plays an important role in triggering the catabolism caspase cascade.28 Caspase-mediated apoptosis changes following exposure of xenobiotics have been reported in cell lines.29,30 The conversion of apoptosis to necrosis has been demonstrated following exposure of high concentration of toxicants for a short time period and a lower concentration for a longer time period.31 Our results indicated that metabolic activation triggered the caspase activity that led to apoptosis and adrenal toxicity. Metabolic activation of mitotane by adrenal mitochondria and covalent binding to proteins directly correlated to the degree of adrenocorticolytical activity when compared in dog, rabbits, rats, and guinea pigs as well as in human in vitro.32 In addition to bioactivation and covalent binding to proteins, mitochondrial P450 enzymes can produce reactive oxygen species and play a role in the induction of mitochondrial apoptosis.33 Although steroidogenic enzymes have high substrate specificity and are in general believed not to involve in metabolism of xenobiotics, there are agents that induce adrenal toxicity through metabolism by these steroidogenic enzymes. Table 5 lists the selected adrenal toxicants that affect steroidogenic enzymes and proteins. 7-Hydroxy 7,12dimethylbenz(a)anthracene (7-OHMe DMBA) is formed in the liver from 7,12-dimethyl DMBA and further bioactivated in the adrenal gland via CYP11B1 leading to adrenal toxicity.43 MeSO2-DDE induced adrenal toxicity in mice was dose-

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ASSOCIATED CONTENT

S Supporting Information *

Steroid biosynthesis pathways, detailed information on structural assignment of metabolites not detailed in the main text, radioactivity recovery and profiling data of plasma, urine, feces and bile after BMS-A administration to rats with or without ABT pretreatment, and metabolite profiles after incubation of BMS-A with Y1 cell cultures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and Development, Princeton, NJ 08543. Phone: 609-252-3636. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Lois Lehman-McKeeman for helpful discussions. 569



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steroidogenesis in the H295R human adrenocortical carcinoma cell line. Toxicol. in Vitro 16, 113−121. (17) Muller-Vieira, U., Angotti, M., and Hartman, R. W. (2005) The adrenocortical tumor cell line CI-H295R as an in vitro screening system for the evaluation of CYP11B2 (aldosterone synthase) and CYP11B1 (steroid-11beta-hydroxylase) inhibitors. J. Steroid Biochem. Mol. Biol. 96, 259−270. (18) Ullerås, E., Ohlsson, Å., and Oskarsson, A. (2008) Secretion of cortisol and aldosterone as a vulnerable target for adrenal endocrine disruption  screening of 30 selected chemicals in the human H295R cell model. J. Applied Toxicol. 28, 1045−1053. (19) Routhier, M. E., Bournat, P., and Ramiraz, L. C. (1995) Aldosterone synthase activity in the Y-1 adrenal cell line. J. Ster. Biochem. Mol. Biol. 52 (6), 581−585. (20) Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. H. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265−275. (21) Oskarsson, A, Ulleras, E, Plant, K. E., Hinson, J. P., and Goldfarb, P. S. (2006) Steroidogenic gene expression in H295R cells and the human adrenal: adrenotoxic effects of lindane in vitro. J. Appl. Toxicol. 26, 484−492. (22) Kashyap, M. P., Singh, A. K., Siddiqui, M. A., Kumar, V., Tripathi, V. K., Khanna, V. K., Yadav, S., Jain, S. K., and Pant, A. B. (2010) Caspase cascade regulated mitochondria mediated apoptosis in monocrotophos exposued PC12 cells. Chem. Res. Toxicol. 23, 1663− 1672. (23) Perrier, D., and Gibaldi, M. (1982) General derivation of the equation for time to reach a certain fraction of steady state. J. Pharm. Sci. 71, 474−475. (24) Harvey, P. W., and Everett, D. J. (2003) The adrenal cortex and steroidogenesis as cellular and molecular target for toxicity: Critical omission from regulatory endocrine disrupter screening strategies for human health? J. Appl. Toxicol. 23, 81−87. (25) Hinson, J. P., and Raven, P. W. (2006) Effects of endocrinedisrupting chemicals on adrenal function. Clin. Endocrinol. Metab. 20, 111−120. (26) Jung-Testas, I., Hu, Z. Y., Baulieu, E. E., and Robel, P. (1989) Steroid synthesis in rat brain cell culture. J. Steroid Biochem. 34, 511− 519. (27) Subramanian, R., Lin, C. C., Ho, J. Z., Pitzenberger, S. M., SilvaElipe, M. V., Gibson, C. R., Braun, M. P., Yu, X, Yergey, J. L., and Singh, R (2003) Bioactivation of the 3-amino-6-chloropyrazinone ring in a thrombin inhibitor leads to novel dihydro-imidazole and imidazolidine derivatives: structures and mechanism using 13C-labels, mass spectrometry, and NMR. Drug Metab. Dispos. 31, 1437−1447. (28) Porter, A. G., and Jänicke, R. U. (1999) Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6, 99−104. (29) Sarabia, L., Maurer, I., and Bustos-Obregon, E. (2009) Melatonin prevents damage elicited by the organophosphorous pesticide diazinon on the mouse testis. Ecotoxicol. Environ. Saf. 72, 938−942. (30) Zhao, M., Zhang, Y., Wang, C., Fu, Z., Liu, W., and Gan, J. (2009) Induction of macrophage apoptosis by an organochlorine insecticide acetofenate. Chem. Res. Toxicol. 22, 504−510. (31) Saini, K. S., Thompson, C., Winterford, C. M., Walker, N. I., and Cameron, D. P. (1996) Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic beta cell line, INS-1. Biochem. Mol. Biol. Int. 39, 1229−1236. (32) Martz, F., and Straw, J. A. (1980) Metabolism and covalent binding of 1-(O-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloro ethane (O,P′-DDD). Correlation between adrenocorticolytical activity and metabolism by adrenocortical mitochondria. Drug Metab. Dispos. 8, 127−130. (33) Derouet-Humbert, E., Roemer, K., and Bureik, M. (2005) Adrenodoxin (Adx) and CYP11A1 (P450scc) induce apoptosis by the generation of reactive oxygen species in mitochondria. Biol. Chem. 386, 453−61. (34) Raven, P. W., and Hinson, J. P. (1996) Transport, Action, And Metabolism of Adrenal Hormones and Pathology and Pharmacology

ABBREVIATIONS ACN, acetonitrile; ACTH, adrenocorticotrophic hormone; ABT, 1-aminobenzotriazole; R-AGT, R-(+)-aminoglutethimide; BDC, bile duct cannulated rats; CPM, counts per minute; GSH, reduced glutathione; KTZ, ketoconazole; LC/MS/MS, liquid chromatography tandem mass spectrometry; MTY, metyrapone; P450, cytochrome P450.

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